dhplc principles an introduction to denaturing high performance liquid chromatography pooria gill...
TRANSCRIPT
DHPLC PrinciplesAn Introduction to
Denaturing High Performance Liquid Chromatography
Pooria GillPhD of Nanobiotechnology
pooriagillyahoocom
In The Name of Allah
Nucleic Acids
bull DNAndash Linear
bull Nature-madendash High Molecular
Weightndash Low Molecular
Weightbull Man-made
ndash High Molecular Weight
ndash Low Molecular Weight
ndash Circularbull Nature-made
ndash High Molecular Weight
ndash Low Molecular Weight
bull Man-madendash High Molecular
Weightndash Low Molecular
Weight
bull RNAbull Nature-made
ndash High Molecular Weight
ndash Low Molecular Weight
bull Man-madendash High Molecular
Weightndash Low Molecular
Weight
Nucleic Acids OMICS
bull Genomicsndash Various analyses of DNAs
bull Qualitativebull Quantitative
bull Transcriptomicsndash Various analyses of RNAs
bull Qualitativebull Quantitative
Genomics Variations as MutationPolymorphism
bull Scanning Procedures for Unknown Mutation Detections those simple methods which rely on differences in electrophoretic properties being generated between mutant and wild-type nucleic acid by point mutations (these methods cannot as currently used detect all mutations do not localize them within the fragment and can only be applied to DNA fragments hundreds of bases long)
bull Screening Procedures for Known Mutation Detections those group which have the potential to detect all mutations Sequencing is more frequently used to detect unknown mutations than it is for diagnostic purposes
The Principles for Gene Variation Analysis
bull Bioinformatics
bull Biothermodynamics Biophysical-Chemistry
bull Spectroscopics
bull Electrophoretics
bull Chromatographics
Scanning Procedures
ndash Ribonuclease cleavage (RNAase)
ndash Denaturing gradient-gel electrophoresis
(DGGE) and related techniques
ndash Carbodiimide modification (CDI)
ndash Chemical cleavage of mismatch (CCM)
ndash Single-strand conformation polymorphism
(SSCP)
ndash Heteroduplex analysis (HET)
ndash Direct sequencing (DS)
Scanning Procedures
RGH Cotton Mutation Research 285 (1993) 125-
144
Screening Procedures
bull Allele-specific oligonucleotide (ASO)
bull Allele-specific amplification (ASA)
bull Ligation (LIG)
bull Primer extension (PEX)
bull Artificial introduction of restriction sites
(AIRS)
Screening Procedures
RGH Cotton Mutation Research 285 (1993) 125-144
Heteroduplex analysis (HET) Keen J et al (1991) Rapid detection of single base mismatches as
heteroduplexes on hydrolink gels Trends Genet 7 5
bull Heteroduplexes containing single base-pair mismatches can be accurately separated from related heteroduplexes on nondenaturing gels
bull Others performed separation of heteroduplexes on normal gels which detected deletions but their method probably would not detect point mutations and can thus be considered a different method of lesser sensitivity
bull Thus far there have been few modifications The main advantage of the HET method is simplicity (as for SSCP) but its application has not been so widespread partly because of its later description and partly because of the need for Hydrolink gels in the initial description
bull The main disadvantage is the lack of 100 detection Like SSCP the HET method can only be applied to fragments hundreds of base pairs long (for example 200-300 bp)
Technological Improvement of HET to eg DHPLC
bull Patent Information1 Column matrix
ndash 1Bonn G Huber C Oefner P (1994) Verfahren zur Trennung von Nucleinsaeuren Austrian Patent No 398 973 Vienna Austria
ndash 2Bonn G Huber C Oefner P (1996) Nucleic Acid Separation on Alkylated Nonporous Polymer Beads US Patent No 5585236
ndash Currently exclusively licensed to Transgenomic Inc Omaha NE USA
DHPLCndash 3Oefner PJ Underhill PA (1998) Detection of Nucleic Acid Heteroduplex
Molecules by Denaturing High-Performance Liquid Chromatography and Methods for Comparative Sequencing US Patent 5795976 [Stanford Reference] [USPTO]
ndash 4Hansen NF Oefner PJ (1997) Software to Determine Optimum Temperature for DHPLC Given DNA Sequence Stanford University Invention Disclosure S97-175 Tangible Research Property in conjunction with US Patent 5795976 [Stanford Reference]
ndash Currently licensed to Transgenomic Inc Omaha NE USA Agilent Palo Alto CA USA and Varian Walnut Creek CA USA
ndash 5Oefner PJ (1999) Detection of Polymorphisms by Denaturing High-Performance Liquid Chromatography US Patent 6453244 [Stanford Reference] [USPTO]
ndash 6Huber CG OKeefe M Oberacher H Oefner PJ Premstaller A Xiao W Temperature-Modulated Array High-Performance Liquid Chromatography Provisional Patent filed [Stanford Reference]
Technological Improvement of HET to eg DHPLCbull Patent Information2 DNA shearing
ndash 7Oefner PJ Hunicke-Smith S (1998) Apparatus and Methods for Shear Breakage of Poly-nucleotides US Patent 5846832 [Stanford Reference] [USPTO]
ndash Licensed to Gene Machines Redwood City CA USA
DNA markersndash 8Oefner PJ Underhill PA Human Y Chromosome Specific
Single Nucleotide Polymorphisms US Patent Pending [Stanford Reference]
Principle of DHPLC
Rapid denaturation of DNA by heating re-annealing by slow cooling
Heteroduplexes form in the presence of two different alleles
DHPLCGraphical Scheme
Software1 HSM (Hitachi) and WAVEMAKER 41 (Transgenomic) software2 Navigator (Transgenomic)
Nucleic Acid Amplifications eg PCR
PCR Amplification for Mutation Analysis Using the Transgenomic WAVEreg System
from Invitrogen
ndash Introduction Discoverasetrade dHPLC DNA Polymerase is an enzyme mixture
composed of recombinant Taq DNA polymerase and Pyrococcus species GB-D
polymerase Pyrococcus species GB-D polymerase possesses a proofreading
ability by virtue of its 3rsquo to 5rsquo exonuclease activity Mixture of the proofreading
enzyme with Taq DNA polymerase at an optimized ratio increases fidelity
approximately eight times over that of Taq DNA polymerase alone and allows
amplification of simple and complex DNA templates The enzyme mixture is
provided with an optimized buffer that improves enzyme fidelity
ndash The Discoverasetrade dHPLC DNA Polymerase enzyme mixture and buffer
formulation have been optimized for use with denaturing high-performance
liquid chromatography (dHPLC) systems They were developed and tested using
the Transgenomic WAVEreg SystemDiscoverasetrade dHPLC DNA Polymerase is
supplied at 1 unit per microl
Equipments
1 Thermocycler (with heated lid)
2 DHPLC WAVE DNA fragment analysis system
(Transgenomic)
3 DNA Sep HT cartridge (cat no DNA-99-3710
Transgenomic)
4 Transmit silicone sealing mats (see Note 2) (cat no
172024 Transgenomic)
5 PCR plates (see Note 3) (cat no 0030 127307
Eppendorf)
6 Chemical waste container
Solutions1 Optimase polymerase (supplied with Mg2+-free
buffer and separate Mg2+ solution)(cat no 703045 Transgenomic)2 100 mM dNTP set PCR grade (cat no 10297-117
Invitrogen)3 Mutation control standards low-range mutation
standard (cat no 560077) and high-range mutation standard (cat no 562001 Transgenomic)
4 DNA sizing standard (cat no 560078 Transgenomic)5 Acetonitrile 9993+ HPLC grade (cat no 27 071-7
Sigma-Aldrich)6 WAVE ion-pairing agent 2 M triethylammonium
acetate (TEAA) (cat no 553303 Transgenomic)7 Water (eg MilliQ water) use 18 M1048625 resistance or
better8 WAVE optimized buffers (optional) Buffers A B D
and syringe solution (cat nos 553401 553402 553404 and 553403 Transgenomic)
HomoduplexesHeteroduplex
es
Chromatographic Separation Reverse-phase ion-pair system
Gradient elution with acetonitrilewater
UV detection
Column oven temperature selected for partial denaturation of heteroduplexes and thus earlier elution
Results
Example of elution profiles for a wild-type and a mutant sample The mutant sample contains an insertiondeletion mutation in exon 8 of the RET gene
Example of elution profiles obtained for a wild-type and a mutant sample The mutant sample contains a stop codon mutation in exon 15 of the RET gene
Applications DHPLC Systems (eg WAVE Systems) can
be used for a wide range of applications includingndash Mutation Detectionndash Sizingndash Oligonucleotide Purification and Analysisndash Forensicsndash Microbial Analysisndash RNA Isolation and Purificationndash Single Base Extensionndash Methylation
With the WAVE System you can
Scan fragments for both known and novel
mutationsSNPs without extensive resequencing
Detect low-abundance mutations in heterogeneous
samples
Enrich for low-abundance alleles
Fractionate complex mixtures of related fragments
based on differences in sequence content
Perform targeted mutationSNP scoring via primer
extension assays
Analyze end-point products from allele-specific
ProbePrimer-based Amplification Technologies
ProbePrimer-based Amplification Technologies
bull Primer-based Amplificationsndash Non-Isothermal Amplifications eg PCR-based
Methodsndash Isothermal AmplificationsNon-PCR-based
Amplificationsndash NASBAndash HDAndash SDAndash and more than 45 NAIA Technologies
bull Probe-based Amplificationsndash For example Ligase Chain Reactions
CARTRIDGE dependent Applications eg via WAVE Systems
DNA-99-3510 DNASep Cartridge 46 mm x 50 mm for variety of
DNA applications including Sizing and Mutation Detection load
capacity of 2microg
DNA-99-3710 DNASep HT Cartridge 65 mm x 37 mm for variety
of DNA application but especially for Rapid Mutation Detection
load capacity of 4microg
NUC-99-3550 OligoSepreg Cartridge 46 mm x 50 mm for Oligo
Purification work only
NUC-99-3860 OligoSep Prep HC Cartridge 78 mm x 50 mm for
Oligo Purification load capacity of 5000microg
RNA-99-3810 RNASepreg Prep Cartridge 78 mm x 50 mm for
variety of RNA applications load capacity of 50microg total RNA -OR-
1000ng of specific RNA
Chromatograms and pedigree of patients 3 and 16 A DHPLC analysis in normal control patients 3 and 16 Both patients show abnormal DHPLC peak patterns with two peaks in comparison to normal control B Sequencing data in patient 3 and his family members C Sequencing data in patient 16 and his family members
Example of Oligo Analysis by DHPLC
Thanks for Your AttentionsReference
Thanks for Your Attentions
ReferenceMethods in Molecular Medicine Vol 108
Hypertension Methods and Protocols Edited by J P Fennell and A H Baker copy Humana Press Inc Totowa NJ Chapter 13
- DHPLC Principles An Introduction to Denaturing High Performanc
- Nucleic Acids
- Nucleic Acids OMICS
- Genomics Variations as MutationPolymorphism
- The Principles for Gene Variation Analysis
- Scanning Procedures
- Scanning Procedures (2)
- Screening Procedures
- Screening Procedures (2)
- Heteroduplex analysis (HET) Keen J et al (1991) Rapid dete
- Technological Improvement of HET to eg DHPLC
- Technological Improvement of HET to eg DHPLC (2)
- Slide 13
- DHPLC Graphical Scheme
- Software 1 HSM (Hitachi) and WAVEMAKER 41 (Transgenomic) soft
- Slide 16
- Slide 17
- Nucleic Acid Amplifications eg PCR
- Equipments
- Solutions
- Slide 21
- Results
- Applications
- With the WAVE System you can
- ProbePrimer-based Amplification Technologies
- CARTRIDGE dependent Applications eg via WAVE Systems
- Slide 27
- Slide 28
- Slide 29
- Chromatograms and pedigree of patients 3 and 16 A DHPLC analy
- Example of Oligo Analysis by DHPLC
- Slide 32
- Thanks for Your Attentions Reference
- Thanks for Your Attentions
-
Nucleic Acids
bull DNAndash Linear
bull Nature-madendash High Molecular
Weightndash Low Molecular
Weightbull Man-made
ndash High Molecular Weight
ndash Low Molecular Weight
ndash Circularbull Nature-made
ndash High Molecular Weight
ndash Low Molecular Weight
bull Man-madendash High Molecular
Weightndash Low Molecular
Weight
bull RNAbull Nature-made
ndash High Molecular Weight
ndash Low Molecular Weight
bull Man-madendash High Molecular
Weightndash Low Molecular
Weight
Nucleic Acids OMICS
bull Genomicsndash Various analyses of DNAs
bull Qualitativebull Quantitative
bull Transcriptomicsndash Various analyses of RNAs
bull Qualitativebull Quantitative
Genomics Variations as MutationPolymorphism
bull Scanning Procedures for Unknown Mutation Detections those simple methods which rely on differences in electrophoretic properties being generated between mutant and wild-type nucleic acid by point mutations (these methods cannot as currently used detect all mutations do not localize them within the fragment and can only be applied to DNA fragments hundreds of bases long)
bull Screening Procedures for Known Mutation Detections those group which have the potential to detect all mutations Sequencing is more frequently used to detect unknown mutations than it is for diagnostic purposes
The Principles for Gene Variation Analysis
bull Bioinformatics
bull Biothermodynamics Biophysical-Chemistry
bull Spectroscopics
bull Electrophoretics
bull Chromatographics
Scanning Procedures
ndash Ribonuclease cleavage (RNAase)
ndash Denaturing gradient-gel electrophoresis
(DGGE) and related techniques
ndash Carbodiimide modification (CDI)
ndash Chemical cleavage of mismatch (CCM)
ndash Single-strand conformation polymorphism
(SSCP)
ndash Heteroduplex analysis (HET)
ndash Direct sequencing (DS)
Scanning Procedures
RGH Cotton Mutation Research 285 (1993) 125-
144
Screening Procedures
bull Allele-specific oligonucleotide (ASO)
bull Allele-specific amplification (ASA)
bull Ligation (LIG)
bull Primer extension (PEX)
bull Artificial introduction of restriction sites
(AIRS)
Screening Procedures
RGH Cotton Mutation Research 285 (1993) 125-144
Heteroduplex analysis (HET) Keen J et al (1991) Rapid detection of single base mismatches as
heteroduplexes on hydrolink gels Trends Genet 7 5
bull Heteroduplexes containing single base-pair mismatches can be accurately separated from related heteroduplexes on nondenaturing gels
bull Others performed separation of heteroduplexes on normal gels which detected deletions but their method probably would not detect point mutations and can thus be considered a different method of lesser sensitivity
bull Thus far there have been few modifications The main advantage of the HET method is simplicity (as for SSCP) but its application has not been so widespread partly because of its later description and partly because of the need for Hydrolink gels in the initial description
bull The main disadvantage is the lack of 100 detection Like SSCP the HET method can only be applied to fragments hundreds of base pairs long (for example 200-300 bp)
Technological Improvement of HET to eg DHPLC
bull Patent Information1 Column matrix
ndash 1Bonn G Huber C Oefner P (1994) Verfahren zur Trennung von Nucleinsaeuren Austrian Patent No 398 973 Vienna Austria
ndash 2Bonn G Huber C Oefner P (1996) Nucleic Acid Separation on Alkylated Nonporous Polymer Beads US Patent No 5585236
ndash Currently exclusively licensed to Transgenomic Inc Omaha NE USA
DHPLCndash 3Oefner PJ Underhill PA (1998) Detection of Nucleic Acid Heteroduplex
Molecules by Denaturing High-Performance Liquid Chromatography and Methods for Comparative Sequencing US Patent 5795976 [Stanford Reference] [USPTO]
ndash 4Hansen NF Oefner PJ (1997) Software to Determine Optimum Temperature for DHPLC Given DNA Sequence Stanford University Invention Disclosure S97-175 Tangible Research Property in conjunction with US Patent 5795976 [Stanford Reference]
ndash Currently licensed to Transgenomic Inc Omaha NE USA Agilent Palo Alto CA USA and Varian Walnut Creek CA USA
ndash 5Oefner PJ (1999) Detection of Polymorphisms by Denaturing High-Performance Liquid Chromatography US Patent 6453244 [Stanford Reference] [USPTO]
ndash 6Huber CG OKeefe M Oberacher H Oefner PJ Premstaller A Xiao W Temperature-Modulated Array High-Performance Liquid Chromatography Provisional Patent filed [Stanford Reference]
Technological Improvement of HET to eg DHPLCbull Patent Information2 DNA shearing
ndash 7Oefner PJ Hunicke-Smith S (1998) Apparatus and Methods for Shear Breakage of Poly-nucleotides US Patent 5846832 [Stanford Reference] [USPTO]
ndash Licensed to Gene Machines Redwood City CA USA
DNA markersndash 8Oefner PJ Underhill PA Human Y Chromosome Specific
Single Nucleotide Polymorphisms US Patent Pending [Stanford Reference]
Principle of DHPLC
Rapid denaturation of DNA by heating re-annealing by slow cooling
Heteroduplexes form in the presence of two different alleles
DHPLCGraphical Scheme
Software1 HSM (Hitachi) and WAVEMAKER 41 (Transgenomic) software2 Navigator (Transgenomic)
Nucleic Acid Amplifications eg PCR
PCR Amplification for Mutation Analysis Using the Transgenomic WAVEreg System
from Invitrogen
ndash Introduction Discoverasetrade dHPLC DNA Polymerase is an enzyme mixture
composed of recombinant Taq DNA polymerase and Pyrococcus species GB-D
polymerase Pyrococcus species GB-D polymerase possesses a proofreading
ability by virtue of its 3rsquo to 5rsquo exonuclease activity Mixture of the proofreading
enzyme with Taq DNA polymerase at an optimized ratio increases fidelity
approximately eight times over that of Taq DNA polymerase alone and allows
amplification of simple and complex DNA templates The enzyme mixture is
provided with an optimized buffer that improves enzyme fidelity
ndash The Discoverasetrade dHPLC DNA Polymerase enzyme mixture and buffer
formulation have been optimized for use with denaturing high-performance
liquid chromatography (dHPLC) systems They were developed and tested using
the Transgenomic WAVEreg SystemDiscoverasetrade dHPLC DNA Polymerase is
supplied at 1 unit per microl
Equipments
1 Thermocycler (with heated lid)
2 DHPLC WAVE DNA fragment analysis system
(Transgenomic)
3 DNA Sep HT cartridge (cat no DNA-99-3710
Transgenomic)
4 Transmit silicone sealing mats (see Note 2) (cat no
172024 Transgenomic)
5 PCR plates (see Note 3) (cat no 0030 127307
Eppendorf)
6 Chemical waste container
Solutions1 Optimase polymerase (supplied with Mg2+-free
buffer and separate Mg2+ solution)(cat no 703045 Transgenomic)2 100 mM dNTP set PCR grade (cat no 10297-117
Invitrogen)3 Mutation control standards low-range mutation
standard (cat no 560077) and high-range mutation standard (cat no 562001 Transgenomic)
4 DNA sizing standard (cat no 560078 Transgenomic)5 Acetonitrile 9993+ HPLC grade (cat no 27 071-7
Sigma-Aldrich)6 WAVE ion-pairing agent 2 M triethylammonium
acetate (TEAA) (cat no 553303 Transgenomic)7 Water (eg MilliQ water) use 18 M1048625 resistance or
better8 WAVE optimized buffers (optional) Buffers A B D
and syringe solution (cat nos 553401 553402 553404 and 553403 Transgenomic)
HomoduplexesHeteroduplex
es
Chromatographic Separation Reverse-phase ion-pair system
Gradient elution with acetonitrilewater
UV detection
Column oven temperature selected for partial denaturation of heteroduplexes and thus earlier elution
Results
Example of elution profiles for a wild-type and a mutant sample The mutant sample contains an insertiondeletion mutation in exon 8 of the RET gene
Example of elution profiles obtained for a wild-type and a mutant sample The mutant sample contains a stop codon mutation in exon 15 of the RET gene
Applications DHPLC Systems (eg WAVE Systems) can
be used for a wide range of applications includingndash Mutation Detectionndash Sizingndash Oligonucleotide Purification and Analysisndash Forensicsndash Microbial Analysisndash RNA Isolation and Purificationndash Single Base Extensionndash Methylation
With the WAVE System you can
Scan fragments for both known and novel
mutationsSNPs without extensive resequencing
Detect low-abundance mutations in heterogeneous
samples
Enrich for low-abundance alleles
Fractionate complex mixtures of related fragments
based on differences in sequence content
Perform targeted mutationSNP scoring via primer
extension assays
Analyze end-point products from allele-specific
ProbePrimer-based Amplification Technologies
ProbePrimer-based Amplification Technologies
bull Primer-based Amplificationsndash Non-Isothermal Amplifications eg PCR-based
Methodsndash Isothermal AmplificationsNon-PCR-based
Amplificationsndash NASBAndash HDAndash SDAndash and more than 45 NAIA Technologies
bull Probe-based Amplificationsndash For example Ligase Chain Reactions
CARTRIDGE dependent Applications eg via WAVE Systems
DNA-99-3510 DNASep Cartridge 46 mm x 50 mm for variety of
DNA applications including Sizing and Mutation Detection load
capacity of 2microg
DNA-99-3710 DNASep HT Cartridge 65 mm x 37 mm for variety
of DNA application but especially for Rapid Mutation Detection
load capacity of 4microg
NUC-99-3550 OligoSepreg Cartridge 46 mm x 50 mm for Oligo
Purification work only
NUC-99-3860 OligoSep Prep HC Cartridge 78 mm x 50 mm for
Oligo Purification load capacity of 5000microg
RNA-99-3810 RNASepreg Prep Cartridge 78 mm x 50 mm for
variety of RNA applications load capacity of 50microg total RNA -OR-
1000ng of specific RNA
Chromatograms and pedigree of patients 3 and 16 A DHPLC analysis in normal control patients 3 and 16 Both patients show abnormal DHPLC peak patterns with two peaks in comparison to normal control B Sequencing data in patient 3 and his family members C Sequencing data in patient 16 and his family members
Example of Oligo Analysis by DHPLC
Thanks for Your AttentionsReference
Thanks for Your Attentions
ReferenceMethods in Molecular Medicine Vol 108
Hypertension Methods and Protocols Edited by J P Fennell and A H Baker copy Humana Press Inc Totowa NJ Chapter 13
- DHPLC Principles An Introduction to Denaturing High Performanc
- Nucleic Acids
- Nucleic Acids OMICS
- Genomics Variations as MutationPolymorphism
- The Principles for Gene Variation Analysis
- Scanning Procedures
- Scanning Procedures (2)
- Screening Procedures
- Screening Procedures (2)
- Heteroduplex analysis (HET) Keen J et al (1991) Rapid dete
- Technological Improvement of HET to eg DHPLC
- Technological Improvement of HET to eg DHPLC (2)
- Slide 13
- DHPLC Graphical Scheme
- Software 1 HSM (Hitachi) and WAVEMAKER 41 (Transgenomic) soft
- Slide 16
- Slide 17
- Nucleic Acid Amplifications eg PCR
- Equipments
- Solutions
- Slide 21
- Results
- Applications
- With the WAVE System you can
- ProbePrimer-based Amplification Technologies
- CARTRIDGE dependent Applications eg via WAVE Systems
- Slide 27
- Slide 28
- Slide 29
- Chromatograms and pedigree of patients 3 and 16 A DHPLC analy
- Example of Oligo Analysis by DHPLC
- Slide 32
- Thanks for Your Attentions Reference
- Thanks for Your Attentions
-
Nucleic Acids OMICS
bull Genomicsndash Various analyses of DNAs
bull Qualitativebull Quantitative
bull Transcriptomicsndash Various analyses of RNAs
bull Qualitativebull Quantitative
Genomics Variations as MutationPolymorphism
bull Scanning Procedures for Unknown Mutation Detections those simple methods which rely on differences in electrophoretic properties being generated between mutant and wild-type nucleic acid by point mutations (these methods cannot as currently used detect all mutations do not localize them within the fragment and can only be applied to DNA fragments hundreds of bases long)
bull Screening Procedures for Known Mutation Detections those group which have the potential to detect all mutations Sequencing is more frequently used to detect unknown mutations than it is for diagnostic purposes
The Principles for Gene Variation Analysis
bull Bioinformatics
bull Biothermodynamics Biophysical-Chemistry
bull Spectroscopics
bull Electrophoretics
bull Chromatographics
Scanning Procedures
ndash Ribonuclease cleavage (RNAase)
ndash Denaturing gradient-gel electrophoresis
(DGGE) and related techniques
ndash Carbodiimide modification (CDI)
ndash Chemical cleavage of mismatch (CCM)
ndash Single-strand conformation polymorphism
(SSCP)
ndash Heteroduplex analysis (HET)
ndash Direct sequencing (DS)
Scanning Procedures
RGH Cotton Mutation Research 285 (1993) 125-
144
Screening Procedures
bull Allele-specific oligonucleotide (ASO)
bull Allele-specific amplification (ASA)
bull Ligation (LIG)
bull Primer extension (PEX)
bull Artificial introduction of restriction sites
(AIRS)
Screening Procedures
RGH Cotton Mutation Research 285 (1993) 125-144
Heteroduplex analysis (HET) Keen J et al (1991) Rapid detection of single base mismatches as
heteroduplexes on hydrolink gels Trends Genet 7 5
bull Heteroduplexes containing single base-pair mismatches can be accurately separated from related heteroduplexes on nondenaturing gels
bull Others performed separation of heteroduplexes on normal gels which detected deletions but their method probably would not detect point mutations and can thus be considered a different method of lesser sensitivity
bull Thus far there have been few modifications The main advantage of the HET method is simplicity (as for SSCP) but its application has not been so widespread partly because of its later description and partly because of the need for Hydrolink gels in the initial description
bull The main disadvantage is the lack of 100 detection Like SSCP the HET method can only be applied to fragments hundreds of base pairs long (for example 200-300 bp)
Technological Improvement of HET to eg DHPLC
bull Patent Information1 Column matrix
ndash 1Bonn G Huber C Oefner P (1994) Verfahren zur Trennung von Nucleinsaeuren Austrian Patent No 398 973 Vienna Austria
ndash 2Bonn G Huber C Oefner P (1996) Nucleic Acid Separation on Alkylated Nonporous Polymer Beads US Patent No 5585236
ndash Currently exclusively licensed to Transgenomic Inc Omaha NE USA
DHPLCndash 3Oefner PJ Underhill PA (1998) Detection of Nucleic Acid Heteroduplex
Molecules by Denaturing High-Performance Liquid Chromatography and Methods for Comparative Sequencing US Patent 5795976 [Stanford Reference] [USPTO]
ndash 4Hansen NF Oefner PJ (1997) Software to Determine Optimum Temperature for DHPLC Given DNA Sequence Stanford University Invention Disclosure S97-175 Tangible Research Property in conjunction with US Patent 5795976 [Stanford Reference]
ndash Currently licensed to Transgenomic Inc Omaha NE USA Agilent Palo Alto CA USA and Varian Walnut Creek CA USA
ndash 5Oefner PJ (1999) Detection of Polymorphisms by Denaturing High-Performance Liquid Chromatography US Patent 6453244 [Stanford Reference] [USPTO]
ndash 6Huber CG OKeefe M Oberacher H Oefner PJ Premstaller A Xiao W Temperature-Modulated Array High-Performance Liquid Chromatography Provisional Patent filed [Stanford Reference]
Technological Improvement of HET to eg DHPLCbull Patent Information2 DNA shearing
ndash 7Oefner PJ Hunicke-Smith S (1998) Apparatus and Methods for Shear Breakage of Poly-nucleotides US Patent 5846832 [Stanford Reference] [USPTO]
ndash Licensed to Gene Machines Redwood City CA USA
DNA markersndash 8Oefner PJ Underhill PA Human Y Chromosome Specific
Single Nucleotide Polymorphisms US Patent Pending [Stanford Reference]
Principle of DHPLC
Rapid denaturation of DNA by heating re-annealing by slow cooling
Heteroduplexes form in the presence of two different alleles
DHPLCGraphical Scheme
Software1 HSM (Hitachi) and WAVEMAKER 41 (Transgenomic) software2 Navigator (Transgenomic)
Nucleic Acid Amplifications eg PCR
PCR Amplification for Mutation Analysis Using the Transgenomic WAVEreg System
from Invitrogen
ndash Introduction Discoverasetrade dHPLC DNA Polymerase is an enzyme mixture
composed of recombinant Taq DNA polymerase and Pyrococcus species GB-D
polymerase Pyrococcus species GB-D polymerase possesses a proofreading
ability by virtue of its 3rsquo to 5rsquo exonuclease activity Mixture of the proofreading
enzyme with Taq DNA polymerase at an optimized ratio increases fidelity
approximately eight times over that of Taq DNA polymerase alone and allows
amplification of simple and complex DNA templates The enzyme mixture is
provided with an optimized buffer that improves enzyme fidelity
ndash The Discoverasetrade dHPLC DNA Polymerase enzyme mixture and buffer
formulation have been optimized for use with denaturing high-performance
liquid chromatography (dHPLC) systems They were developed and tested using
the Transgenomic WAVEreg SystemDiscoverasetrade dHPLC DNA Polymerase is
supplied at 1 unit per microl
Equipments
1 Thermocycler (with heated lid)
2 DHPLC WAVE DNA fragment analysis system
(Transgenomic)
3 DNA Sep HT cartridge (cat no DNA-99-3710
Transgenomic)
4 Transmit silicone sealing mats (see Note 2) (cat no
172024 Transgenomic)
5 PCR plates (see Note 3) (cat no 0030 127307
Eppendorf)
6 Chemical waste container
Solutions1 Optimase polymerase (supplied with Mg2+-free
buffer and separate Mg2+ solution)(cat no 703045 Transgenomic)2 100 mM dNTP set PCR grade (cat no 10297-117
Invitrogen)3 Mutation control standards low-range mutation
standard (cat no 560077) and high-range mutation standard (cat no 562001 Transgenomic)
4 DNA sizing standard (cat no 560078 Transgenomic)5 Acetonitrile 9993+ HPLC grade (cat no 27 071-7
Sigma-Aldrich)6 WAVE ion-pairing agent 2 M triethylammonium
acetate (TEAA) (cat no 553303 Transgenomic)7 Water (eg MilliQ water) use 18 M1048625 resistance or
better8 WAVE optimized buffers (optional) Buffers A B D
and syringe solution (cat nos 553401 553402 553404 and 553403 Transgenomic)
HomoduplexesHeteroduplex
es
Chromatographic Separation Reverse-phase ion-pair system
Gradient elution with acetonitrilewater
UV detection
Column oven temperature selected for partial denaturation of heteroduplexes and thus earlier elution
Results
Example of elution profiles for a wild-type and a mutant sample The mutant sample contains an insertiondeletion mutation in exon 8 of the RET gene
Example of elution profiles obtained for a wild-type and a mutant sample The mutant sample contains a stop codon mutation in exon 15 of the RET gene
Applications DHPLC Systems (eg WAVE Systems) can
be used for a wide range of applications includingndash Mutation Detectionndash Sizingndash Oligonucleotide Purification and Analysisndash Forensicsndash Microbial Analysisndash RNA Isolation and Purificationndash Single Base Extensionndash Methylation
With the WAVE System you can
Scan fragments for both known and novel
mutationsSNPs without extensive resequencing
Detect low-abundance mutations in heterogeneous
samples
Enrich for low-abundance alleles
Fractionate complex mixtures of related fragments
based on differences in sequence content
Perform targeted mutationSNP scoring via primer
extension assays
Analyze end-point products from allele-specific
ProbePrimer-based Amplification Technologies
ProbePrimer-based Amplification Technologies
bull Primer-based Amplificationsndash Non-Isothermal Amplifications eg PCR-based
Methodsndash Isothermal AmplificationsNon-PCR-based
Amplificationsndash NASBAndash HDAndash SDAndash and more than 45 NAIA Technologies
bull Probe-based Amplificationsndash For example Ligase Chain Reactions
CARTRIDGE dependent Applications eg via WAVE Systems
DNA-99-3510 DNASep Cartridge 46 mm x 50 mm for variety of
DNA applications including Sizing and Mutation Detection load
capacity of 2microg
DNA-99-3710 DNASep HT Cartridge 65 mm x 37 mm for variety
of DNA application but especially for Rapid Mutation Detection
load capacity of 4microg
NUC-99-3550 OligoSepreg Cartridge 46 mm x 50 mm for Oligo
Purification work only
NUC-99-3860 OligoSep Prep HC Cartridge 78 mm x 50 mm for
Oligo Purification load capacity of 5000microg
RNA-99-3810 RNASepreg Prep Cartridge 78 mm x 50 mm for
variety of RNA applications load capacity of 50microg total RNA -OR-
1000ng of specific RNA
Chromatograms and pedigree of patients 3 and 16 A DHPLC analysis in normal control patients 3 and 16 Both patients show abnormal DHPLC peak patterns with two peaks in comparison to normal control B Sequencing data in patient 3 and his family members C Sequencing data in patient 16 and his family members
Example of Oligo Analysis by DHPLC
Thanks for Your AttentionsReference
Thanks for Your Attentions
ReferenceMethods in Molecular Medicine Vol 108
Hypertension Methods and Protocols Edited by J P Fennell and A H Baker copy Humana Press Inc Totowa NJ Chapter 13
- DHPLC Principles An Introduction to Denaturing High Performanc
- Nucleic Acids
- Nucleic Acids OMICS
- Genomics Variations as MutationPolymorphism
- The Principles for Gene Variation Analysis
- Scanning Procedures
- Scanning Procedures (2)
- Screening Procedures
- Screening Procedures (2)
- Heteroduplex analysis (HET) Keen J et al (1991) Rapid dete
- Technological Improvement of HET to eg DHPLC
- Technological Improvement of HET to eg DHPLC (2)
- Slide 13
- DHPLC Graphical Scheme
- Software 1 HSM (Hitachi) and WAVEMAKER 41 (Transgenomic) soft
- Slide 16
- Slide 17
- Nucleic Acid Amplifications eg PCR
- Equipments
- Solutions
- Slide 21
- Results
- Applications
- With the WAVE System you can
- ProbePrimer-based Amplification Technologies
- CARTRIDGE dependent Applications eg via WAVE Systems
- Slide 27
- Slide 28
- Slide 29
- Chromatograms and pedigree of patients 3 and 16 A DHPLC analy
- Example of Oligo Analysis by DHPLC
- Slide 32
- Thanks for Your Attentions Reference
- Thanks for Your Attentions
-
Genomics Variations as MutationPolymorphism
bull Scanning Procedures for Unknown Mutation Detections those simple methods which rely on differences in electrophoretic properties being generated between mutant and wild-type nucleic acid by point mutations (these methods cannot as currently used detect all mutations do not localize them within the fragment and can only be applied to DNA fragments hundreds of bases long)
bull Screening Procedures for Known Mutation Detections those group which have the potential to detect all mutations Sequencing is more frequently used to detect unknown mutations than it is for diagnostic purposes
The Principles for Gene Variation Analysis
bull Bioinformatics
bull Biothermodynamics Biophysical-Chemistry
bull Spectroscopics
bull Electrophoretics
bull Chromatographics
Scanning Procedures
ndash Ribonuclease cleavage (RNAase)
ndash Denaturing gradient-gel electrophoresis
(DGGE) and related techniques
ndash Carbodiimide modification (CDI)
ndash Chemical cleavage of mismatch (CCM)
ndash Single-strand conformation polymorphism
(SSCP)
ndash Heteroduplex analysis (HET)
ndash Direct sequencing (DS)
Scanning Procedures
RGH Cotton Mutation Research 285 (1993) 125-
144
Screening Procedures
bull Allele-specific oligonucleotide (ASO)
bull Allele-specific amplification (ASA)
bull Ligation (LIG)
bull Primer extension (PEX)
bull Artificial introduction of restriction sites
(AIRS)
Screening Procedures
RGH Cotton Mutation Research 285 (1993) 125-144
Heteroduplex analysis (HET) Keen J et al (1991) Rapid detection of single base mismatches as
heteroduplexes on hydrolink gels Trends Genet 7 5
bull Heteroduplexes containing single base-pair mismatches can be accurately separated from related heteroduplexes on nondenaturing gels
bull Others performed separation of heteroduplexes on normal gels which detected deletions but their method probably would not detect point mutations and can thus be considered a different method of lesser sensitivity
bull Thus far there have been few modifications The main advantage of the HET method is simplicity (as for SSCP) but its application has not been so widespread partly because of its later description and partly because of the need for Hydrolink gels in the initial description
bull The main disadvantage is the lack of 100 detection Like SSCP the HET method can only be applied to fragments hundreds of base pairs long (for example 200-300 bp)
Technological Improvement of HET to eg DHPLC
bull Patent Information1 Column matrix
ndash 1Bonn G Huber C Oefner P (1994) Verfahren zur Trennung von Nucleinsaeuren Austrian Patent No 398 973 Vienna Austria
ndash 2Bonn G Huber C Oefner P (1996) Nucleic Acid Separation on Alkylated Nonporous Polymer Beads US Patent No 5585236
ndash Currently exclusively licensed to Transgenomic Inc Omaha NE USA
DHPLCndash 3Oefner PJ Underhill PA (1998) Detection of Nucleic Acid Heteroduplex
Molecules by Denaturing High-Performance Liquid Chromatography and Methods for Comparative Sequencing US Patent 5795976 [Stanford Reference] [USPTO]
ndash 4Hansen NF Oefner PJ (1997) Software to Determine Optimum Temperature for DHPLC Given DNA Sequence Stanford University Invention Disclosure S97-175 Tangible Research Property in conjunction with US Patent 5795976 [Stanford Reference]
ndash Currently licensed to Transgenomic Inc Omaha NE USA Agilent Palo Alto CA USA and Varian Walnut Creek CA USA
ndash 5Oefner PJ (1999) Detection of Polymorphisms by Denaturing High-Performance Liquid Chromatography US Patent 6453244 [Stanford Reference] [USPTO]
ndash 6Huber CG OKeefe M Oberacher H Oefner PJ Premstaller A Xiao W Temperature-Modulated Array High-Performance Liquid Chromatography Provisional Patent filed [Stanford Reference]
Technological Improvement of HET to eg DHPLCbull Patent Information2 DNA shearing
ndash 7Oefner PJ Hunicke-Smith S (1998) Apparatus and Methods for Shear Breakage of Poly-nucleotides US Patent 5846832 [Stanford Reference] [USPTO]
ndash Licensed to Gene Machines Redwood City CA USA
DNA markersndash 8Oefner PJ Underhill PA Human Y Chromosome Specific
Single Nucleotide Polymorphisms US Patent Pending [Stanford Reference]
Principle of DHPLC
Rapid denaturation of DNA by heating re-annealing by slow cooling
Heteroduplexes form in the presence of two different alleles
DHPLCGraphical Scheme
Software1 HSM (Hitachi) and WAVEMAKER 41 (Transgenomic) software2 Navigator (Transgenomic)
Nucleic Acid Amplifications eg PCR
PCR Amplification for Mutation Analysis Using the Transgenomic WAVEreg System
from Invitrogen
ndash Introduction Discoverasetrade dHPLC DNA Polymerase is an enzyme mixture
composed of recombinant Taq DNA polymerase and Pyrococcus species GB-D
polymerase Pyrococcus species GB-D polymerase possesses a proofreading
ability by virtue of its 3rsquo to 5rsquo exonuclease activity Mixture of the proofreading
enzyme with Taq DNA polymerase at an optimized ratio increases fidelity
approximately eight times over that of Taq DNA polymerase alone and allows
amplification of simple and complex DNA templates The enzyme mixture is
provided with an optimized buffer that improves enzyme fidelity
ndash The Discoverasetrade dHPLC DNA Polymerase enzyme mixture and buffer
formulation have been optimized for use with denaturing high-performance
liquid chromatography (dHPLC) systems They were developed and tested using
the Transgenomic WAVEreg SystemDiscoverasetrade dHPLC DNA Polymerase is
supplied at 1 unit per microl
Equipments
1 Thermocycler (with heated lid)
2 DHPLC WAVE DNA fragment analysis system
(Transgenomic)
3 DNA Sep HT cartridge (cat no DNA-99-3710
Transgenomic)
4 Transmit silicone sealing mats (see Note 2) (cat no
172024 Transgenomic)
5 PCR plates (see Note 3) (cat no 0030 127307
Eppendorf)
6 Chemical waste container
Solutions1 Optimase polymerase (supplied with Mg2+-free
buffer and separate Mg2+ solution)(cat no 703045 Transgenomic)2 100 mM dNTP set PCR grade (cat no 10297-117
Invitrogen)3 Mutation control standards low-range mutation
standard (cat no 560077) and high-range mutation standard (cat no 562001 Transgenomic)
4 DNA sizing standard (cat no 560078 Transgenomic)5 Acetonitrile 9993+ HPLC grade (cat no 27 071-7
Sigma-Aldrich)6 WAVE ion-pairing agent 2 M triethylammonium
acetate (TEAA) (cat no 553303 Transgenomic)7 Water (eg MilliQ water) use 18 M1048625 resistance or
better8 WAVE optimized buffers (optional) Buffers A B D
and syringe solution (cat nos 553401 553402 553404 and 553403 Transgenomic)
HomoduplexesHeteroduplex
es
Chromatographic Separation Reverse-phase ion-pair system
Gradient elution with acetonitrilewater
UV detection
Column oven temperature selected for partial denaturation of heteroduplexes and thus earlier elution
Results
Example of elution profiles for a wild-type and a mutant sample The mutant sample contains an insertiondeletion mutation in exon 8 of the RET gene
Example of elution profiles obtained for a wild-type and a mutant sample The mutant sample contains a stop codon mutation in exon 15 of the RET gene
Applications DHPLC Systems (eg WAVE Systems) can
be used for a wide range of applications includingndash Mutation Detectionndash Sizingndash Oligonucleotide Purification and Analysisndash Forensicsndash Microbial Analysisndash RNA Isolation and Purificationndash Single Base Extensionndash Methylation
With the WAVE System you can
Scan fragments for both known and novel
mutationsSNPs without extensive resequencing
Detect low-abundance mutations in heterogeneous
samples
Enrich for low-abundance alleles
Fractionate complex mixtures of related fragments
based on differences in sequence content
Perform targeted mutationSNP scoring via primer
extension assays
Analyze end-point products from allele-specific
ProbePrimer-based Amplification Technologies
ProbePrimer-based Amplification Technologies
bull Primer-based Amplificationsndash Non-Isothermal Amplifications eg PCR-based
Methodsndash Isothermal AmplificationsNon-PCR-based
Amplificationsndash NASBAndash HDAndash SDAndash and more than 45 NAIA Technologies
bull Probe-based Amplificationsndash For example Ligase Chain Reactions
CARTRIDGE dependent Applications eg via WAVE Systems
DNA-99-3510 DNASep Cartridge 46 mm x 50 mm for variety of
DNA applications including Sizing and Mutation Detection load
capacity of 2microg
DNA-99-3710 DNASep HT Cartridge 65 mm x 37 mm for variety
of DNA application but especially for Rapid Mutation Detection
load capacity of 4microg
NUC-99-3550 OligoSepreg Cartridge 46 mm x 50 mm for Oligo
Purification work only
NUC-99-3860 OligoSep Prep HC Cartridge 78 mm x 50 mm for
Oligo Purification load capacity of 5000microg
RNA-99-3810 RNASepreg Prep Cartridge 78 mm x 50 mm for
variety of RNA applications load capacity of 50microg total RNA -OR-
1000ng of specific RNA
Chromatograms and pedigree of patients 3 and 16 A DHPLC analysis in normal control patients 3 and 16 Both patients show abnormal DHPLC peak patterns with two peaks in comparison to normal control B Sequencing data in patient 3 and his family members C Sequencing data in patient 16 and his family members
Example of Oligo Analysis by DHPLC
Thanks for Your AttentionsReference
Thanks for Your Attentions
ReferenceMethods in Molecular Medicine Vol 108
Hypertension Methods and Protocols Edited by J P Fennell and A H Baker copy Humana Press Inc Totowa NJ Chapter 13
- DHPLC Principles An Introduction to Denaturing High Performanc
- Nucleic Acids
- Nucleic Acids OMICS
- Genomics Variations as MutationPolymorphism
- The Principles for Gene Variation Analysis
- Scanning Procedures
- Scanning Procedures (2)
- Screening Procedures
- Screening Procedures (2)
- Heteroduplex analysis (HET) Keen J et al (1991) Rapid dete
- Technological Improvement of HET to eg DHPLC
- Technological Improvement of HET to eg DHPLC (2)
- Slide 13
- DHPLC Graphical Scheme
- Software 1 HSM (Hitachi) and WAVEMAKER 41 (Transgenomic) soft
- Slide 16
- Slide 17
- Nucleic Acid Amplifications eg PCR
- Equipments
- Solutions
- Slide 21
- Results
- Applications
- With the WAVE System you can
- ProbePrimer-based Amplification Technologies
- CARTRIDGE dependent Applications eg via WAVE Systems
- Slide 27
- Slide 28
- Slide 29
- Chromatograms and pedigree of patients 3 and 16 A DHPLC analy
- Example of Oligo Analysis by DHPLC
- Slide 32
- Thanks for Your Attentions Reference
- Thanks for Your Attentions
-
The Principles for Gene Variation Analysis
bull Bioinformatics
bull Biothermodynamics Biophysical-Chemistry
bull Spectroscopics
bull Electrophoretics
bull Chromatographics
Scanning Procedures
ndash Ribonuclease cleavage (RNAase)
ndash Denaturing gradient-gel electrophoresis
(DGGE) and related techniques
ndash Carbodiimide modification (CDI)
ndash Chemical cleavage of mismatch (CCM)
ndash Single-strand conformation polymorphism
(SSCP)
ndash Heteroduplex analysis (HET)
ndash Direct sequencing (DS)
Scanning Procedures
RGH Cotton Mutation Research 285 (1993) 125-
144
Screening Procedures
bull Allele-specific oligonucleotide (ASO)
bull Allele-specific amplification (ASA)
bull Ligation (LIG)
bull Primer extension (PEX)
bull Artificial introduction of restriction sites
(AIRS)
Screening Procedures
RGH Cotton Mutation Research 285 (1993) 125-144
Heteroduplex analysis (HET) Keen J et al (1991) Rapid detection of single base mismatches as
heteroduplexes on hydrolink gels Trends Genet 7 5
bull Heteroduplexes containing single base-pair mismatches can be accurately separated from related heteroduplexes on nondenaturing gels
bull Others performed separation of heteroduplexes on normal gels which detected deletions but their method probably would not detect point mutations and can thus be considered a different method of lesser sensitivity
bull Thus far there have been few modifications The main advantage of the HET method is simplicity (as for SSCP) but its application has not been so widespread partly because of its later description and partly because of the need for Hydrolink gels in the initial description
bull The main disadvantage is the lack of 100 detection Like SSCP the HET method can only be applied to fragments hundreds of base pairs long (for example 200-300 bp)
Technological Improvement of HET to eg DHPLC
bull Patent Information1 Column matrix
ndash 1Bonn G Huber C Oefner P (1994) Verfahren zur Trennung von Nucleinsaeuren Austrian Patent No 398 973 Vienna Austria
ndash 2Bonn G Huber C Oefner P (1996) Nucleic Acid Separation on Alkylated Nonporous Polymer Beads US Patent No 5585236
ndash Currently exclusively licensed to Transgenomic Inc Omaha NE USA
DHPLCndash 3Oefner PJ Underhill PA (1998) Detection of Nucleic Acid Heteroduplex
Molecules by Denaturing High-Performance Liquid Chromatography and Methods for Comparative Sequencing US Patent 5795976 [Stanford Reference] [USPTO]
ndash 4Hansen NF Oefner PJ (1997) Software to Determine Optimum Temperature for DHPLC Given DNA Sequence Stanford University Invention Disclosure S97-175 Tangible Research Property in conjunction with US Patent 5795976 [Stanford Reference]
ndash Currently licensed to Transgenomic Inc Omaha NE USA Agilent Palo Alto CA USA and Varian Walnut Creek CA USA
ndash 5Oefner PJ (1999) Detection of Polymorphisms by Denaturing High-Performance Liquid Chromatography US Patent 6453244 [Stanford Reference] [USPTO]
ndash 6Huber CG OKeefe M Oberacher H Oefner PJ Premstaller A Xiao W Temperature-Modulated Array High-Performance Liquid Chromatography Provisional Patent filed [Stanford Reference]
Technological Improvement of HET to eg DHPLCbull Patent Information2 DNA shearing
ndash 7Oefner PJ Hunicke-Smith S (1998) Apparatus and Methods for Shear Breakage of Poly-nucleotides US Patent 5846832 [Stanford Reference] [USPTO]
ndash Licensed to Gene Machines Redwood City CA USA
DNA markersndash 8Oefner PJ Underhill PA Human Y Chromosome Specific
Single Nucleotide Polymorphisms US Patent Pending [Stanford Reference]
Principle of DHPLC
Rapid denaturation of DNA by heating re-annealing by slow cooling
Heteroduplexes form in the presence of two different alleles
DHPLCGraphical Scheme
Software1 HSM (Hitachi) and WAVEMAKER 41 (Transgenomic) software2 Navigator (Transgenomic)
Nucleic Acid Amplifications eg PCR
PCR Amplification for Mutation Analysis Using the Transgenomic WAVEreg System
from Invitrogen
ndash Introduction Discoverasetrade dHPLC DNA Polymerase is an enzyme mixture
composed of recombinant Taq DNA polymerase and Pyrococcus species GB-D
polymerase Pyrococcus species GB-D polymerase possesses a proofreading
ability by virtue of its 3rsquo to 5rsquo exonuclease activity Mixture of the proofreading
enzyme with Taq DNA polymerase at an optimized ratio increases fidelity
approximately eight times over that of Taq DNA polymerase alone and allows
amplification of simple and complex DNA templates The enzyme mixture is
provided with an optimized buffer that improves enzyme fidelity
ndash The Discoverasetrade dHPLC DNA Polymerase enzyme mixture and buffer
formulation have been optimized for use with denaturing high-performance
liquid chromatography (dHPLC) systems They were developed and tested using
the Transgenomic WAVEreg SystemDiscoverasetrade dHPLC DNA Polymerase is
supplied at 1 unit per microl
Equipments
1 Thermocycler (with heated lid)
2 DHPLC WAVE DNA fragment analysis system
(Transgenomic)
3 DNA Sep HT cartridge (cat no DNA-99-3710
Transgenomic)
4 Transmit silicone sealing mats (see Note 2) (cat no
172024 Transgenomic)
5 PCR plates (see Note 3) (cat no 0030 127307
Eppendorf)
6 Chemical waste container
Solutions1 Optimase polymerase (supplied with Mg2+-free
buffer and separate Mg2+ solution)(cat no 703045 Transgenomic)2 100 mM dNTP set PCR grade (cat no 10297-117
Invitrogen)3 Mutation control standards low-range mutation
standard (cat no 560077) and high-range mutation standard (cat no 562001 Transgenomic)
4 DNA sizing standard (cat no 560078 Transgenomic)5 Acetonitrile 9993+ HPLC grade (cat no 27 071-7
Sigma-Aldrich)6 WAVE ion-pairing agent 2 M triethylammonium
acetate (TEAA) (cat no 553303 Transgenomic)7 Water (eg MilliQ water) use 18 M1048625 resistance or
better8 WAVE optimized buffers (optional) Buffers A B D
and syringe solution (cat nos 553401 553402 553404 and 553403 Transgenomic)
HomoduplexesHeteroduplex
es
Chromatographic Separation Reverse-phase ion-pair system
Gradient elution with acetonitrilewater
UV detection
Column oven temperature selected for partial denaturation of heteroduplexes and thus earlier elution
Results
Example of elution profiles for a wild-type and a mutant sample The mutant sample contains an insertiondeletion mutation in exon 8 of the RET gene
Example of elution profiles obtained for a wild-type and a mutant sample The mutant sample contains a stop codon mutation in exon 15 of the RET gene
Applications DHPLC Systems (eg WAVE Systems) can
be used for a wide range of applications includingndash Mutation Detectionndash Sizingndash Oligonucleotide Purification and Analysisndash Forensicsndash Microbial Analysisndash RNA Isolation and Purificationndash Single Base Extensionndash Methylation
With the WAVE System you can
Scan fragments for both known and novel
mutationsSNPs without extensive resequencing
Detect low-abundance mutations in heterogeneous
samples
Enrich for low-abundance alleles
Fractionate complex mixtures of related fragments
based on differences in sequence content
Perform targeted mutationSNP scoring via primer
extension assays
Analyze end-point products from allele-specific
ProbePrimer-based Amplification Technologies
ProbePrimer-based Amplification Technologies
bull Primer-based Amplificationsndash Non-Isothermal Amplifications eg PCR-based
Methodsndash Isothermal AmplificationsNon-PCR-based
Amplificationsndash NASBAndash HDAndash SDAndash and more than 45 NAIA Technologies
bull Probe-based Amplificationsndash For example Ligase Chain Reactions
CARTRIDGE dependent Applications eg via WAVE Systems
DNA-99-3510 DNASep Cartridge 46 mm x 50 mm for variety of
DNA applications including Sizing and Mutation Detection load
capacity of 2microg
DNA-99-3710 DNASep HT Cartridge 65 mm x 37 mm for variety
of DNA application but especially for Rapid Mutation Detection
load capacity of 4microg
NUC-99-3550 OligoSepreg Cartridge 46 mm x 50 mm for Oligo
Purification work only
NUC-99-3860 OligoSep Prep HC Cartridge 78 mm x 50 mm for
Oligo Purification load capacity of 5000microg
RNA-99-3810 RNASepreg Prep Cartridge 78 mm x 50 mm for
variety of RNA applications load capacity of 50microg total RNA -OR-
1000ng of specific RNA
Chromatograms and pedigree of patients 3 and 16 A DHPLC analysis in normal control patients 3 and 16 Both patients show abnormal DHPLC peak patterns with two peaks in comparison to normal control B Sequencing data in patient 3 and his family members C Sequencing data in patient 16 and his family members
Example of Oligo Analysis by DHPLC
Thanks for Your AttentionsReference
Thanks for Your Attentions
ReferenceMethods in Molecular Medicine Vol 108
Hypertension Methods and Protocols Edited by J P Fennell and A H Baker copy Humana Press Inc Totowa NJ Chapter 13
- DHPLC Principles An Introduction to Denaturing High Performanc
- Nucleic Acids
- Nucleic Acids OMICS
- Genomics Variations as MutationPolymorphism
- The Principles for Gene Variation Analysis
- Scanning Procedures
- Scanning Procedures (2)
- Screening Procedures
- Screening Procedures (2)
- Heteroduplex analysis (HET) Keen J et al (1991) Rapid dete
- Technological Improvement of HET to eg DHPLC
- Technological Improvement of HET to eg DHPLC (2)
- Slide 13
- DHPLC Graphical Scheme
- Software 1 HSM (Hitachi) and WAVEMAKER 41 (Transgenomic) soft
- Slide 16
- Slide 17
- Nucleic Acid Amplifications eg PCR
- Equipments
- Solutions
- Slide 21
- Results
- Applications
- With the WAVE System you can
- ProbePrimer-based Amplification Technologies
- CARTRIDGE dependent Applications eg via WAVE Systems
- Slide 27
- Slide 28
- Slide 29
- Chromatograms and pedigree of patients 3 and 16 A DHPLC analy
- Example of Oligo Analysis by DHPLC
- Slide 32
- Thanks for Your Attentions Reference
- Thanks for Your Attentions
-
Scanning Procedures
ndash Ribonuclease cleavage (RNAase)
ndash Denaturing gradient-gel electrophoresis
(DGGE) and related techniques
ndash Carbodiimide modification (CDI)
ndash Chemical cleavage of mismatch (CCM)
ndash Single-strand conformation polymorphism
(SSCP)
ndash Heteroduplex analysis (HET)
ndash Direct sequencing (DS)
Scanning Procedures
RGH Cotton Mutation Research 285 (1993) 125-
144
Screening Procedures
bull Allele-specific oligonucleotide (ASO)
bull Allele-specific amplification (ASA)
bull Ligation (LIG)
bull Primer extension (PEX)
bull Artificial introduction of restriction sites
(AIRS)
Screening Procedures
RGH Cotton Mutation Research 285 (1993) 125-144
Heteroduplex analysis (HET) Keen J et al (1991) Rapid detection of single base mismatches as
heteroduplexes on hydrolink gels Trends Genet 7 5
bull Heteroduplexes containing single base-pair mismatches can be accurately separated from related heteroduplexes on nondenaturing gels
bull Others performed separation of heteroduplexes on normal gels which detected deletions but their method probably would not detect point mutations and can thus be considered a different method of lesser sensitivity
bull Thus far there have been few modifications The main advantage of the HET method is simplicity (as for SSCP) but its application has not been so widespread partly because of its later description and partly because of the need for Hydrolink gels in the initial description
bull The main disadvantage is the lack of 100 detection Like SSCP the HET method can only be applied to fragments hundreds of base pairs long (for example 200-300 bp)
Technological Improvement of HET to eg DHPLC
bull Patent Information1 Column matrix
ndash 1Bonn G Huber C Oefner P (1994) Verfahren zur Trennung von Nucleinsaeuren Austrian Patent No 398 973 Vienna Austria
ndash 2Bonn G Huber C Oefner P (1996) Nucleic Acid Separation on Alkylated Nonporous Polymer Beads US Patent No 5585236
ndash Currently exclusively licensed to Transgenomic Inc Omaha NE USA
DHPLCndash 3Oefner PJ Underhill PA (1998) Detection of Nucleic Acid Heteroduplex
Molecules by Denaturing High-Performance Liquid Chromatography and Methods for Comparative Sequencing US Patent 5795976 [Stanford Reference] [USPTO]
ndash 4Hansen NF Oefner PJ (1997) Software to Determine Optimum Temperature for DHPLC Given DNA Sequence Stanford University Invention Disclosure S97-175 Tangible Research Property in conjunction with US Patent 5795976 [Stanford Reference]
ndash Currently licensed to Transgenomic Inc Omaha NE USA Agilent Palo Alto CA USA and Varian Walnut Creek CA USA
ndash 5Oefner PJ (1999) Detection of Polymorphisms by Denaturing High-Performance Liquid Chromatography US Patent 6453244 [Stanford Reference] [USPTO]
ndash 6Huber CG OKeefe M Oberacher H Oefner PJ Premstaller A Xiao W Temperature-Modulated Array High-Performance Liquid Chromatography Provisional Patent filed [Stanford Reference]
Technological Improvement of HET to eg DHPLCbull Patent Information2 DNA shearing
ndash 7Oefner PJ Hunicke-Smith S (1998) Apparatus and Methods for Shear Breakage of Poly-nucleotides US Patent 5846832 [Stanford Reference] [USPTO]
ndash Licensed to Gene Machines Redwood City CA USA
DNA markersndash 8Oefner PJ Underhill PA Human Y Chromosome Specific
Single Nucleotide Polymorphisms US Patent Pending [Stanford Reference]
Principle of DHPLC
Rapid denaturation of DNA by heating re-annealing by slow cooling
Heteroduplexes form in the presence of two different alleles
DHPLCGraphical Scheme
Software1 HSM (Hitachi) and WAVEMAKER 41 (Transgenomic) software2 Navigator (Transgenomic)
Nucleic Acid Amplifications eg PCR
PCR Amplification for Mutation Analysis Using the Transgenomic WAVEreg System
from Invitrogen
ndash Introduction Discoverasetrade dHPLC DNA Polymerase is an enzyme mixture
composed of recombinant Taq DNA polymerase and Pyrococcus species GB-D
polymerase Pyrococcus species GB-D polymerase possesses a proofreading
ability by virtue of its 3rsquo to 5rsquo exonuclease activity Mixture of the proofreading
enzyme with Taq DNA polymerase at an optimized ratio increases fidelity
approximately eight times over that of Taq DNA polymerase alone and allows
amplification of simple and complex DNA templates The enzyme mixture is
provided with an optimized buffer that improves enzyme fidelity
ndash The Discoverasetrade dHPLC DNA Polymerase enzyme mixture and buffer
formulation have been optimized for use with denaturing high-performance
liquid chromatography (dHPLC) systems They were developed and tested using
the Transgenomic WAVEreg SystemDiscoverasetrade dHPLC DNA Polymerase is
supplied at 1 unit per microl
Equipments
1 Thermocycler (with heated lid)
2 DHPLC WAVE DNA fragment analysis system
(Transgenomic)
3 DNA Sep HT cartridge (cat no DNA-99-3710
Transgenomic)
4 Transmit silicone sealing mats (see Note 2) (cat no
172024 Transgenomic)
5 PCR plates (see Note 3) (cat no 0030 127307
Eppendorf)
6 Chemical waste container
Solutions1 Optimase polymerase (supplied with Mg2+-free
buffer and separate Mg2+ solution)(cat no 703045 Transgenomic)2 100 mM dNTP set PCR grade (cat no 10297-117
Invitrogen)3 Mutation control standards low-range mutation
standard (cat no 560077) and high-range mutation standard (cat no 562001 Transgenomic)
4 DNA sizing standard (cat no 560078 Transgenomic)5 Acetonitrile 9993+ HPLC grade (cat no 27 071-7
Sigma-Aldrich)6 WAVE ion-pairing agent 2 M triethylammonium
acetate (TEAA) (cat no 553303 Transgenomic)7 Water (eg MilliQ water) use 18 M1048625 resistance or
better8 WAVE optimized buffers (optional) Buffers A B D
and syringe solution (cat nos 553401 553402 553404 and 553403 Transgenomic)
HomoduplexesHeteroduplex
es
Chromatographic Separation Reverse-phase ion-pair system
Gradient elution with acetonitrilewater
UV detection
Column oven temperature selected for partial denaturation of heteroduplexes and thus earlier elution
Results
Example of elution profiles for a wild-type and a mutant sample The mutant sample contains an insertiondeletion mutation in exon 8 of the RET gene
Example of elution profiles obtained for a wild-type and a mutant sample The mutant sample contains a stop codon mutation in exon 15 of the RET gene
Applications DHPLC Systems (eg WAVE Systems) can
be used for a wide range of applications includingndash Mutation Detectionndash Sizingndash Oligonucleotide Purification and Analysisndash Forensicsndash Microbial Analysisndash RNA Isolation and Purificationndash Single Base Extensionndash Methylation
With the WAVE System you can
Scan fragments for both known and novel
mutationsSNPs without extensive resequencing
Detect low-abundance mutations in heterogeneous
samples
Enrich for low-abundance alleles
Fractionate complex mixtures of related fragments
based on differences in sequence content
Perform targeted mutationSNP scoring via primer
extension assays
Analyze end-point products from allele-specific
ProbePrimer-based Amplification Technologies
ProbePrimer-based Amplification Technologies
bull Primer-based Amplificationsndash Non-Isothermal Amplifications eg PCR-based
Methodsndash Isothermal AmplificationsNon-PCR-based
Amplificationsndash NASBAndash HDAndash SDAndash and more than 45 NAIA Technologies
bull Probe-based Amplificationsndash For example Ligase Chain Reactions
CARTRIDGE dependent Applications eg via WAVE Systems
DNA-99-3510 DNASep Cartridge 46 mm x 50 mm for variety of
DNA applications including Sizing and Mutation Detection load
capacity of 2microg
DNA-99-3710 DNASep HT Cartridge 65 mm x 37 mm for variety
of DNA application but especially for Rapid Mutation Detection
load capacity of 4microg
NUC-99-3550 OligoSepreg Cartridge 46 mm x 50 mm for Oligo
Purification work only
NUC-99-3860 OligoSep Prep HC Cartridge 78 mm x 50 mm for
Oligo Purification load capacity of 5000microg
RNA-99-3810 RNASepreg Prep Cartridge 78 mm x 50 mm for
variety of RNA applications load capacity of 50microg total RNA -OR-
1000ng of specific RNA
Chromatograms and pedigree of patients 3 and 16 A DHPLC analysis in normal control patients 3 and 16 Both patients show abnormal DHPLC peak patterns with two peaks in comparison to normal control B Sequencing data in patient 3 and his family members C Sequencing data in patient 16 and his family members
Example of Oligo Analysis by DHPLC
Thanks for Your AttentionsReference
Thanks for Your Attentions
ReferenceMethods in Molecular Medicine Vol 108
Hypertension Methods and Protocols Edited by J P Fennell and A H Baker copy Humana Press Inc Totowa NJ Chapter 13
- DHPLC Principles An Introduction to Denaturing High Performanc
- Nucleic Acids
- Nucleic Acids OMICS
- Genomics Variations as MutationPolymorphism
- The Principles for Gene Variation Analysis
- Scanning Procedures
- Scanning Procedures (2)
- Screening Procedures
- Screening Procedures (2)
- Heteroduplex analysis (HET) Keen J et al (1991) Rapid dete
- Technological Improvement of HET to eg DHPLC
- Technological Improvement of HET to eg DHPLC (2)
- Slide 13
- DHPLC Graphical Scheme
- Software 1 HSM (Hitachi) and WAVEMAKER 41 (Transgenomic) soft
- Slide 16
- Slide 17
- Nucleic Acid Amplifications eg PCR
- Equipments
- Solutions
- Slide 21
- Results
- Applications
- With the WAVE System you can
- ProbePrimer-based Amplification Technologies
- CARTRIDGE dependent Applications eg via WAVE Systems
- Slide 27
- Slide 28
- Slide 29
- Chromatograms and pedigree of patients 3 and 16 A DHPLC analy
- Example of Oligo Analysis by DHPLC
- Slide 32
- Thanks for Your Attentions Reference
- Thanks for Your Attentions
-
Scanning Procedures
RGH Cotton Mutation Research 285 (1993) 125-
144
Screening Procedures
bull Allele-specific oligonucleotide (ASO)
bull Allele-specific amplification (ASA)
bull Ligation (LIG)
bull Primer extension (PEX)
bull Artificial introduction of restriction sites
(AIRS)
Screening Procedures
RGH Cotton Mutation Research 285 (1993) 125-144
Heteroduplex analysis (HET) Keen J et al (1991) Rapid detection of single base mismatches as
heteroduplexes on hydrolink gels Trends Genet 7 5
bull Heteroduplexes containing single base-pair mismatches can be accurately separated from related heteroduplexes on nondenaturing gels
bull Others performed separation of heteroduplexes on normal gels which detected deletions but their method probably would not detect point mutations and can thus be considered a different method of lesser sensitivity
bull Thus far there have been few modifications The main advantage of the HET method is simplicity (as for SSCP) but its application has not been so widespread partly because of its later description and partly because of the need for Hydrolink gels in the initial description
bull The main disadvantage is the lack of 100 detection Like SSCP the HET method can only be applied to fragments hundreds of base pairs long (for example 200-300 bp)
Technological Improvement of HET to eg DHPLC
bull Patent Information1 Column matrix
ndash 1Bonn G Huber C Oefner P (1994) Verfahren zur Trennung von Nucleinsaeuren Austrian Patent No 398 973 Vienna Austria
ndash 2Bonn G Huber C Oefner P (1996) Nucleic Acid Separation on Alkylated Nonporous Polymer Beads US Patent No 5585236
ndash Currently exclusively licensed to Transgenomic Inc Omaha NE USA
DHPLCndash 3Oefner PJ Underhill PA (1998) Detection of Nucleic Acid Heteroduplex
Molecules by Denaturing High-Performance Liquid Chromatography and Methods for Comparative Sequencing US Patent 5795976 [Stanford Reference] [USPTO]
ndash 4Hansen NF Oefner PJ (1997) Software to Determine Optimum Temperature for DHPLC Given DNA Sequence Stanford University Invention Disclosure S97-175 Tangible Research Property in conjunction with US Patent 5795976 [Stanford Reference]
ndash Currently licensed to Transgenomic Inc Omaha NE USA Agilent Palo Alto CA USA and Varian Walnut Creek CA USA
ndash 5Oefner PJ (1999) Detection of Polymorphisms by Denaturing High-Performance Liquid Chromatography US Patent 6453244 [Stanford Reference] [USPTO]
ndash 6Huber CG OKeefe M Oberacher H Oefner PJ Premstaller A Xiao W Temperature-Modulated Array High-Performance Liquid Chromatography Provisional Patent filed [Stanford Reference]
Technological Improvement of HET to eg DHPLCbull Patent Information2 DNA shearing
ndash 7Oefner PJ Hunicke-Smith S (1998) Apparatus and Methods for Shear Breakage of Poly-nucleotides US Patent 5846832 [Stanford Reference] [USPTO]
ndash Licensed to Gene Machines Redwood City CA USA
DNA markersndash 8Oefner PJ Underhill PA Human Y Chromosome Specific
Single Nucleotide Polymorphisms US Patent Pending [Stanford Reference]
Principle of DHPLC
Rapid denaturation of DNA by heating re-annealing by slow cooling
Heteroduplexes form in the presence of two different alleles
DHPLCGraphical Scheme
Software1 HSM (Hitachi) and WAVEMAKER 41 (Transgenomic) software2 Navigator (Transgenomic)
Nucleic Acid Amplifications eg PCR
PCR Amplification for Mutation Analysis Using the Transgenomic WAVEreg System
from Invitrogen
ndash Introduction Discoverasetrade dHPLC DNA Polymerase is an enzyme mixture
composed of recombinant Taq DNA polymerase and Pyrococcus species GB-D
polymerase Pyrococcus species GB-D polymerase possesses a proofreading
ability by virtue of its 3rsquo to 5rsquo exonuclease activity Mixture of the proofreading
enzyme with Taq DNA polymerase at an optimized ratio increases fidelity
approximately eight times over that of Taq DNA polymerase alone and allows
amplification of simple and complex DNA templates The enzyme mixture is
provided with an optimized buffer that improves enzyme fidelity
ndash The Discoverasetrade dHPLC DNA Polymerase enzyme mixture and buffer
formulation have been optimized for use with denaturing high-performance
liquid chromatography (dHPLC) systems They were developed and tested using
the Transgenomic WAVEreg SystemDiscoverasetrade dHPLC DNA Polymerase is
supplied at 1 unit per microl
Equipments
1 Thermocycler (with heated lid)
2 DHPLC WAVE DNA fragment analysis system
(Transgenomic)
3 DNA Sep HT cartridge (cat no DNA-99-3710
Transgenomic)
4 Transmit silicone sealing mats (see Note 2) (cat no
172024 Transgenomic)
5 PCR plates (see Note 3) (cat no 0030 127307
Eppendorf)
6 Chemical waste container
Solutions1 Optimase polymerase (supplied with Mg2+-free
buffer and separate Mg2+ solution)(cat no 703045 Transgenomic)2 100 mM dNTP set PCR grade (cat no 10297-117
Invitrogen)3 Mutation control standards low-range mutation
standard (cat no 560077) and high-range mutation standard (cat no 562001 Transgenomic)
4 DNA sizing standard (cat no 560078 Transgenomic)5 Acetonitrile 9993+ HPLC grade (cat no 27 071-7
Sigma-Aldrich)6 WAVE ion-pairing agent 2 M triethylammonium
acetate (TEAA) (cat no 553303 Transgenomic)7 Water (eg MilliQ water) use 18 M1048625 resistance or
better8 WAVE optimized buffers (optional) Buffers A B D
and syringe solution (cat nos 553401 553402 553404 and 553403 Transgenomic)
HomoduplexesHeteroduplex
es
Chromatographic Separation Reverse-phase ion-pair system
Gradient elution with acetonitrilewater
UV detection
Column oven temperature selected for partial denaturation of heteroduplexes and thus earlier elution
Results
Example of elution profiles for a wild-type and a mutant sample The mutant sample contains an insertiondeletion mutation in exon 8 of the RET gene
Example of elution profiles obtained for a wild-type and a mutant sample The mutant sample contains a stop codon mutation in exon 15 of the RET gene
Applications DHPLC Systems (eg WAVE Systems) can
be used for a wide range of applications includingndash Mutation Detectionndash Sizingndash Oligonucleotide Purification and Analysisndash Forensicsndash Microbial Analysisndash RNA Isolation and Purificationndash Single Base Extensionndash Methylation
With the WAVE System you can
Scan fragments for both known and novel
mutationsSNPs without extensive resequencing
Detect low-abundance mutations in heterogeneous
samples
Enrich for low-abundance alleles
Fractionate complex mixtures of related fragments
based on differences in sequence content
Perform targeted mutationSNP scoring via primer
extension assays
Analyze end-point products from allele-specific
ProbePrimer-based Amplification Technologies
ProbePrimer-based Amplification Technologies
bull Primer-based Amplificationsndash Non-Isothermal Amplifications eg PCR-based
Methodsndash Isothermal AmplificationsNon-PCR-based
Amplificationsndash NASBAndash HDAndash SDAndash and more than 45 NAIA Technologies
bull Probe-based Amplificationsndash For example Ligase Chain Reactions
CARTRIDGE dependent Applications eg via WAVE Systems
DNA-99-3510 DNASep Cartridge 46 mm x 50 mm for variety of
DNA applications including Sizing and Mutation Detection load
capacity of 2microg
DNA-99-3710 DNASep HT Cartridge 65 mm x 37 mm for variety
of DNA application but especially for Rapid Mutation Detection
load capacity of 4microg
NUC-99-3550 OligoSepreg Cartridge 46 mm x 50 mm for Oligo
Purification work only
NUC-99-3860 OligoSep Prep HC Cartridge 78 mm x 50 mm for
Oligo Purification load capacity of 5000microg
RNA-99-3810 RNASepreg Prep Cartridge 78 mm x 50 mm for
variety of RNA applications load capacity of 50microg total RNA -OR-
1000ng of specific RNA
Chromatograms and pedigree of patients 3 and 16 A DHPLC analysis in normal control patients 3 and 16 Both patients show abnormal DHPLC peak patterns with two peaks in comparison to normal control B Sequencing data in patient 3 and his family members C Sequencing data in patient 16 and his family members
Example of Oligo Analysis by DHPLC
Thanks for Your AttentionsReference
Thanks for Your Attentions
ReferenceMethods in Molecular Medicine Vol 108
Hypertension Methods and Protocols Edited by J P Fennell and A H Baker copy Humana Press Inc Totowa NJ Chapter 13
- DHPLC Principles An Introduction to Denaturing High Performanc
- Nucleic Acids
- Nucleic Acids OMICS
- Genomics Variations as MutationPolymorphism
- The Principles for Gene Variation Analysis
- Scanning Procedures
- Scanning Procedures (2)
- Screening Procedures
- Screening Procedures (2)
- Heteroduplex analysis (HET) Keen J et al (1991) Rapid dete
- Technological Improvement of HET to eg DHPLC
- Technological Improvement of HET to eg DHPLC (2)
- Slide 13
- DHPLC Graphical Scheme
- Software 1 HSM (Hitachi) and WAVEMAKER 41 (Transgenomic) soft
- Slide 16
- Slide 17
- Nucleic Acid Amplifications eg PCR
- Equipments
- Solutions
- Slide 21
- Results
- Applications
- With the WAVE System you can
- ProbePrimer-based Amplification Technologies
- CARTRIDGE dependent Applications eg via WAVE Systems
- Slide 27
- Slide 28
- Slide 29
- Chromatograms and pedigree of patients 3 and 16 A DHPLC analy
- Example of Oligo Analysis by DHPLC
- Slide 32
- Thanks for Your Attentions Reference
- Thanks for Your Attentions
-
Screening Procedures
bull Allele-specific oligonucleotide (ASO)
bull Allele-specific amplification (ASA)
bull Ligation (LIG)
bull Primer extension (PEX)
bull Artificial introduction of restriction sites
(AIRS)
Screening Procedures
RGH Cotton Mutation Research 285 (1993) 125-144
Heteroduplex analysis (HET) Keen J et al (1991) Rapid detection of single base mismatches as
heteroduplexes on hydrolink gels Trends Genet 7 5
bull Heteroduplexes containing single base-pair mismatches can be accurately separated from related heteroduplexes on nondenaturing gels
bull Others performed separation of heteroduplexes on normal gels which detected deletions but their method probably would not detect point mutations and can thus be considered a different method of lesser sensitivity
bull Thus far there have been few modifications The main advantage of the HET method is simplicity (as for SSCP) but its application has not been so widespread partly because of its later description and partly because of the need for Hydrolink gels in the initial description
bull The main disadvantage is the lack of 100 detection Like SSCP the HET method can only be applied to fragments hundreds of base pairs long (for example 200-300 bp)
Technological Improvement of HET to eg DHPLC
bull Patent Information1 Column matrix
ndash 1Bonn G Huber C Oefner P (1994) Verfahren zur Trennung von Nucleinsaeuren Austrian Patent No 398 973 Vienna Austria
ndash 2Bonn G Huber C Oefner P (1996) Nucleic Acid Separation on Alkylated Nonporous Polymer Beads US Patent No 5585236
ndash Currently exclusively licensed to Transgenomic Inc Omaha NE USA
DHPLCndash 3Oefner PJ Underhill PA (1998) Detection of Nucleic Acid Heteroduplex
Molecules by Denaturing High-Performance Liquid Chromatography and Methods for Comparative Sequencing US Patent 5795976 [Stanford Reference] [USPTO]
ndash 4Hansen NF Oefner PJ (1997) Software to Determine Optimum Temperature for DHPLC Given DNA Sequence Stanford University Invention Disclosure S97-175 Tangible Research Property in conjunction with US Patent 5795976 [Stanford Reference]
ndash Currently licensed to Transgenomic Inc Omaha NE USA Agilent Palo Alto CA USA and Varian Walnut Creek CA USA
ndash 5Oefner PJ (1999) Detection of Polymorphisms by Denaturing High-Performance Liquid Chromatography US Patent 6453244 [Stanford Reference] [USPTO]
ndash 6Huber CG OKeefe M Oberacher H Oefner PJ Premstaller A Xiao W Temperature-Modulated Array High-Performance Liquid Chromatography Provisional Patent filed [Stanford Reference]
Technological Improvement of HET to eg DHPLCbull Patent Information2 DNA shearing
ndash 7Oefner PJ Hunicke-Smith S (1998) Apparatus and Methods for Shear Breakage of Poly-nucleotides US Patent 5846832 [Stanford Reference] [USPTO]
ndash Licensed to Gene Machines Redwood City CA USA
DNA markersndash 8Oefner PJ Underhill PA Human Y Chromosome Specific
Single Nucleotide Polymorphisms US Patent Pending [Stanford Reference]
Principle of DHPLC
Rapid denaturation of DNA by heating re-annealing by slow cooling
Heteroduplexes form in the presence of two different alleles
DHPLCGraphical Scheme
Software1 HSM (Hitachi) and WAVEMAKER 41 (Transgenomic) software2 Navigator (Transgenomic)
Nucleic Acid Amplifications eg PCR
PCR Amplification for Mutation Analysis Using the Transgenomic WAVEreg System
from Invitrogen
ndash Introduction Discoverasetrade dHPLC DNA Polymerase is an enzyme mixture
composed of recombinant Taq DNA polymerase and Pyrococcus species GB-D
polymerase Pyrococcus species GB-D polymerase possesses a proofreading
ability by virtue of its 3rsquo to 5rsquo exonuclease activity Mixture of the proofreading
enzyme with Taq DNA polymerase at an optimized ratio increases fidelity
approximately eight times over that of Taq DNA polymerase alone and allows
amplification of simple and complex DNA templates The enzyme mixture is
provided with an optimized buffer that improves enzyme fidelity
ndash The Discoverasetrade dHPLC DNA Polymerase enzyme mixture and buffer
formulation have been optimized for use with denaturing high-performance
liquid chromatography (dHPLC) systems They were developed and tested using
the Transgenomic WAVEreg SystemDiscoverasetrade dHPLC DNA Polymerase is
supplied at 1 unit per microl
Equipments
1 Thermocycler (with heated lid)
2 DHPLC WAVE DNA fragment analysis system
(Transgenomic)
3 DNA Sep HT cartridge (cat no DNA-99-3710
Transgenomic)
4 Transmit silicone sealing mats (see Note 2) (cat no
172024 Transgenomic)
5 PCR plates (see Note 3) (cat no 0030 127307
Eppendorf)
6 Chemical waste container
Solutions1 Optimase polymerase (supplied with Mg2+-free
buffer and separate Mg2+ solution)(cat no 703045 Transgenomic)2 100 mM dNTP set PCR grade (cat no 10297-117
Invitrogen)3 Mutation control standards low-range mutation
standard (cat no 560077) and high-range mutation standard (cat no 562001 Transgenomic)
4 DNA sizing standard (cat no 560078 Transgenomic)5 Acetonitrile 9993+ HPLC grade (cat no 27 071-7
Sigma-Aldrich)6 WAVE ion-pairing agent 2 M triethylammonium
acetate (TEAA) (cat no 553303 Transgenomic)7 Water (eg MilliQ water) use 18 M1048625 resistance or
better8 WAVE optimized buffers (optional) Buffers A B D
and syringe solution (cat nos 553401 553402 553404 and 553403 Transgenomic)
HomoduplexesHeteroduplex
es
Chromatographic Separation Reverse-phase ion-pair system
Gradient elution with acetonitrilewater
UV detection
Column oven temperature selected for partial denaturation of heteroduplexes and thus earlier elution
Results
Example of elution profiles for a wild-type and a mutant sample The mutant sample contains an insertiondeletion mutation in exon 8 of the RET gene
Example of elution profiles obtained for a wild-type and a mutant sample The mutant sample contains a stop codon mutation in exon 15 of the RET gene
Applications DHPLC Systems (eg WAVE Systems) can
be used for a wide range of applications includingndash Mutation Detectionndash Sizingndash Oligonucleotide Purification and Analysisndash Forensicsndash Microbial Analysisndash RNA Isolation and Purificationndash Single Base Extensionndash Methylation
With the WAVE System you can
Scan fragments for both known and novel
mutationsSNPs without extensive resequencing
Detect low-abundance mutations in heterogeneous
samples
Enrich for low-abundance alleles
Fractionate complex mixtures of related fragments
based on differences in sequence content
Perform targeted mutationSNP scoring via primer
extension assays
Analyze end-point products from allele-specific
ProbePrimer-based Amplification Technologies
ProbePrimer-based Amplification Technologies
bull Primer-based Amplificationsndash Non-Isothermal Amplifications eg PCR-based
Methodsndash Isothermal AmplificationsNon-PCR-based
Amplificationsndash NASBAndash HDAndash SDAndash and more than 45 NAIA Technologies
bull Probe-based Amplificationsndash For example Ligase Chain Reactions
CARTRIDGE dependent Applications eg via WAVE Systems
DNA-99-3510 DNASep Cartridge 46 mm x 50 mm for variety of
DNA applications including Sizing and Mutation Detection load
capacity of 2microg
DNA-99-3710 DNASep HT Cartridge 65 mm x 37 mm for variety
of DNA application but especially for Rapid Mutation Detection
load capacity of 4microg
NUC-99-3550 OligoSepreg Cartridge 46 mm x 50 mm for Oligo
Purification work only
NUC-99-3860 OligoSep Prep HC Cartridge 78 mm x 50 mm for
Oligo Purification load capacity of 5000microg
RNA-99-3810 RNASepreg Prep Cartridge 78 mm x 50 mm for
variety of RNA applications load capacity of 50microg total RNA -OR-
1000ng of specific RNA
Chromatograms and pedigree of patients 3 and 16 A DHPLC analysis in normal control patients 3 and 16 Both patients show abnormal DHPLC peak patterns with two peaks in comparison to normal control B Sequencing data in patient 3 and his family members C Sequencing data in patient 16 and his family members
Example of Oligo Analysis by DHPLC
Thanks for Your AttentionsReference
Thanks for Your Attentions
ReferenceMethods in Molecular Medicine Vol 108
Hypertension Methods and Protocols Edited by J P Fennell and A H Baker copy Humana Press Inc Totowa NJ Chapter 13
- DHPLC Principles An Introduction to Denaturing High Performanc
- Nucleic Acids
- Nucleic Acids OMICS
- Genomics Variations as MutationPolymorphism
- The Principles for Gene Variation Analysis
- Scanning Procedures
- Scanning Procedures (2)
- Screening Procedures
- Screening Procedures (2)
- Heteroduplex analysis (HET) Keen J et al (1991) Rapid dete
- Technological Improvement of HET to eg DHPLC
- Technological Improvement of HET to eg DHPLC (2)
- Slide 13
- DHPLC Graphical Scheme
- Software 1 HSM (Hitachi) and WAVEMAKER 41 (Transgenomic) soft
- Slide 16
- Slide 17
- Nucleic Acid Amplifications eg PCR
- Equipments
- Solutions
- Slide 21
- Results
- Applications
- With the WAVE System you can
- ProbePrimer-based Amplification Technologies
- CARTRIDGE dependent Applications eg via WAVE Systems
- Slide 27
- Slide 28
- Slide 29
- Chromatograms and pedigree of patients 3 and 16 A DHPLC analy
- Example of Oligo Analysis by DHPLC
- Slide 32
- Thanks for Your Attentions Reference
- Thanks for Your Attentions
-
Screening Procedures
RGH Cotton Mutation Research 285 (1993) 125-144
Heteroduplex analysis (HET) Keen J et al (1991) Rapid detection of single base mismatches as
heteroduplexes on hydrolink gels Trends Genet 7 5
bull Heteroduplexes containing single base-pair mismatches can be accurately separated from related heteroduplexes on nondenaturing gels
bull Others performed separation of heteroduplexes on normal gels which detected deletions but their method probably would not detect point mutations and can thus be considered a different method of lesser sensitivity
bull Thus far there have been few modifications The main advantage of the HET method is simplicity (as for SSCP) but its application has not been so widespread partly because of its later description and partly because of the need for Hydrolink gels in the initial description
bull The main disadvantage is the lack of 100 detection Like SSCP the HET method can only be applied to fragments hundreds of base pairs long (for example 200-300 bp)
Technological Improvement of HET to eg DHPLC
bull Patent Information1 Column matrix
ndash 1Bonn G Huber C Oefner P (1994) Verfahren zur Trennung von Nucleinsaeuren Austrian Patent No 398 973 Vienna Austria
ndash 2Bonn G Huber C Oefner P (1996) Nucleic Acid Separation on Alkylated Nonporous Polymer Beads US Patent No 5585236
ndash Currently exclusively licensed to Transgenomic Inc Omaha NE USA
DHPLCndash 3Oefner PJ Underhill PA (1998) Detection of Nucleic Acid Heteroduplex
Molecules by Denaturing High-Performance Liquid Chromatography and Methods for Comparative Sequencing US Patent 5795976 [Stanford Reference] [USPTO]
ndash 4Hansen NF Oefner PJ (1997) Software to Determine Optimum Temperature for DHPLC Given DNA Sequence Stanford University Invention Disclosure S97-175 Tangible Research Property in conjunction with US Patent 5795976 [Stanford Reference]
ndash Currently licensed to Transgenomic Inc Omaha NE USA Agilent Palo Alto CA USA and Varian Walnut Creek CA USA
ndash 5Oefner PJ (1999) Detection of Polymorphisms by Denaturing High-Performance Liquid Chromatography US Patent 6453244 [Stanford Reference] [USPTO]
ndash 6Huber CG OKeefe M Oberacher H Oefner PJ Premstaller A Xiao W Temperature-Modulated Array High-Performance Liquid Chromatography Provisional Patent filed [Stanford Reference]
Technological Improvement of HET to eg DHPLCbull Patent Information2 DNA shearing
ndash 7Oefner PJ Hunicke-Smith S (1998) Apparatus and Methods for Shear Breakage of Poly-nucleotides US Patent 5846832 [Stanford Reference] [USPTO]
ndash Licensed to Gene Machines Redwood City CA USA
DNA markersndash 8Oefner PJ Underhill PA Human Y Chromosome Specific
Single Nucleotide Polymorphisms US Patent Pending [Stanford Reference]
Principle of DHPLC
Rapid denaturation of DNA by heating re-annealing by slow cooling
Heteroduplexes form in the presence of two different alleles
DHPLCGraphical Scheme
Software1 HSM (Hitachi) and WAVEMAKER 41 (Transgenomic) software2 Navigator (Transgenomic)
Nucleic Acid Amplifications eg PCR
PCR Amplification for Mutation Analysis Using the Transgenomic WAVEreg System
from Invitrogen
ndash Introduction Discoverasetrade dHPLC DNA Polymerase is an enzyme mixture
composed of recombinant Taq DNA polymerase and Pyrococcus species GB-D
polymerase Pyrococcus species GB-D polymerase possesses a proofreading
ability by virtue of its 3rsquo to 5rsquo exonuclease activity Mixture of the proofreading
enzyme with Taq DNA polymerase at an optimized ratio increases fidelity
approximately eight times over that of Taq DNA polymerase alone and allows
amplification of simple and complex DNA templates The enzyme mixture is
provided with an optimized buffer that improves enzyme fidelity
ndash The Discoverasetrade dHPLC DNA Polymerase enzyme mixture and buffer
formulation have been optimized for use with denaturing high-performance
liquid chromatography (dHPLC) systems They were developed and tested using
the Transgenomic WAVEreg SystemDiscoverasetrade dHPLC DNA Polymerase is
supplied at 1 unit per microl
Equipments
1 Thermocycler (with heated lid)
2 DHPLC WAVE DNA fragment analysis system
(Transgenomic)
3 DNA Sep HT cartridge (cat no DNA-99-3710
Transgenomic)
4 Transmit silicone sealing mats (see Note 2) (cat no
172024 Transgenomic)
5 PCR plates (see Note 3) (cat no 0030 127307
Eppendorf)
6 Chemical waste container
Solutions1 Optimase polymerase (supplied with Mg2+-free
buffer and separate Mg2+ solution)(cat no 703045 Transgenomic)2 100 mM dNTP set PCR grade (cat no 10297-117
Invitrogen)3 Mutation control standards low-range mutation
standard (cat no 560077) and high-range mutation standard (cat no 562001 Transgenomic)
4 DNA sizing standard (cat no 560078 Transgenomic)5 Acetonitrile 9993+ HPLC grade (cat no 27 071-7
Sigma-Aldrich)6 WAVE ion-pairing agent 2 M triethylammonium
acetate (TEAA) (cat no 553303 Transgenomic)7 Water (eg MilliQ water) use 18 M1048625 resistance or
better8 WAVE optimized buffers (optional) Buffers A B D
and syringe solution (cat nos 553401 553402 553404 and 553403 Transgenomic)
HomoduplexesHeteroduplex
es
Chromatographic Separation Reverse-phase ion-pair system
Gradient elution with acetonitrilewater
UV detection
Column oven temperature selected for partial denaturation of heteroduplexes and thus earlier elution
Results
Example of elution profiles for a wild-type and a mutant sample The mutant sample contains an insertiondeletion mutation in exon 8 of the RET gene
Example of elution profiles obtained for a wild-type and a mutant sample The mutant sample contains a stop codon mutation in exon 15 of the RET gene
Applications DHPLC Systems (eg WAVE Systems) can
be used for a wide range of applications includingndash Mutation Detectionndash Sizingndash Oligonucleotide Purification and Analysisndash Forensicsndash Microbial Analysisndash RNA Isolation and Purificationndash Single Base Extensionndash Methylation
With the WAVE System you can
Scan fragments for both known and novel
mutationsSNPs without extensive resequencing
Detect low-abundance mutations in heterogeneous
samples
Enrich for low-abundance alleles
Fractionate complex mixtures of related fragments
based on differences in sequence content
Perform targeted mutationSNP scoring via primer
extension assays
Analyze end-point products from allele-specific
ProbePrimer-based Amplification Technologies
ProbePrimer-based Amplification Technologies
bull Primer-based Amplificationsndash Non-Isothermal Amplifications eg PCR-based
Methodsndash Isothermal AmplificationsNon-PCR-based
Amplificationsndash NASBAndash HDAndash SDAndash and more than 45 NAIA Technologies
bull Probe-based Amplificationsndash For example Ligase Chain Reactions
CARTRIDGE dependent Applications eg via WAVE Systems
DNA-99-3510 DNASep Cartridge 46 mm x 50 mm for variety of
DNA applications including Sizing and Mutation Detection load
capacity of 2microg
DNA-99-3710 DNASep HT Cartridge 65 mm x 37 mm for variety
of DNA application but especially for Rapid Mutation Detection
load capacity of 4microg
NUC-99-3550 OligoSepreg Cartridge 46 mm x 50 mm for Oligo
Purification work only
NUC-99-3860 OligoSep Prep HC Cartridge 78 mm x 50 mm for
Oligo Purification load capacity of 5000microg
RNA-99-3810 RNASepreg Prep Cartridge 78 mm x 50 mm for
variety of RNA applications load capacity of 50microg total RNA -OR-
1000ng of specific RNA
Chromatograms and pedigree of patients 3 and 16 A DHPLC analysis in normal control patients 3 and 16 Both patients show abnormal DHPLC peak patterns with two peaks in comparison to normal control B Sequencing data in patient 3 and his family members C Sequencing data in patient 16 and his family members
Example of Oligo Analysis by DHPLC
Thanks for Your AttentionsReference
Thanks for Your Attentions
ReferenceMethods in Molecular Medicine Vol 108
Hypertension Methods and Protocols Edited by J P Fennell and A H Baker copy Humana Press Inc Totowa NJ Chapter 13
- DHPLC Principles An Introduction to Denaturing High Performanc
- Nucleic Acids
- Nucleic Acids OMICS
- Genomics Variations as MutationPolymorphism
- The Principles for Gene Variation Analysis
- Scanning Procedures
- Scanning Procedures (2)
- Screening Procedures
- Screening Procedures (2)
- Heteroduplex analysis (HET) Keen J et al (1991) Rapid dete
- Technological Improvement of HET to eg DHPLC
- Technological Improvement of HET to eg DHPLC (2)
- Slide 13
- DHPLC Graphical Scheme
- Software 1 HSM (Hitachi) and WAVEMAKER 41 (Transgenomic) soft
- Slide 16
- Slide 17
- Nucleic Acid Amplifications eg PCR
- Equipments
- Solutions
- Slide 21
- Results
- Applications
- With the WAVE System you can
- ProbePrimer-based Amplification Technologies
- CARTRIDGE dependent Applications eg via WAVE Systems
- Slide 27
- Slide 28
- Slide 29
- Chromatograms and pedigree of patients 3 and 16 A DHPLC analy
- Example of Oligo Analysis by DHPLC
- Slide 32
- Thanks for Your Attentions Reference
- Thanks for Your Attentions
-
Heteroduplex analysis (HET) Keen J et al (1991) Rapid detection of single base mismatches as
heteroduplexes on hydrolink gels Trends Genet 7 5
bull Heteroduplexes containing single base-pair mismatches can be accurately separated from related heteroduplexes on nondenaturing gels
bull Others performed separation of heteroduplexes on normal gels which detected deletions but their method probably would not detect point mutations and can thus be considered a different method of lesser sensitivity
bull Thus far there have been few modifications The main advantage of the HET method is simplicity (as for SSCP) but its application has not been so widespread partly because of its later description and partly because of the need for Hydrolink gels in the initial description
bull The main disadvantage is the lack of 100 detection Like SSCP the HET method can only be applied to fragments hundreds of base pairs long (for example 200-300 bp)
Technological Improvement of HET to eg DHPLC
bull Patent Information1 Column matrix
ndash 1Bonn G Huber C Oefner P (1994) Verfahren zur Trennung von Nucleinsaeuren Austrian Patent No 398 973 Vienna Austria
ndash 2Bonn G Huber C Oefner P (1996) Nucleic Acid Separation on Alkylated Nonporous Polymer Beads US Patent No 5585236
ndash Currently exclusively licensed to Transgenomic Inc Omaha NE USA
DHPLCndash 3Oefner PJ Underhill PA (1998) Detection of Nucleic Acid Heteroduplex
Molecules by Denaturing High-Performance Liquid Chromatography and Methods for Comparative Sequencing US Patent 5795976 [Stanford Reference] [USPTO]
ndash 4Hansen NF Oefner PJ (1997) Software to Determine Optimum Temperature for DHPLC Given DNA Sequence Stanford University Invention Disclosure S97-175 Tangible Research Property in conjunction with US Patent 5795976 [Stanford Reference]
ndash Currently licensed to Transgenomic Inc Omaha NE USA Agilent Palo Alto CA USA and Varian Walnut Creek CA USA
ndash 5Oefner PJ (1999) Detection of Polymorphisms by Denaturing High-Performance Liquid Chromatography US Patent 6453244 [Stanford Reference] [USPTO]
ndash 6Huber CG OKeefe M Oberacher H Oefner PJ Premstaller A Xiao W Temperature-Modulated Array High-Performance Liquid Chromatography Provisional Patent filed [Stanford Reference]
Technological Improvement of HET to eg DHPLCbull Patent Information2 DNA shearing
ndash 7Oefner PJ Hunicke-Smith S (1998) Apparatus and Methods for Shear Breakage of Poly-nucleotides US Patent 5846832 [Stanford Reference] [USPTO]
ndash Licensed to Gene Machines Redwood City CA USA
DNA markersndash 8Oefner PJ Underhill PA Human Y Chromosome Specific
Single Nucleotide Polymorphisms US Patent Pending [Stanford Reference]
Principle of DHPLC
Rapid denaturation of DNA by heating re-annealing by slow cooling
Heteroduplexes form in the presence of two different alleles
DHPLCGraphical Scheme
Software1 HSM (Hitachi) and WAVEMAKER 41 (Transgenomic) software2 Navigator (Transgenomic)
Nucleic Acid Amplifications eg PCR
PCR Amplification for Mutation Analysis Using the Transgenomic WAVEreg System
from Invitrogen
ndash Introduction Discoverasetrade dHPLC DNA Polymerase is an enzyme mixture
composed of recombinant Taq DNA polymerase and Pyrococcus species GB-D
polymerase Pyrococcus species GB-D polymerase possesses a proofreading
ability by virtue of its 3rsquo to 5rsquo exonuclease activity Mixture of the proofreading
enzyme with Taq DNA polymerase at an optimized ratio increases fidelity
approximately eight times over that of Taq DNA polymerase alone and allows
amplification of simple and complex DNA templates The enzyme mixture is
provided with an optimized buffer that improves enzyme fidelity
ndash The Discoverasetrade dHPLC DNA Polymerase enzyme mixture and buffer
formulation have been optimized for use with denaturing high-performance
liquid chromatography (dHPLC) systems They were developed and tested using
the Transgenomic WAVEreg SystemDiscoverasetrade dHPLC DNA Polymerase is
supplied at 1 unit per microl
Equipments
1 Thermocycler (with heated lid)
2 DHPLC WAVE DNA fragment analysis system
(Transgenomic)
3 DNA Sep HT cartridge (cat no DNA-99-3710
Transgenomic)
4 Transmit silicone sealing mats (see Note 2) (cat no
172024 Transgenomic)
5 PCR plates (see Note 3) (cat no 0030 127307
Eppendorf)
6 Chemical waste container
Solutions1 Optimase polymerase (supplied with Mg2+-free
buffer and separate Mg2+ solution)(cat no 703045 Transgenomic)2 100 mM dNTP set PCR grade (cat no 10297-117
Invitrogen)3 Mutation control standards low-range mutation
standard (cat no 560077) and high-range mutation standard (cat no 562001 Transgenomic)
4 DNA sizing standard (cat no 560078 Transgenomic)5 Acetonitrile 9993+ HPLC grade (cat no 27 071-7
Sigma-Aldrich)6 WAVE ion-pairing agent 2 M triethylammonium
acetate (TEAA) (cat no 553303 Transgenomic)7 Water (eg MilliQ water) use 18 M1048625 resistance or
better8 WAVE optimized buffers (optional) Buffers A B D
and syringe solution (cat nos 553401 553402 553404 and 553403 Transgenomic)
HomoduplexesHeteroduplex
es
Chromatographic Separation Reverse-phase ion-pair system
Gradient elution with acetonitrilewater
UV detection
Column oven temperature selected for partial denaturation of heteroduplexes and thus earlier elution
Results
Example of elution profiles for a wild-type and a mutant sample The mutant sample contains an insertiondeletion mutation in exon 8 of the RET gene
Example of elution profiles obtained for a wild-type and a mutant sample The mutant sample contains a stop codon mutation in exon 15 of the RET gene
Applications DHPLC Systems (eg WAVE Systems) can
be used for a wide range of applications includingndash Mutation Detectionndash Sizingndash Oligonucleotide Purification and Analysisndash Forensicsndash Microbial Analysisndash RNA Isolation and Purificationndash Single Base Extensionndash Methylation
With the WAVE System you can
Scan fragments for both known and novel
mutationsSNPs without extensive resequencing
Detect low-abundance mutations in heterogeneous
samples
Enrich for low-abundance alleles
Fractionate complex mixtures of related fragments
based on differences in sequence content
Perform targeted mutationSNP scoring via primer
extension assays
Analyze end-point products from allele-specific
ProbePrimer-based Amplification Technologies
ProbePrimer-based Amplification Technologies
bull Primer-based Amplificationsndash Non-Isothermal Amplifications eg PCR-based
Methodsndash Isothermal AmplificationsNon-PCR-based
Amplificationsndash NASBAndash HDAndash SDAndash and more than 45 NAIA Technologies
bull Probe-based Amplificationsndash For example Ligase Chain Reactions
CARTRIDGE dependent Applications eg via WAVE Systems
DNA-99-3510 DNASep Cartridge 46 mm x 50 mm for variety of
DNA applications including Sizing and Mutation Detection load
capacity of 2microg
DNA-99-3710 DNASep HT Cartridge 65 mm x 37 mm for variety
of DNA application but especially for Rapid Mutation Detection
load capacity of 4microg
NUC-99-3550 OligoSepreg Cartridge 46 mm x 50 mm for Oligo
Purification work only
NUC-99-3860 OligoSep Prep HC Cartridge 78 mm x 50 mm for
Oligo Purification load capacity of 5000microg
RNA-99-3810 RNASepreg Prep Cartridge 78 mm x 50 mm for
variety of RNA applications load capacity of 50microg total RNA -OR-
1000ng of specific RNA
Chromatograms and pedigree of patients 3 and 16 A DHPLC analysis in normal control patients 3 and 16 Both patients show abnormal DHPLC peak patterns with two peaks in comparison to normal control B Sequencing data in patient 3 and his family members C Sequencing data in patient 16 and his family members
Example of Oligo Analysis by DHPLC
Thanks for Your AttentionsReference
Thanks for Your Attentions
ReferenceMethods in Molecular Medicine Vol 108
Hypertension Methods and Protocols Edited by J P Fennell and A H Baker copy Humana Press Inc Totowa NJ Chapter 13
- DHPLC Principles An Introduction to Denaturing High Performanc
- Nucleic Acids
- Nucleic Acids OMICS
- Genomics Variations as MutationPolymorphism
- The Principles for Gene Variation Analysis
- Scanning Procedures
- Scanning Procedures (2)
- Screening Procedures
- Screening Procedures (2)
- Heteroduplex analysis (HET) Keen J et al (1991) Rapid dete
- Technological Improvement of HET to eg DHPLC
- Technological Improvement of HET to eg DHPLC (2)
- Slide 13
- DHPLC Graphical Scheme
- Software 1 HSM (Hitachi) and WAVEMAKER 41 (Transgenomic) soft
- Slide 16
- Slide 17
- Nucleic Acid Amplifications eg PCR
- Equipments
- Solutions
- Slide 21
- Results
- Applications
- With the WAVE System you can
- ProbePrimer-based Amplification Technologies
- CARTRIDGE dependent Applications eg via WAVE Systems
- Slide 27
- Slide 28
- Slide 29
- Chromatograms and pedigree of patients 3 and 16 A DHPLC analy
- Example of Oligo Analysis by DHPLC
- Slide 32
- Thanks for Your Attentions Reference
- Thanks for Your Attentions
-
Technological Improvement of HET to eg DHPLC
bull Patent Information1 Column matrix
ndash 1Bonn G Huber C Oefner P (1994) Verfahren zur Trennung von Nucleinsaeuren Austrian Patent No 398 973 Vienna Austria
ndash 2Bonn G Huber C Oefner P (1996) Nucleic Acid Separation on Alkylated Nonporous Polymer Beads US Patent No 5585236
ndash Currently exclusively licensed to Transgenomic Inc Omaha NE USA
DHPLCndash 3Oefner PJ Underhill PA (1998) Detection of Nucleic Acid Heteroduplex
Molecules by Denaturing High-Performance Liquid Chromatography and Methods for Comparative Sequencing US Patent 5795976 [Stanford Reference] [USPTO]
ndash 4Hansen NF Oefner PJ (1997) Software to Determine Optimum Temperature for DHPLC Given DNA Sequence Stanford University Invention Disclosure S97-175 Tangible Research Property in conjunction with US Patent 5795976 [Stanford Reference]
ndash Currently licensed to Transgenomic Inc Omaha NE USA Agilent Palo Alto CA USA and Varian Walnut Creek CA USA
ndash 5Oefner PJ (1999) Detection of Polymorphisms by Denaturing High-Performance Liquid Chromatography US Patent 6453244 [Stanford Reference] [USPTO]
ndash 6Huber CG OKeefe M Oberacher H Oefner PJ Premstaller A Xiao W Temperature-Modulated Array High-Performance Liquid Chromatography Provisional Patent filed [Stanford Reference]
Technological Improvement of HET to eg DHPLCbull Patent Information2 DNA shearing
ndash 7Oefner PJ Hunicke-Smith S (1998) Apparatus and Methods for Shear Breakage of Poly-nucleotides US Patent 5846832 [Stanford Reference] [USPTO]
ndash Licensed to Gene Machines Redwood City CA USA
DNA markersndash 8Oefner PJ Underhill PA Human Y Chromosome Specific
Single Nucleotide Polymorphisms US Patent Pending [Stanford Reference]
Principle of DHPLC
Rapid denaturation of DNA by heating re-annealing by slow cooling
Heteroduplexes form in the presence of two different alleles
DHPLCGraphical Scheme
Software1 HSM (Hitachi) and WAVEMAKER 41 (Transgenomic) software2 Navigator (Transgenomic)
Nucleic Acid Amplifications eg PCR
PCR Amplification for Mutation Analysis Using the Transgenomic WAVEreg System
from Invitrogen
ndash Introduction Discoverasetrade dHPLC DNA Polymerase is an enzyme mixture
composed of recombinant Taq DNA polymerase and Pyrococcus species GB-D
polymerase Pyrococcus species GB-D polymerase possesses a proofreading
ability by virtue of its 3rsquo to 5rsquo exonuclease activity Mixture of the proofreading
enzyme with Taq DNA polymerase at an optimized ratio increases fidelity
approximately eight times over that of Taq DNA polymerase alone and allows
amplification of simple and complex DNA templates The enzyme mixture is
provided with an optimized buffer that improves enzyme fidelity
ndash The Discoverasetrade dHPLC DNA Polymerase enzyme mixture and buffer
formulation have been optimized for use with denaturing high-performance
liquid chromatography (dHPLC) systems They were developed and tested using
the Transgenomic WAVEreg SystemDiscoverasetrade dHPLC DNA Polymerase is
supplied at 1 unit per microl
Equipments
1 Thermocycler (with heated lid)
2 DHPLC WAVE DNA fragment analysis system
(Transgenomic)
3 DNA Sep HT cartridge (cat no DNA-99-3710
Transgenomic)
4 Transmit silicone sealing mats (see Note 2) (cat no
172024 Transgenomic)
5 PCR plates (see Note 3) (cat no 0030 127307
Eppendorf)
6 Chemical waste container
Solutions1 Optimase polymerase (supplied with Mg2+-free
buffer and separate Mg2+ solution)(cat no 703045 Transgenomic)2 100 mM dNTP set PCR grade (cat no 10297-117
Invitrogen)3 Mutation control standards low-range mutation
standard (cat no 560077) and high-range mutation standard (cat no 562001 Transgenomic)
4 DNA sizing standard (cat no 560078 Transgenomic)5 Acetonitrile 9993+ HPLC grade (cat no 27 071-7
Sigma-Aldrich)6 WAVE ion-pairing agent 2 M triethylammonium
acetate (TEAA) (cat no 553303 Transgenomic)7 Water (eg MilliQ water) use 18 M1048625 resistance or
better8 WAVE optimized buffers (optional) Buffers A B D
and syringe solution (cat nos 553401 553402 553404 and 553403 Transgenomic)
HomoduplexesHeteroduplex
es
Chromatographic Separation Reverse-phase ion-pair system
Gradient elution with acetonitrilewater
UV detection
Column oven temperature selected for partial denaturation of heteroduplexes and thus earlier elution
Results
Example of elution profiles for a wild-type and a mutant sample The mutant sample contains an insertiondeletion mutation in exon 8 of the RET gene
Example of elution profiles obtained for a wild-type and a mutant sample The mutant sample contains a stop codon mutation in exon 15 of the RET gene
Applications DHPLC Systems (eg WAVE Systems) can
be used for a wide range of applications includingndash Mutation Detectionndash Sizingndash Oligonucleotide Purification and Analysisndash Forensicsndash Microbial Analysisndash RNA Isolation and Purificationndash Single Base Extensionndash Methylation
With the WAVE System you can
Scan fragments for both known and novel
mutationsSNPs without extensive resequencing
Detect low-abundance mutations in heterogeneous
samples
Enrich for low-abundance alleles
Fractionate complex mixtures of related fragments
based on differences in sequence content
Perform targeted mutationSNP scoring via primer
extension assays
Analyze end-point products from allele-specific
ProbePrimer-based Amplification Technologies
ProbePrimer-based Amplification Technologies
bull Primer-based Amplificationsndash Non-Isothermal Amplifications eg PCR-based
Methodsndash Isothermal AmplificationsNon-PCR-based
Amplificationsndash NASBAndash HDAndash SDAndash and more than 45 NAIA Technologies
bull Probe-based Amplificationsndash For example Ligase Chain Reactions
CARTRIDGE dependent Applications eg via WAVE Systems
DNA-99-3510 DNASep Cartridge 46 mm x 50 mm for variety of
DNA applications including Sizing and Mutation Detection load
capacity of 2microg
DNA-99-3710 DNASep HT Cartridge 65 mm x 37 mm for variety
of DNA application but especially for Rapid Mutation Detection
load capacity of 4microg
NUC-99-3550 OligoSepreg Cartridge 46 mm x 50 mm for Oligo
Purification work only
NUC-99-3860 OligoSep Prep HC Cartridge 78 mm x 50 mm for
Oligo Purification load capacity of 5000microg
RNA-99-3810 RNASepreg Prep Cartridge 78 mm x 50 mm for
variety of RNA applications load capacity of 50microg total RNA -OR-
1000ng of specific RNA
Chromatograms and pedigree of patients 3 and 16 A DHPLC analysis in normal control patients 3 and 16 Both patients show abnormal DHPLC peak patterns with two peaks in comparison to normal control B Sequencing data in patient 3 and his family members C Sequencing data in patient 16 and his family members
Example of Oligo Analysis by DHPLC
Thanks for Your AttentionsReference
Thanks for Your Attentions
ReferenceMethods in Molecular Medicine Vol 108
Hypertension Methods and Protocols Edited by J P Fennell and A H Baker copy Humana Press Inc Totowa NJ Chapter 13
- DHPLC Principles An Introduction to Denaturing High Performanc
- Nucleic Acids
- Nucleic Acids OMICS
- Genomics Variations as MutationPolymorphism
- The Principles for Gene Variation Analysis
- Scanning Procedures
- Scanning Procedures (2)
- Screening Procedures
- Screening Procedures (2)
- Heteroduplex analysis (HET) Keen J et al (1991) Rapid dete
- Technological Improvement of HET to eg DHPLC
- Technological Improvement of HET to eg DHPLC (2)
- Slide 13
- DHPLC Graphical Scheme
- Software 1 HSM (Hitachi) and WAVEMAKER 41 (Transgenomic) soft
- Slide 16
- Slide 17
- Nucleic Acid Amplifications eg PCR
- Equipments
- Solutions
- Slide 21
- Results
- Applications
- With the WAVE System you can
- ProbePrimer-based Amplification Technologies
- CARTRIDGE dependent Applications eg via WAVE Systems
- Slide 27
- Slide 28
- Slide 29
- Chromatograms and pedigree of patients 3 and 16 A DHPLC analy
- Example of Oligo Analysis by DHPLC
- Slide 32
- Thanks for Your Attentions Reference
- Thanks for Your Attentions
-
Technological Improvement of HET to eg DHPLCbull Patent Information2 DNA shearing
ndash 7Oefner PJ Hunicke-Smith S (1998) Apparatus and Methods for Shear Breakage of Poly-nucleotides US Patent 5846832 [Stanford Reference] [USPTO]
ndash Licensed to Gene Machines Redwood City CA USA
DNA markersndash 8Oefner PJ Underhill PA Human Y Chromosome Specific
Single Nucleotide Polymorphisms US Patent Pending [Stanford Reference]
Principle of DHPLC
Rapid denaturation of DNA by heating re-annealing by slow cooling
Heteroduplexes form in the presence of two different alleles
DHPLCGraphical Scheme
Software1 HSM (Hitachi) and WAVEMAKER 41 (Transgenomic) software2 Navigator (Transgenomic)
Nucleic Acid Amplifications eg PCR
PCR Amplification for Mutation Analysis Using the Transgenomic WAVEreg System
from Invitrogen
ndash Introduction Discoverasetrade dHPLC DNA Polymerase is an enzyme mixture
composed of recombinant Taq DNA polymerase and Pyrococcus species GB-D
polymerase Pyrococcus species GB-D polymerase possesses a proofreading
ability by virtue of its 3rsquo to 5rsquo exonuclease activity Mixture of the proofreading
enzyme with Taq DNA polymerase at an optimized ratio increases fidelity
approximately eight times over that of Taq DNA polymerase alone and allows
amplification of simple and complex DNA templates The enzyme mixture is
provided with an optimized buffer that improves enzyme fidelity
ndash The Discoverasetrade dHPLC DNA Polymerase enzyme mixture and buffer
formulation have been optimized for use with denaturing high-performance
liquid chromatography (dHPLC) systems They were developed and tested using
the Transgenomic WAVEreg SystemDiscoverasetrade dHPLC DNA Polymerase is
supplied at 1 unit per microl
Equipments
1 Thermocycler (with heated lid)
2 DHPLC WAVE DNA fragment analysis system
(Transgenomic)
3 DNA Sep HT cartridge (cat no DNA-99-3710
Transgenomic)
4 Transmit silicone sealing mats (see Note 2) (cat no
172024 Transgenomic)
5 PCR plates (see Note 3) (cat no 0030 127307
Eppendorf)
6 Chemical waste container
Solutions1 Optimase polymerase (supplied with Mg2+-free
buffer and separate Mg2+ solution)(cat no 703045 Transgenomic)2 100 mM dNTP set PCR grade (cat no 10297-117
Invitrogen)3 Mutation control standards low-range mutation
standard (cat no 560077) and high-range mutation standard (cat no 562001 Transgenomic)
4 DNA sizing standard (cat no 560078 Transgenomic)5 Acetonitrile 9993+ HPLC grade (cat no 27 071-7
Sigma-Aldrich)6 WAVE ion-pairing agent 2 M triethylammonium
acetate (TEAA) (cat no 553303 Transgenomic)7 Water (eg MilliQ water) use 18 M1048625 resistance or
better8 WAVE optimized buffers (optional) Buffers A B D
and syringe solution (cat nos 553401 553402 553404 and 553403 Transgenomic)
HomoduplexesHeteroduplex
es
Chromatographic Separation Reverse-phase ion-pair system
Gradient elution with acetonitrilewater
UV detection
Column oven temperature selected for partial denaturation of heteroduplexes and thus earlier elution
Results
Example of elution profiles for a wild-type and a mutant sample The mutant sample contains an insertiondeletion mutation in exon 8 of the RET gene
Example of elution profiles obtained for a wild-type and a mutant sample The mutant sample contains a stop codon mutation in exon 15 of the RET gene
Applications DHPLC Systems (eg WAVE Systems) can
be used for a wide range of applications includingndash Mutation Detectionndash Sizingndash Oligonucleotide Purification and Analysisndash Forensicsndash Microbial Analysisndash RNA Isolation and Purificationndash Single Base Extensionndash Methylation
With the WAVE System you can
Scan fragments for both known and novel
mutationsSNPs without extensive resequencing
Detect low-abundance mutations in heterogeneous
samples
Enrich for low-abundance alleles
Fractionate complex mixtures of related fragments
based on differences in sequence content
Perform targeted mutationSNP scoring via primer
extension assays
Analyze end-point products from allele-specific
ProbePrimer-based Amplification Technologies
ProbePrimer-based Amplification Technologies
bull Primer-based Amplificationsndash Non-Isothermal Amplifications eg PCR-based
Methodsndash Isothermal AmplificationsNon-PCR-based
Amplificationsndash NASBAndash HDAndash SDAndash and more than 45 NAIA Technologies
bull Probe-based Amplificationsndash For example Ligase Chain Reactions
CARTRIDGE dependent Applications eg via WAVE Systems
DNA-99-3510 DNASep Cartridge 46 mm x 50 mm for variety of
DNA applications including Sizing and Mutation Detection load
capacity of 2microg
DNA-99-3710 DNASep HT Cartridge 65 mm x 37 mm for variety
of DNA application but especially for Rapid Mutation Detection
load capacity of 4microg
NUC-99-3550 OligoSepreg Cartridge 46 mm x 50 mm for Oligo
Purification work only
NUC-99-3860 OligoSep Prep HC Cartridge 78 mm x 50 mm for
Oligo Purification load capacity of 5000microg
RNA-99-3810 RNASepreg Prep Cartridge 78 mm x 50 mm for
variety of RNA applications load capacity of 50microg total RNA -OR-
1000ng of specific RNA
Chromatograms and pedigree of patients 3 and 16 A DHPLC analysis in normal control patients 3 and 16 Both patients show abnormal DHPLC peak patterns with two peaks in comparison to normal control B Sequencing data in patient 3 and his family members C Sequencing data in patient 16 and his family members
Example of Oligo Analysis by DHPLC
Thanks for Your AttentionsReference
Thanks for Your Attentions
ReferenceMethods in Molecular Medicine Vol 108
Hypertension Methods and Protocols Edited by J P Fennell and A H Baker copy Humana Press Inc Totowa NJ Chapter 13
- DHPLC Principles An Introduction to Denaturing High Performanc
- Nucleic Acids
- Nucleic Acids OMICS
- Genomics Variations as MutationPolymorphism
- The Principles for Gene Variation Analysis
- Scanning Procedures
- Scanning Procedures (2)
- Screening Procedures
- Screening Procedures (2)
- Heteroduplex analysis (HET) Keen J et al (1991) Rapid dete
- Technological Improvement of HET to eg DHPLC
- Technological Improvement of HET to eg DHPLC (2)
- Slide 13
- DHPLC Graphical Scheme
- Software 1 HSM (Hitachi) and WAVEMAKER 41 (Transgenomic) soft
- Slide 16
- Slide 17
- Nucleic Acid Amplifications eg PCR
- Equipments
- Solutions
- Slide 21
- Results
- Applications
- With the WAVE System you can
- ProbePrimer-based Amplification Technologies
- CARTRIDGE dependent Applications eg via WAVE Systems
- Slide 27
- Slide 28
- Slide 29
- Chromatograms and pedigree of patients 3 and 16 A DHPLC analy
- Example of Oligo Analysis by DHPLC
- Slide 32
- Thanks for Your Attentions Reference
- Thanks for Your Attentions
-
Principle of DHPLC
Rapid denaturation of DNA by heating re-annealing by slow cooling
Heteroduplexes form in the presence of two different alleles
DHPLCGraphical Scheme
Software1 HSM (Hitachi) and WAVEMAKER 41 (Transgenomic) software2 Navigator (Transgenomic)
Nucleic Acid Amplifications eg PCR
PCR Amplification for Mutation Analysis Using the Transgenomic WAVEreg System
from Invitrogen
ndash Introduction Discoverasetrade dHPLC DNA Polymerase is an enzyme mixture
composed of recombinant Taq DNA polymerase and Pyrococcus species GB-D
polymerase Pyrococcus species GB-D polymerase possesses a proofreading
ability by virtue of its 3rsquo to 5rsquo exonuclease activity Mixture of the proofreading
enzyme with Taq DNA polymerase at an optimized ratio increases fidelity
approximately eight times over that of Taq DNA polymerase alone and allows
amplification of simple and complex DNA templates The enzyme mixture is
provided with an optimized buffer that improves enzyme fidelity
ndash The Discoverasetrade dHPLC DNA Polymerase enzyme mixture and buffer
formulation have been optimized for use with denaturing high-performance
liquid chromatography (dHPLC) systems They were developed and tested using
the Transgenomic WAVEreg SystemDiscoverasetrade dHPLC DNA Polymerase is
supplied at 1 unit per microl
Equipments
1 Thermocycler (with heated lid)
2 DHPLC WAVE DNA fragment analysis system
(Transgenomic)
3 DNA Sep HT cartridge (cat no DNA-99-3710
Transgenomic)
4 Transmit silicone sealing mats (see Note 2) (cat no
172024 Transgenomic)
5 PCR plates (see Note 3) (cat no 0030 127307
Eppendorf)
6 Chemical waste container
Solutions1 Optimase polymerase (supplied with Mg2+-free
buffer and separate Mg2+ solution)(cat no 703045 Transgenomic)2 100 mM dNTP set PCR grade (cat no 10297-117
Invitrogen)3 Mutation control standards low-range mutation
standard (cat no 560077) and high-range mutation standard (cat no 562001 Transgenomic)
4 DNA sizing standard (cat no 560078 Transgenomic)5 Acetonitrile 9993+ HPLC grade (cat no 27 071-7
Sigma-Aldrich)6 WAVE ion-pairing agent 2 M triethylammonium
acetate (TEAA) (cat no 553303 Transgenomic)7 Water (eg MilliQ water) use 18 M1048625 resistance or
better8 WAVE optimized buffers (optional) Buffers A B D
and syringe solution (cat nos 553401 553402 553404 and 553403 Transgenomic)
HomoduplexesHeteroduplex
es
Chromatographic Separation Reverse-phase ion-pair system
Gradient elution with acetonitrilewater
UV detection
Column oven temperature selected for partial denaturation of heteroduplexes and thus earlier elution
Results
Example of elution profiles for a wild-type and a mutant sample The mutant sample contains an insertiondeletion mutation in exon 8 of the RET gene
Example of elution profiles obtained for a wild-type and a mutant sample The mutant sample contains a stop codon mutation in exon 15 of the RET gene
Applications DHPLC Systems (eg WAVE Systems) can
be used for a wide range of applications includingndash Mutation Detectionndash Sizingndash Oligonucleotide Purification and Analysisndash Forensicsndash Microbial Analysisndash RNA Isolation and Purificationndash Single Base Extensionndash Methylation
With the WAVE System you can
Scan fragments for both known and novel
mutationsSNPs without extensive resequencing
Detect low-abundance mutations in heterogeneous
samples
Enrich for low-abundance alleles
Fractionate complex mixtures of related fragments
based on differences in sequence content
Perform targeted mutationSNP scoring via primer
extension assays
Analyze end-point products from allele-specific
ProbePrimer-based Amplification Technologies
ProbePrimer-based Amplification Technologies
bull Primer-based Amplificationsndash Non-Isothermal Amplifications eg PCR-based
Methodsndash Isothermal AmplificationsNon-PCR-based
Amplificationsndash NASBAndash HDAndash SDAndash and more than 45 NAIA Technologies
bull Probe-based Amplificationsndash For example Ligase Chain Reactions
CARTRIDGE dependent Applications eg via WAVE Systems
DNA-99-3510 DNASep Cartridge 46 mm x 50 mm for variety of
DNA applications including Sizing and Mutation Detection load
capacity of 2microg
DNA-99-3710 DNASep HT Cartridge 65 mm x 37 mm for variety
of DNA application but especially for Rapid Mutation Detection
load capacity of 4microg
NUC-99-3550 OligoSepreg Cartridge 46 mm x 50 mm for Oligo
Purification work only
NUC-99-3860 OligoSep Prep HC Cartridge 78 mm x 50 mm for
Oligo Purification load capacity of 5000microg
RNA-99-3810 RNASepreg Prep Cartridge 78 mm x 50 mm for
variety of RNA applications load capacity of 50microg total RNA -OR-
1000ng of specific RNA
Chromatograms and pedigree of patients 3 and 16 A DHPLC analysis in normal control patients 3 and 16 Both patients show abnormal DHPLC peak patterns with two peaks in comparison to normal control B Sequencing data in patient 3 and his family members C Sequencing data in patient 16 and his family members
Example of Oligo Analysis by DHPLC
Thanks for Your AttentionsReference
Thanks for Your Attentions
ReferenceMethods in Molecular Medicine Vol 108
Hypertension Methods and Protocols Edited by J P Fennell and A H Baker copy Humana Press Inc Totowa NJ Chapter 13
- DHPLC Principles An Introduction to Denaturing High Performanc
- Nucleic Acids
- Nucleic Acids OMICS
- Genomics Variations as MutationPolymorphism
- The Principles for Gene Variation Analysis
- Scanning Procedures
- Scanning Procedures (2)
- Screening Procedures
- Screening Procedures (2)
- Heteroduplex analysis (HET) Keen J et al (1991) Rapid dete
- Technological Improvement of HET to eg DHPLC
- Technological Improvement of HET to eg DHPLC (2)
- Slide 13
- DHPLC Graphical Scheme
- Software 1 HSM (Hitachi) and WAVEMAKER 41 (Transgenomic) soft
- Slide 16
- Slide 17
- Nucleic Acid Amplifications eg PCR
- Equipments
- Solutions
- Slide 21
- Results
- Applications
- With the WAVE System you can
- ProbePrimer-based Amplification Technologies
- CARTRIDGE dependent Applications eg via WAVE Systems
- Slide 27
- Slide 28
- Slide 29
- Chromatograms and pedigree of patients 3 and 16 A DHPLC analy
- Example of Oligo Analysis by DHPLC
- Slide 32
- Thanks for Your Attentions Reference
- Thanks for Your Attentions
-
DHPLCGraphical Scheme
Software1 HSM (Hitachi) and WAVEMAKER 41 (Transgenomic) software2 Navigator (Transgenomic)
Nucleic Acid Amplifications eg PCR
PCR Amplification for Mutation Analysis Using the Transgenomic WAVEreg System
from Invitrogen
ndash Introduction Discoverasetrade dHPLC DNA Polymerase is an enzyme mixture
composed of recombinant Taq DNA polymerase and Pyrococcus species GB-D
polymerase Pyrococcus species GB-D polymerase possesses a proofreading
ability by virtue of its 3rsquo to 5rsquo exonuclease activity Mixture of the proofreading
enzyme with Taq DNA polymerase at an optimized ratio increases fidelity
approximately eight times over that of Taq DNA polymerase alone and allows
amplification of simple and complex DNA templates The enzyme mixture is
provided with an optimized buffer that improves enzyme fidelity
ndash The Discoverasetrade dHPLC DNA Polymerase enzyme mixture and buffer
formulation have been optimized for use with denaturing high-performance
liquid chromatography (dHPLC) systems They were developed and tested using
the Transgenomic WAVEreg SystemDiscoverasetrade dHPLC DNA Polymerase is
supplied at 1 unit per microl
Equipments
1 Thermocycler (with heated lid)
2 DHPLC WAVE DNA fragment analysis system
(Transgenomic)
3 DNA Sep HT cartridge (cat no DNA-99-3710
Transgenomic)
4 Transmit silicone sealing mats (see Note 2) (cat no
172024 Transgenomic)
5 PCR plates (see Note 3) (cat no 0030 127307
Eppendorf)
6 Chemical waste container
Solutions1 Optimase polymerase (supplied with Mg2+-free
buffer and separate Mg2+ solution)(cat no 703045 Transgenomic)2 100 mM dNTP set PCR grade (cat no 10297-117
Invitrogen)3 Mutation control standards low-range mutation
standard (cat no 560077) and high-range mutation standard (cat no 562001 Transgenomic)
4 DNA sizing standard (cat no 560078 Transgenomic)5 Acetonitrile 9993+ HPLC grade (cat no 27 071-7
Sigma-Aldrich)6 WAVE ion-pairing agent 2 M triethylammonium
acetate (TEAA) (cat no 553303 Transgenomic)7 Water (eg MilliQ water) use 18 M1048625 resistance or
better8 WAVE optimized buffers (optional) Buffers A B D
and syringe solution (cat nos 553401 553402 553404 and 553403 Transgenomic)
HomoduplexesHeteroduplex
es
Chromatographic Separation Reverse-phase ion-pair system
Gradient elution with acetonitrilewater
UV detection
Column oven temperature selected for partial denaturation of heteroduplexes and thus earlier elution
Results
Example of elution profiles for a wild-type and a mutant sample The mutant sample contains an insertiondeletion mutation in exon 8 of the RET gene
Example of elution profiles obtained for a wild-type and a mutant sample The mutant sample contains a stop codon mutation in exon 15 of the RET gene
Applications DHPLC Systems (eg WAVE Systems) can
be used for a wide range of applications includingndash Mutation Detectionndash Sizingndash Oligonucleotide Purification and Analysisndash Forensicsndash Microbial Analysisndash RNA Isolation and Purificationndash Single Base Extensionndash Methylation
With the WAVE System you can
Scan fragments for both known and novel
mutationsSNPs without extensive resequencing
Detect low-abundance mutations in heterogeneous
samples
Enrich for low-abundance alleles
Fractionate complex mixtures of related fragments
based on differences in sequence content
Perform targeted mutationSNP scoring via primer
extension assays
Analyze end-point products from allele-specific
ProbePrimer-based Amplification Technologies
ProbePrimer-based Amplification Technologies
bull Primer-based Amplificationsndash Non-Isothermal Amplifications eg PCR-based
Methodsndash Isothermal AmplificationsNon-PCR-based
Amplificationsndash NASBAndash HDAndash SDAndash and more than 45 NAIA Technologies
bull Probe-based Amplificationsndash For example Ligase Chain Reactions
CARTRIDGE dependent Applications eg via WAVE Systems
DNA-99-3510 DNASep Cartridge 46 mm x 50 mm for variety of
DNA applications including Sizing and Mutation Detection load
capacity of 2microg
DNA-99-3710 DNASep HT Cartridge 65 mm x 37 mm for variety
of DNA application but especially for Rapid Mutation Detection
load capacity of 4microg
NUC-99-3550 OligoSepreg Cartridge 46 mm x 50 mm for Oligo
Purification work only
NUC-99-3860 OligoSep Prep HC Cartridge 78 mm x 50 mm for
Oligo Purification load capacity of 5000microg
RNA-99-3810 RNASepreg Prep Cartridge 78 mm x 50 mm for
variety of RNA applications load capacity of 50microg total RNA -OR-
1000ng of specific RNA
Chromatograms and pedigree of patients 3 and 16 A DHPLC analysis in normal control patients 3 and 16 Both patients show abnormal DHPLC peak patterns with two peaks in comparison to normal control B Sequencing data in patient 3 and his family members C Sequencing data in patient 16 and his family members
Example of Oligo Analysis by DHPLC
Thanks for Your AttentionsReference
Thanks for Your Attentions
ReferenceMethods in Molecular Medicine Vol 108
Hypertension Methods and Protocols Edited by J P Fennell and A H Baker copy Humana Press Inc Totowa NJ Chapter 13
- DHPLC Principles An Introduction to Denaturing High Performanc
- Nucleic Acids
- Nucleic Acids OMICS
- Genomics Variations as MutationPolymorphism
- The Principles for Gene Variation Analysis
- Scanning Procedures
- Scanning Procedures (2)
- Screening Procedures
- Screening Procedures (2)
- Heteroduplex analysis (HET) Keen J et al (1991) Rapid dete
- Technological Improvement of HET to eg DHPLC
- Technological Improvement of HET to eg DHPLC (2)
- Slide 13
- DHPLC Graphical Scheme
- Software 1 HSM (Hitachi) and WAVEMAKER 41 (Transgenomic) soft
- Slide 16
- Slide 17
- Nucleic Acid Amplifications eg PCR
- Equipments
- Solutions
- Slide 21
- Results
- Applications
- With the WAVE System you can
- ProbePrimer-based Amplification Technologies
- CARTRIDGE dependent Applications eg via WAVE Systems
- Slide 27
- Slide 28
- Slide 29
- Chromatograms and pedigree of patients 3 and 16 A DHPLC analy
- Example of Oligo Analysis by DHPLC
- Slide 32
- Thanks for Your Attentions Reference
- Thanks for Your Attentions
-
Software1 HSM (Hitachi) and WAVEMAKER 41 (Transgenomic) software2 Navigator (Transgenomic)
Nucleic Acid Amplifications eg PCR
PCR Amplification for Mutation Analysis Using the Transgenomic WAVEreg System
from Invitrogen
ndash Introduction Discoverasetrade dHPLC DNA Polymerase is an enzyme mixture
composed of recombinant Taq DNA polymerase and Pyrococcus species GB-D
polymerase Pyrococcus species GB-D polymerase possesses a proofreading
ability by virtue of its 3rsquo to 5rsquo exonuclease activity Mixture of the proofreading
enzyme with Taq DNA polymerase at an optimized ratio increases fidelity
approximately eight times over that of Taq DNA polymerase alone and allows
amplification of simple and complex DNA templates The enzyme mixture is
provided with an optimized buffer that improves enzyme fidelity
ndash The Discoverasetrade dHPLC DNA Polymerase enzyme mixture and buffer
formulation have been optimized for use with denaturing high-performance
liquid chromatography (dHPLC) systems They were developed and tested using
the Transgenomic WAVEreg SystemDiscoverasetrade dHPLC DNA Polymerase is
supplied at 1 unit per microl
Equipments
1 Thermocycler (with heated lid)
2 DHPLC WAVE DNA fragment analysis system
(Transgenomic)
3 DNA Sep HT cartridge (cat no DNA-99-3710
Transgenomic)
4 Transmit silicone sealing mats (see Note 2) (cat no
172024 Transgenomic)
5 PCR plates (see Note 3) (cat no 0030 127307
Eppendorf)
6 Chemical waste container
Solutions1 Optimase polymerase (supplied with Mg2+-free
buffer and separate Mg2+ solution)(cat no 703045 Transgenomic)2 100 mM dNTP set PCR grade (cat no 10297-117
Invitrogen)3 Mutation control standards low-range mutation
standard (cat no 560077) and high-range mutation standard (cat no 562001 Transgenomic)
4 DNA sizing standard (cat no 560078 Transgenomic)5 Acetonitrile 9993+ HPLC grade (cat no 27 071-7
Sigma-Aldrich)6 WAVE ion-pairing agent 2 M triethylammonium
acetate (TEAA) (cat no 553303 Transgenomic)7 Water (eg MilliQ water) use 18 M1048625 resistance or
better8 WAVE optimized buffers (optional) Buffers A B D
and syringe solution (cat nos 553401 553402 553404 and 553403 Transgenomic)
HomoduplexesHeteroduplex
es
Chromatographic Separation Reverse-phase ion-pair system
Gradient elution with acetonitrilewater
UV detection
Column oven temperature selected for partial denaturation of heteroduplexes and thus earlier elution
Results
Example of elution profiles for a wild-type and a mutant sample The mutant sample contains an insertiondeletion mutation in exon 8 of the RET gene
Example of elution profiles obtained for a wild-type and a mutant sample The mutant sample contains a stop codon mutation in exon 15 of the RET gene
Applications DHPLC Systems (eg WAVE Systems) can
be used for a wide range of applications includingndash Mutation Detectionndash Sizingndash Oligonucleotide Purification and Analysisndash Forensicsndash Microbial Analysisndash RNA Isolation and Purificationndash Single Base Extensionndash Methylation
With the WAVE System you can
Scan fragments for both known and novel
mutationsSNPs without extensive resequencing
Detect low-abundance mutations in heterogeneous
samples
Enrich for low-abundance alleles
Fractionate complex mixtures of related fragments
based on differences in sequence content
Perform targeted mutationSNP scoring via primer
extension assays
Analyze end-point products from allele-specific
ProbePrimer-based Amplification Technologies
ProbePrimer-based Amplification Technologies
bull Primer-based Amplificationsndash Non-Isothermal Amplifications eg PCR-based
Methodsndash Isothermal AmplificationsNon-PCR-based
Amplificationsndash NASBAndash HDAndash SDAndash and more than 45 NAIA Technologies
bull Probe-based Amplificationsndash For example Ligase Chain Reactions
CARTRIDGE dependent Applications eg via WAVE Systems
DNA-99-3510 DNASep Cartridge 46 mm x 50 mm for variety of
DNA applications including Sizing and Mutation Detection load
capacity of 2microg
DNA-99-3710 DNASep HT Cartridge 65 mm x 37 mm for variety
of DNA application but especially for Rapid Mutation Detection
load capacity of 4microg
NUC-99-3550 OligoSepreg Cartridge 46 mm x 50 mm for Oligo
Purification work only
NUC-99-3860 OligoSep Prep HC Cartridge 78 mm x 50 mm for
Oligo Purification load capacity of 5000microg
RNA-99-3810 RNASepreg Prep Cartridge 78 mm x 50 mm for
variety of RNA applications load capacity of 50microg total RNA -OR-
1000ng of specific RNA
Chromatograms and pedigree of patients 3 and 16 A DHPLC analysis in normal control patients 3 and 16 Both patients show abnormal DHPLC peak patterns with two peaks in comparison to normal control B Sequencing data in patient 3 and his family members C Sequencing data in patient 16 and his family members
Example of Oligo Analysis by DHPLC
Thanks for Your AttentionsReference
Thanks for Your Attentions
ReferenceMethods in Molecular Medicine Vol 108
Hypertension Methods and Protocols Edited by J P Fennell and A H Baker copy Humana Press Inc Totowa NJ Chapter 13
- DHPLC Principles An Introduction to Denaturing High Performanc
- Nucleic Acids
- Nucleic Acids OMICS
- Genomics Variations as MutationPolymorphism
- The Principles for Gene Variation Analysis
- Scanning Procedures
- Scanning Procedures (2)
- Screening Procedures
- Screening Procedures (2)
- Heteroduplex analysis (HET) Keen J et al (1991) Rapid dete
- Technological Improvement of HET to eg DHPLC
- Technological Improvement of HET to eg DHPLC (2)
- Slide 13
- DHPLC Graphical Scheme
- Software 1 HSM (Hitachi) and WAVEMAKER 41 (Transgenomic) soft
- Slide 16
- Slide 17
- Nucleic Acid Amplifications eg PCR
- Equipments
- Solutions
- Slide 21
- Results
- Applications
- With the WAVE System you can
- ProbePrimer-based Amplification Technologies
- CARTRIDGE dependent Applications eg via WAVE Systems
- Slide 27
- Slide 28
- Slide 29
- Chromatograms and pedigree of patients 3 and 16 A DHPLC analy
- Example of Oligo Analysis by DHPLC
- Slide 32
- Thanks for Your Attentions Reference
- Thanks for Your Attentions
-
Nucleic Acid Amplifications eg PCR
PCR Amplification for Mutation Analysis Using the Transgenomic WAVEreg System
from Invitrogen
ndash Introduction Discoverasetrade dHPLC DNA Polymerase is an enzyme mixture
composed of recombinant Taq DNA polymerase and Pyrococcus species GB-D
polymerase Pyrococcus species GB-D polymerase possesses a proofreading
ability by virtue of its 3rsquo to 5rsquo exonuclease activity Mixture of the proofreading
enzyme with Taq DNA polymerase at an optimized ratio increases fidelity
approximately eight times over that of Taq DNA polymerase alone and allows
amplification of simple and complex DNA templates The enzyme mixture is
provided with an optimized buffer that improves enzyme fidelity
ndash The Discoverasetrade dHPLC DNA Polymerase enzyme mixture and buffer
formulation have been optimized for use with denaturing high-performance
liquid chromatography (dHPLC) systems They were developed and tested using
the Transgenomic WAVEreg SystemDiscoverasetrade dHPLC DNA Polymerase is
supplied at 1 unit per microl
Equipments
1 Thermocycler (with heated lid)
2 DHPLC WAVE DNA fragment analysis system
(Transgenomic)
3 DNA Sep HT cartridge (cat no DNA-99-3710
Transgenomic)
4 Transmit silicone sealing mats (see Note 2) (cat no
172024 Transgenomic)
5 PCR plates (see Note 3) (cat no 0030 127307
Eppendorf)
6 Chemical waste container
Solutions1 Optimase polymerase (supplied with Mg2+-free
buffer and separate Mg2+ solution)(cat no 703045 Transgenomic)2 100 mM dNTP set PCR grade (cat no 10297-117
Invitrogen)3 Mutation control standards low-range mutation
standard (cat no 560077) and high-range mutation standard (cat no 562001 Transgenomic)
4 DNA sizing standard (cat no 560078 Transgenomic)5 Acetonitrile 9993+ HPLC grade (cat no 27 071-7
Sigma-Aldrich)6 WAVE ion-pairing agent 2 M triethylammonium
acetate (TEAA) (cat no 553303 Transgenomic)7 Water (eg MilliQ water) use 18 M1048625 resistance or
better8 WAVE optimized buffers (optional) Buffers A B D
and syringe solution (cat nos 553401 553402 553404 and 553403 Transgenomic)
HomoduplexesHeteroduplex
es
Chromatographic Separation Reverse-phase ion-pair system
Gradient elution with acetonitrilewater
UV detection
Column oven temperature selected for partial denaturation of heteroduplexes and thus earlier elution
Results
Example of elution profiles for a wild-type and a mutant sample The mutant sample contains an insertiondeletion mutation in exon 8 of the RET gene
Example of elution profiles obtained for a wild-type and a mutant sample The mutant sample contains a stop codon mutation in exon 15 of the RET gene
Applications DHPLC Systems (eg WAVE Systems) can
be used for a wide range of applications includingndash Mutation Detectionndash Sizingndash Oligonucleotide Purification and Analysisndash Forensicsndash Microbial Analysisndash RNA Isolation and Purificationndash Single Base Extensionndash Methylation
With the WAVE System you can
Scan fragments for both known and novel
mutationsSNPs without extensive resequencing
Detect low-abundance mutations in heterogeneous
samples
Enrich for low-abundance alleles
Fractionate complex mixtures of related fragments
based on differences in sequence content
Perform targeted mutationSNP scoring via primer
extension assays
Analyze end-point products from allele-specific
ProbePrimer-based Amplification Technologies
ProbePrimer-based Amplification Technologies
bull Primer-based Amplificationsndash Non-Isothermal Amplifications eg PCR-based
Methodsndash Isothermal AmplificationsNon-PCR-based
Amplificationsndash NASBAndash HDAndash SDAndash and more than 45 NAIA Technologies
bull Probe-based Amplificationsndash For example Ligase Chain Reactions
CARTRIDGE dependent Applications eg via WAVE Systems
DNA-99-3510 DNASep Cartridge 46 mm x 50 mm for variety of
DNA applications including Sizing and Mutation Detection load
capacity of 2microg
DNA-99-3710 DNASep HT Cartridge 65 mm x 37 mm for variety
of DNA application but especially for Rapid Mutation Detection
load capacity of 4microg
NUC-99-3550 OligoSepreg Cartridge 46 mm x 50 mm for Oligo
Purification work only
NUC-99-3860 OligoSep Prep HC Cartridge 78 mm x 50 mm for
Oligo Purification load capacity of 5000microg
RNA-99-3810 RNASepreg Prep Cartridge 78 mm x 50 mm for
variety of RNA applications load capacity of 50microg total RNA -OR-
1000ng of specific RNA
Chromatograms and pedigree of patients 3 and 16 A DHPLC analysis in normal control patients 3 and 16 Both patients show abnormal DHPLC peak patterns with two peaks in comparison to normal control B Sequencing data in patient 3 and his family members C Sequencing data in patient 16 and his family members
Example of Oligo Analysis by DHPLC
Thanks for Your AttentionsReference
Thanks for Your Attentions
ReferenceMethods in Molecular Medicine Vol 108
Hypertension Methods and Protocols Edited by J P Fennell and A H Baker copy Humana Press Inc Totowa NJ Chapter 13
- DHPLC Principles An Introduction to Denaturing High Performanc
- Nucleic Acids
- Nucleic Acids OMICS
- Genomics Variations as MutationPolymorphism
- The Principles for Gene Variation Analysis
- Scanning Procedures
- Scanning Procedures (2)
- Screening Procedures
- Screening Procedures (2)
- Heteroduplex analysis (HET) Keen J et al (1991) Rapid dete
- Technological Improvement of HET to eg DHPLC
- Technological Improvement of HET to eg DHPLC (2)
- Slide 13
- DHPLC Graphical Scheme
- Software 1 HSM (Hitachi) and WAVEMAKER 41 (Transgenomic) soft
- Slide 16
- Slide 17
- Nucleic Acid Amplifications eg PCR
- Equipments
- Solutions
- Slide 21
- Results
- Applications
- With the WAVE System you can
- ProbePrimer-based Amplification Technologies
- CARTRIDGE dependent Applications eg via WAVE Systems
- Slide 27
- Slide 28
- Slide 29
- Chromatograms and pedigree of patients 3 and 16 A DHPLC analy
- Example of Oligo Analysis by DHPLC
- Slide 32
- Thanks for Your Attentions Reference
- Thanks for Your Attentions
-
Equipments
1 Thermocycler (with heated lid)
2 DHPLC WAVE DNA fragment analysis system
(Transgenomic)
3 DNA Sep HT cartridge (cat no DNA-99-3710
Transgenomic)
4 Transmit silicone sealing mats (see Note 2) (cat no
172024 Transgenomic)
5 PCR plates (see Note 3) (cat no 0030 127307
Eppendorf)
6 Chemical waste container
Solutions1 Optimase polymerase (supplied with Mg2+-free
buffer and separate Mg2+ solution)(cat no 703045 Transgenomic)2 100 mM dNTP set PCR grade (cat no 10297-117
Invitrogen)3 Mutation control standards low-range mutation
standard (cat no 560077) and high-range mutation standard (cat no 562001 Transgenomic)
4 DNA sizing standard (cat no 560078 Transgenomic)5 Acetonitrile 9993+ HPLC grade (cat no 27 071-7
Sigma-Aldrich)6 WAVE ion-pairing agent 2 M triethylammonium
acetate (TEAA) (cat no 553303 Transgenomic)7 Water (eg MilliQ water) use 18 M1048625 resistance or
better8 WAVE optimized buffers (optional) Buffers A B D
and syringe solution (cat nos 553401 553402 553404 and 553403 Transgenomic)
HomoduplexesHeteroduplex
es
Chromatographic Separation Reverse-phase ion-pair system
Gradient elution with acetonitrilewater
UV detection
Column oven temperature selected for partial denaturation of heteroduplexes and thus earlier elution
Results
Example of elution profiles for a wild-type and a mutant sample The mutant sample contains an insertiondeletion mutation in exon 8 of the RET gene
Example of elution profiles obtained for a wild-type and a mutant sample The mutant sample contains a stop codon mutation in exon 15 of the RET gene
Applications DHPLC Systems (eg WAVE Systems) can
be used for a wide range of applications includingndash Mutation Detectionndash Sizingndash Oligonucleotide Purification and Analysisndash Forensicsndash Microbial Analysisndash RNA Isolation and Purificationndash Single Base Extensionndash Methylation
With the WAVE System you can
Scan fragments for both known and novel
mutationsSNPs without extensive resequencing
Detect low-abundance mutations in heterogeneous
samples
Enrich for low-abundance alleles
Fractionate complex mixtures of related fragments
based on differences in sequence content
Perform targeted mutationSNP scoring via primer
extension assays
Analyze end-point products from allele-specific
ProbePrimer-based Amplification Technologies
ProbePrimer-based Amplification Technologies
bull Primer-based Amplificationsndash Non-Isothermal Amplifications eg PCR-based
Methodsndash Isothermal AmplificationsNon-PCR-based
Amplificationsndash NASBAndash HDAndash SDAndash and more than 45 NAIA Technologies
bull Probe-based Amplificationsndash For example Ligase Chain Reactions
CARTRIDGE dependent Applications eg via WAVE Systems
DNA-99-3510 DNASep Cartridge 46 mm x 50 mm for variety of
DNA applications including Sizing and Mutation Detection load
capacity of 2microg
DNA-99-3710 DNASep HT Cartridge 65 mm x 37 mm for variety
of DNA application but especially for Rapid Mutation Detection
load capacity of 4microg
NUC-99-3550 OligoSepreg Cartridge 46 mm x 50 mm for Oligo
Purification work only
NUC-99-3860 OligoSep Prep HC Cartridge 78 mm x 50 mm for
Oligo Purification load capacity of 5000microg
RNA-99-3810 RNASepreg Prep Cartridge 78 mm x 50 mm for
variety of RNA applications load capacity of 50microg total RNA -OR-
1000ng of specific RNA
Chromatograms and pedigree of patients 3 and 16 A DHPLC analysis in normal control patients 3 and 16 Both patients show abnormal DHPLC peak patterns with two peaks in comparison to normal control B Sequencing data in patient 3 and his family members C Sequencing data in patient 16 and his family members
Example of Oligo Analysis by DHPLC
Thanks for Your AttentionsReference
Thanks for Your Attentions
ReferenceMethods in Molecular Medicine Vol 108
Hypertension Methods and Protocols Edited by J P Fennell and A H Baker copy Humana Press Inc Totowa NJ Chapter 13
- DHPLC Principles An Introduction to Denaturing High Performanc
- Nucleic Acids
- Nucleic Acids OMICS
- Genomics Variations as MutationPolymorphism
- The Principles for Gene Variation Analysis
- Scanning Procedures
- Scanning Procedures (2)
- Screening Procedures
- Screening Procedures (2)
- Heteroduplex analysis (HET) Keen J et al (1991) Rapid dete
- Technological Improvement of HET to eg DHPLC
- Technological Improvement of HET to eg DHPLC (2)
- Slide 13
- DHPLC Graphical Scheme
- Software 1 HSM (Hitachi) and WAVEMAKER 41 (Transgenomic) soft
- Slide 16
- Slide 17
- Nucleic Acid Amplifications eg PCR
- Equipments
- Solutions
- Slide 21
- Results
- Applications
- With the WAVE System you can
- ProbePrimer-based Amplification Technologies
- CARTRIDGE dependent Applications eg via WAVE Systems
- Slide 27
- Slide 28
- Slide 29
- Chromatograms and pedigree of patients 3 and 16 A DHPLC analy
- Example of Oligo Analysis by DHPLC
- Slide 32
- Thanks for Your Attentions Reference
- Thanks for Your Attentions
-
Solutions1 Optimase polymerase (supplied with Mg2+-free
buffer and separate Mg2+ solution)(cat no 703045 Transgenomic)2 100 mM dNTP set PCR grade (cat no 10297-117
Invitrogen)3 Mutation control standards low-range mutation
standard (cat no 560077) and high-range mutation standard (cat no 562001 Transgenomic)
4 DNA sizing standard (cat no 560078 Transgenomic)5 Acetonitrile 9993+ HPLC grade (cat no 27 071-7
Sigma-Aldrich)6 WAVE ion-pairing agent 2 M triethylammonium
acetate (TEAA) (cat no 553303 Transgenomic)7 Water (eg MilliQ water) use 18 M1048625 resistance or
better8 WAVE optimized buffers (optional) Buffers A B D
and syringe solution (cat nos 553401 553402 553404 and 553403 Transgenomic)
HomoduplexesHeteroduplex
es
Chromatographic Separation Reverse-phase ion-pair system
Gradient elution with acetonitrilewater
UV detection
Column oven temperature selected for partial denaturation of heteroduplexes and thus earlier elution
Results
Example of elution profiles for a wild-type and a mutant sample The mutant sample contains an insertiondeletion mutation in exon 8 of the RET gene
Example of elution profiles obtained for a wild-type and a mutant sample The mutant sample contains a stop codon mutation in exon 15 of the RET gene
Applications DHPLC Systems (eg WAVE Systems) can
be used for a wide range of applications includingndash Mutation Detectionndash Sizingndash Oligonucleotide Purification and Analysisndash Forensicsndash Microbial Analysisndash RNA Isolation and Purificationndash Single Base Extensionndash Methylation
With the WAVE System you can
Scan fragments for both known and novel
mutationsSNPs without extensive resequencing
Detect low-abundance mutations in heterogeneous
samples
Enrich for low-abundance alleles
Fractionate complex mixtures of related fragments
based on differences in sequence content
Perform targeted mutationSNP scoring via primer
extension assays
Analyze end-point products from allele-specific
ProbePrimer-based Amplification Technologies
ProbePrimer-based Amplification Technologies
bull Primer-based Amplificationsndash Non-Isothermal Amplifications eg PCR-based
Methodsndash Isothermal AmplificationsNon-PCR-based
Amplificationsndash NASBAndash HDAndash SDAndash and more than 45 NAIA Technologies
bull Probe-based Amplificationsndash For example Ligase Chain Reactions
CARTRIDGE dependent Applications eg via WAVE Systems
DNA-99-3510 DNASep Cartridge 46 mm x 50 mm for variety of
DNA applications including Sizing and Mutation Detection load
capacity of 2microg
DNA-99-3710 DNASep HT Cartridge 65 mm x 37 mm for variety
of DNA application but especially for Rapid Mutation Detection
load capacity of 4microg
NUC-99-3550 OligoSepreg Cartridge 46 mm x 50 mm for Oligo
Purification work only
NUC-99-3860 OligoSep Prep HC Cartridge 78 mm x 50 mm for
Oligo Purification load capacity of 5000microg
RNA-99-3810 RNASepreg Prep Cartridge 78 mm x 50 mm for
variety of RNA applications load capacity of 50microg total RNA -OR-
1000ng of specific RNA
Chromatograms and pedigree of patients 3 and 16 A DHPLC analysis in normal control patients 3 and 16 Both patients show abnormal DHPLC peak patterns with two peaks in comparison to normal control B Sequencing data in patient 3 and his family members C Sequencing data in patient 16 and his family members
Example of Oligo Analysis by DHPLC
Thanks for Your AttentionsReference
Thanks for Your Attentions
ReferenceMethods in Molecular Medicine Vol 108
Hypertension Methods and Protocols Edited by J P Fennell and A H Baker copy Humana Press Inc Totowa NJ Chapter 13
- DHPLC Principles An Introduction to Denaturing High Performanc
- Nucleic Acids
- Nucleic Acids OMICS
- Genomics Variations as MutationPolymorphism
- The Principles for Gene Variation Analysis
- Scanning Procedures
- Scanning Procedures (2)
- Screening Procedures
- Screening Procedures (2)
- Heteroduplex analysis (HET) Keen J et al (1991) Rapid dete
- Technological Improvement of HET to eg DHPLC
- Technological Improvement of HET to eg DHPLC (2)
- Slide 13
- DHPLC Graphical Scheme
- Software 1 HSM (Hitachi) and WAVEMAKER 41 (Transgenomic) soft
- Slide 16
- Slide 17
- Nucleic Acid Amplifications eg PCR
- Equipments
- Solutions
- Slide 21
- Results
- Applications
- With the WAVE System you can
- ProbePrimer-based Amplification Technologies
- CARTRIDGE dependent Applications eg via WAVE Systems
- Slide 27
- Slide 28
- Slide 29
- Chromatograms and pedigree of patients 3 and 16 A DHPLC analy
- Example of Oligo Analysis by DHPLC
- Slide 32
- Thanks for Your Attentions Reference
- Thanks for Your Attentions
-
HomoduplexesHeteroduplex
es
Chromatographic Separation Reverse-phase ion-pair system
Gradient elution with acetonitrilewater
UV detection
Column oven temperature selected for partial denaturation of heteroduplexes and thus earlier elution
Results
Example of elution profiles for a wild-type and a mutant sample The mutant sample contains an insertiondeletion mutation in exon 8 of the RET gene
Example of elution profiles obtained for a wild-type and a mutant sample The mutant sample contains a stop codon mutation in exon 15 of the RET gene
Applications DHPLC Systems (eg WAVE Systems) can
be used for a wide range of applications includingndash Mutation Detectionndash Sizingndash Oligonucleotide Purification and Analysisndash Forensicsndash Microbial Analysisndash RNA Isolation and Purificationndash Single Base Extensionndash Methylation
With the WAVE System you can
Scan fragments for both known and novel
mutationsSNPs without extensive resequencing
Detect low-abundance mutations in heterogeneous
samples
Enrich for low-abundance alleles
Fractionate complex mixtures of related fragments
based on differences in sequence content
Perform targeted mutationSNP scoring via primer
extension assays
Analyze end-point products from allele-specific
ProbePrimer-based Amplification Technologies
ProbePrimer-based Amplification Technologies
bull Primer-based Amplificationsndash Non-Isothermal Amplifications eg PCR-based
Methodsndash Isothermal AmplificationsNon-PCR-based
Amplificationsndash NASBAndash HDAndash SDAndash and more than 45 NAIA Technologies
bull Probe-based Amplificationsndash For example Ligase Chain Reactions
CARTRIDGE dependent Applications eg via WAVE Systems
DNA-99-3510 DNASep Cartridge 46 mm x 50 mm for variety of
DNA applications including Sizing and Mutation Detection load
capacity of 2microg
DNA-99-3710 DNASep HT Cartridge 65 mm x 37 mm for variety
of DNA application but especially for Rapid Mutation Detection
load capacity of 4microg
NUC-99-3550 OligoSepreg Cartridge 46 mm x 50 mm for Oligo
Purification work only
NUC-99-3860 OligoSep Prep HC Cartridge 78 mm x 50 mm for
Oligo Purification load capacity of 5000microg
RNA-99-3810 RNASepreg Prep Cartridge 78 mm x 50 mm for
variety of RNA applications load capacity of 50microg total RNA -OR-
1000ng of specific RNA
Chromatograms and pedigree of patients 3 and 16 A DHPLC analysis in normal control patients 3 and 16 Both patients show abnormal DHPLC peak patterns with two peaks in comparison to normal control B Sequencing data in patient 3 and his family members C Sequencing data in patient 16 and his family members
Example of Oligo Analysis by DHPLC
Thanks for Your AttentionsReference
Thanks for Your Attentions
ReferenceMethods in Molecular Medicine Vol 108
Hypertension Methods and Protocols Edited by J P Fennell and A H Baker copy Humana Press Inc Totowa NJ Chapter 13
- DHPLC Principles An Introduction to Denaturing High Performanc
- Nucleic Acids
- Nucleic Acids OMICS
- Genomics Variations as MutationPolymorphism
- The Principles for Gene Variation Analysis
- Scanning Procedures
- Scanning Procedures (2)
- Screening Procedures
- Screening Procedures (2)
- Heteroduplex analysis (HET) Keen J et al (1991) Rapid dete
- Technological Improvement of HET to eg DHPLC
- Technological Improvement of HET to eg DHPLC (2)
- Slide 13
- DHPLC Graphical Scheme
- Software 1 HSM (Hitachi) and WAVEMAKER 41 (Transgenomic) soft
- Slide 16
- Slide 17
- Nucleic Acid Amplifications eg PCR
- Equipments
- Solutions
- Slide 21
- Results
- Applications
- With the WAVE System you can
- ProbePrimer-based Amplification Technologies
- CARTRIDGE dependent Applications eg via WAVE Systems
- Slide 27
- Slide 28
- Slide 29
- Chromatograms and pedigree of patients 3 and 16 A DHPLC analy
- Example of Oligo Analysis by DHPLC
- Slide 32
- Thanks for Your Attentions Reference
- Thanks for Your Attentions
-
Results
Example of elution profiles for a wild-type and a mutant sample The mutant sample contains an insertiondeletion mutation in exon 8 of the RET gene
Example of elution profiles obtained for a wild-type and a mutant sample The mutant sample contains a stop codon mutation in exon 15 of the RET gene
Applications DHPLC Systems (eg WAVE Systems) can
be used for a wide range of applications includingndash Mutation Detectionndash Sizingndash Oligonucleotide Purification and Analysisndash Forensicsndash Microbial Analysisndash RNA Isolation and Purificationndash Single Base Extensionndash Methylation
With the WAVE System you can
Scan fragments for both known and novel
mutationsSNPs without extensive resequencing
Detect low-abundance mutations in heterogeneous
samples
Enrich for low-abundance alleles
Fractionate complex mixtures of related fragments
based on differences in sequence content
Perform targeted mutationSNP scoring via primer
extension assays
Analyze end-point products from allele-specific
ProbePrimer-based Amplification Technologies
ProbePrimer-based Amplification Technologies
bull Primer-based Amplificationsndash Non-Isothermal Amplifications eg PCR-based
Methodsndash Isothermal AmplificationsNon-PCR-based
Amplificationsndash NASBAndash HDAndash SDAndash and more than 45 NAIA Technologies
bull Probe-based Amplificationsndash For example Ligase Chain Reactions
CARTRIDGE dependent Applications eg via WAVE Systems
DNA-99-3510 DNASep Cartridge 46 mm x 50 mm for variety of
DNA applications including Sizing and Mutation Detection load
capacity of 2microg
DNA-99-3710 DNASep HT Cartridge 65 mm x 37 mm for variety
of DNA application but especially for Rapid Mutation Detection
load capacity of 4microg
NUC-99-3550 OligoSepreg Cartridge 46 mm x 50 mm for Oligo
Purification work only
NUC-99-3860 OligoSep Prep HC Cartridge 78 mm x 50 mm for
Oligo Purification load capacity of 5000microg
RNA-99-3810 RNASepreg Prep Cartridge 78 mm x 50 mm for
variety of RNA applications load capacity of 50microg total RNA -OR-
1000ng of specific RNA
Chromatograms and pedigree of patients 3 and 16 A DHPLC analysis in normal control patients 3 and 16 Both patients show abnormal DHPLC peak patterns with two peaks in comparison to normal control B Sequencing data in patient 3 and his family members C Sequencing data in patient 16 and his family members
Example of Oligo Analysis by DHPLC
Thanks for Your AttentionsReference
Thanks for Your Attentions
ReferenceMethods in Molecular Medicine Vol 108
Hypertension Methods and Protocols Edited by J P Fennell and A H Baker copy Humana Press Inc Totowa NJ Chapter 13
- DHPLC Principles An Introduction to Denaturing High Performanc
- Nucleic Acids
- Nucleic Acids OMICS
- Genomics Variations as MutationPolymorphism
- The Principles for Gene Variation Analysis
- Scanning Procedures
- Scanning Procedures (2)
- Screening Procedures
- Screening Procedures (2)
- Heteroduplex analysis (HET) Keen J et al (1991) Rapid dete
- Technological Improvement of HET to eg DHPLC
- Technological Improvement of HET to eg DHPLC (2)
- Slide 13
- DHPLC Graphical Scheme
- Software 1 HSM (Hitachi) and WAVEMAKER 41 (Transgenomic) soft
- Slide 16
- Slide 17
- Nucleic Acid Amplifications eg PCR
- Equipments
- Solutions
- Slide 21
- Results
- Applications
- With the WAVE System you can
- ProbePrimer-based Amplification Technologies
- CARTRIDGE dependent Applications eg via WAVE Systems
- Slide 27
- Slide 28
- Slide 29
- Chromatograms and pedigree of patients 3 and 16 A DHPLC analy
- Example of Oligo Analysis by DHPLC
- Slide 32
- Thanks for Your Attentions Reference
- Thanks for Your Attentions
-
Applications DHPLC Systems (eg WAVE Systems) can
be used for a wide range of applications includingndash Mutation Detectionndash Sizingndash Oligonucleotide Purification and Analysisndash Forensicsndash Microbial Analysisndash RNA Isolation and Purificationndash Single Base Extensionndash Methylation
With the WAVE System you can
Scan fragments for both known and novel
mutationsSNPs without extensive resequencing
Detect low-abundance mutations in heterogeneous
samples
Enrich for low-abundance alleles
Fractionate complex mixtures of related fragments
based on differences in sequence content
Perform targeted mutationSNP scoring via primer
extension assays
Analyze end-point products from allele-specific
ProbePrimer-based Amplification Technologies
ProbePrimer-based Amplification Technologies
bull Primer-based Amplificationsndash Non-Isothermal Amplifications eg PCR-based
Methodsndash Isothermal AmplificationsNon-PCR-based
Amplificationsndash NASBAndash HDAndash SDAndash and more than 45 NAIA Technologies
bull Probe-based Amplificationsndash For example Ligase Chain Reactions
CARTRIDGE dependent Applications eg via WAVE Systems
DNA-99-3510 DNASep Cartridge 46 mm x 50 mm for variety of
DNA applications including Sizing and Mutation Detection load
capacity of 2microg
DNA-99-3710 DNASep HT Cartridge 65 mm x 37 mm for variety
of DNA application but especially for Rapid Mutation Detection
load capacity of 4microg
NUC-99-3550 OligoSepreg Cartridge 46 mm x 50 mm for Oligo
Purification work only
NUC-99-3860 OligoSep Prep HC Cartridge 78 mm x 50 mm for
Oligo Purification load capacity of 5000microg
RNA-99-3810 RNASepreg Prep Cartridge 78 mm x 50 mm for
variety of RNA applications load capacity of 50microg total RNA -OR-
1000ng of specific RNA
Chromatograms and pedigree of patients 3 and 16 A DHPLC analysis in normal control patients 3 and 16 Both patients show abnormal DHPLC peak patterns with two peaks in comparison to normal control B Sequencing data in patient 3 and his family members C Sequencing data in patient 16 and his family members
Example of Oligo Analysis by DHPLC
Thanks for Your AttentionsReference
Thanks for Your Attentions
ReferenceMethods in Molecular Medicine Vol 108
Hypertension Methods and Protocols Edited by J P Fennell and A H Baker copy Humana Press Inc Totowa NJ Chapter 13
- DHPLC Principles An Introduction to Denaturing High Performanc
- Nucleic Acids
- Nucleic Acids OMICS
- Genomics Variations as MutationPolymorphism
- The Principles for Gene Variation Analysis
- Scanning Procedures
- Scanning Procedures (2)
- Screening Procedures
- Screening Procedures (2)
- Heteroduplex analysis (HET) Keen J et al (1991) Rapid dete
- Technological Improvement of HET to eg DHPLC
- Technological Improvement of HET to eg DHPLC (2)
- Slide 13
- DHPLC Graphical Scheme
- Software 1 HSM (Hitachi) and WAVEMAKER 41 (Transgenomic) soft
- Slide 16
- Slide 17
- Nucleic Acid Amplifications eg PCR
- Equipments
- Solutions
- Slide 21
- Results
- Applications
- With the WAVE System you can
- ProbePrimer-based Amplification Technologies
- CARTRIDGE dependent Applications eg via WAVE Systems
- Slide 27
- Slide 28
- Slide 29
- Chromatograms and pedigree of patients 3 and 16 A DHPLC analy
- Example of Oligo Analysis by DHPLC
- Slide 32
- Thanks for Your Attentions Reference
- Thanks for Your Attentions
-
With the WAVE System you can
Scan fragments for both known and novel
mutationsSNPs without extensive resequencing
Detect low-abundance mutations in heterogeneous
samples
Enrich for low-abundance alleles
Fractionate complex mixtures of related fragments
based on differences in sequence content
Perform targeted mutationSNP scoring via primer
extension assays
Analyze end-point products from allele-specific
ProbePrimer-based Amplification Technologies
ProbePrimer-based Amplification Technologies
bull Primer-based Amplificationsndash Non-Isothermal Amplifications eg PCR-based
Methodsndash Isothermal AmplificationsNon-PCR-based
Amplificationsndash NASBAndash HDAndash SDAndash and more than 45 NAIA Technologies
bull Probe-based Amplificationsndash For example Ligase Chain Reactions
CARTRIDGE dependent Applications eg via WAVE Systems
DNA-99-3510 DNASep Cartridge 46 mm x 50 mm for variety of
DNA applications including Sizing and Mutation Detection load
capacity of 2microg
DNA-99-3710 DNASep HT Cartridge 65 mm x 37 mm for variety
of DNA application but especially for Rapid Mutation Detection
load capacity of 4microg
NUC-99-3550 OligoSepreg Cartridge 46 mm x 50 mm for Oligo
Purification work only
NUC-99-3860 OligoSep Prep HC Cartridge 78 mm x 50 mm for
Oligo Purification load capacity of 5000microg
RNA-99-3810 RNASepreg Prep Cartridge 78 mm x 50 mm for
variety of RNA applications load capacity of 50microg total RNA -OR-
1000ng of specific RNA
Chromatograms and pedigree of patients 3 and 16 A DHPLC analysis in normal control patients 3 and 16 Both patients show abnormal DHPLC peak patterns with two peaks in comparison to normal control B Sequencing data in patient 3 and his family members C Sequencing data in patient 16 and his family members
Example of Oligo Analysis by DHPLC
Thanks for Your AttentionsReference
Thanks for Your Attentions
ReferenceMethods in Molecular Medicine Vol 108
Hypertension Methods and Protocols Edited by J P Fennell and A H Baker copy Humana Press Inc Totowa NJ Chapter 13
- DHPLC Principles An Introduction to Denaturing High Performanc
- Nucleic Acids
- Nucleic Acids OMICS
- Genomics Variations as MutationPolymorphism
- The Principles for Gene Variation Analysis
- Scanning Procedures
- Scanning Procedures (2)
- Screening Procedures
- Screening Procedures (2)
- Heteroduplex analysis (HET) Keen J et al (1991) Rapid dete
- Technological Improvement of HET to eg DHPLC
- Technological Improvement of HET to eg DHPLC (2)
- Slide 13
- DHPLC Graphical Scheme
- Software 1 HSM (Hitachi) and WAVEMAKER 41 (Transgenomic) soft
- Slide 16
- Slide 17
- Nucleic Acid Amplifications eg PCR
- Equipments
- Solutions
- Slide 21
- Results
- Applications
- With the WAVE System you can
- ProbePrimer-based Amplification Technologies
- CARTRIDGE dependent Applications eg via WAVE Systems
- Slide 27
- Slide 28
- Slide 29
- Chromatograms and pedigree of patients 3 and 16 A DHPLC analy
- Example of Oligo Analysis by DHPLC
- Slide 32
- Thanks for Your Attentions Reference
- Thanks for Your Attentions
-
ProbePrimer-based Amplification Technologies
bull Primer-based Amplificationsndash Non-Isothermal Amplifications eg PCR-based
Methodsndash Isothermal AmplificationsNon-PCR-based
Amplificationsndash NASBAndash HDAndash SDAndash and more than 45 NAIA Technologies
bull Probe-based Amplificationsndash For example Ligase Chain Reactions
CARTRIDGE dependent Applications eg via WAVE Systems
DNA-99-3510 DNASep Cartridge 46 mm x 50 mm for variety of
DNA applications including Sizing and Mutation Detection load
capacity of 2microg
DNA-99-3710 DNASep HT Cartridge 65 mm x 37 mm for variety
of DNA application but especially for Rapid Mutation Detection
load capacity of 4microg
NUC-99-3550 OligoSepreg Cartridge 46 mm x 50 mm for Oligo
Purification work only
NUC-99-3860 OligoSep Prep HC Cartridge 78 mm x 50 mm for
Oligo Purification load capacity of 5000microg
RNA-99-3810 RNASepreg Prep Cartridge 78 mm x 50 mm for
variety of RNA applications load capacity of 50microg total RNA -OR-
1000ng of specific RNA
Chromatograms and pedigree of patients 3 and 16 A DHPLC analysis in normal control patients 3 and 16 Both patients show abnormal DHPLC peak patterns with two peaks in comparison to normal control B Sequencing data in patient 3 and his family members C Sequencing data in patient 16 and his family members
Example of Oligo Analysis by DHPLC
Thanks for Your AttentionsReference
Thanks for Your Attentions
ReferenceMethods in Molecular Medicine Vol 108
Hypertension Methods and Protocols Edited by J P Fennell and A H Baker copy Humana Press Inc Totowa NJ Chapter 13
- DHPLC Principles An Introduction to Denaturing High Performanc
- Nucleic Acids
- Nucleic Acids OMICS
- Genomics Variations as MutationPolymorphism
- The Principles for Gene Variation Analysis
- Scanning Procedures
- Scanning Procedures (2)
- Screening Procedures
- Screening Procedures (2)
- Heteroduplex analysis (HET) Keen J et al (1991) Rapid dete
- Technological Improvement of HET to eg DHPLC
- Technological Improvement of HET to eg DHPLC (2)
- Slide 13
- DHPLC Graphical Scheme
- Software 1 HSM (Hitachi) and WAVEMAKER 41 (Transgenomic) soft
- Slide 16
- Slide 17
- Nucleic Acid Amplifications eg PCR
- Equipments
- Solutions
- Slide 21
- Results
- Applications
- With the WAVE System you can
- ProbePrimer-based Amplification Technologies
- CARTRIDGE dependent Applications eg via WAVE Systems
- Slide 27
- Slide 28
- Slide 29
- Chromatograms and pedigree of patients 3 and 16 A DHPLC analy
- Example of Oligo Analysis by DHPLC
- Slide 32
- Thanks for Your Attentions Reference
- Thanks for Your Attentions
-
CARTRIDGE dependent Applications eg via WAVE Systems
DNA-99-3510 DNASep Cartridge 46 mm x 50 mm for variety of
DNA applications including Sizing and Mutation Detection load
capacity of 2microg
DNA-99-3710 DNASep HT Cartridge 65 mm x 37 mm for variety
of DNA application but especially for Rapid Mutation Detection
load capacity of 4microg
NUC-99-3550 OligoSepreg Cartridge 46 mm x 50 mm for Oligo
Purification work only
NUC-99-3860 OligoSep Prep HC Cartridge 78 mm x 50 mm for
Oligo Purification load capacity of 5000microg
RNA-99-3810 RNASepreg Prep Cartridge 78 mm x 50 mm for
variety of RNA applications load capacity of 50microg total RNA -OR-
1000ng of specific RNA
Chromatograms and pedigree of patients 3 and 16 A DHPLC analysis in normal control patients 3 and 16 Both patients show abnormal DHPLC peak patterns with two peaks in comparison to normal control B Sequencing data in patient 3 and his family members C Sequencing data in patient 16 and his family members
Example of Oligo Analysis by DHPLC
Thanks for Your AttentionsReference
Thanks for Your Attentions
ReferenceMethods in Molecular Medicine Vol 108
Hypertension Methods and Protocols Edited by J P Fennell and A H Baker copy Humana Press Inc Totowa NJ Chapter 13
- DHPLC Principles An Introduction to Denaturing High Performanc
- Nucleic Acids
- Nucleic Acids OMICS
- Genomics Variations as MutationPolymorphism
- The Principles for Gene Variation Analysis
- Scanning Procedures
- Scanning Procedures (2)
- Screening Procedures
- Screening Procedures (2)
- Heteroduplex analysis (HET) Keen J et al (1991) Rapid dete
- Technological Improvement of HET to eg DHPLC
- Technological Improvement of HET to eg DHPLC (2)
- Slide 13
- DHPLC Graphical Scheme
- Software 1 HSM (Hitachi) and WAVEMAKER 41 (Transgenomic) soft
- Slide 16
- Slide 17
- Nucleic Acid Amplifications eg PCR
- Equipments
- Solutions
- Slide 21
- Results
- Applications
- With the WAVE System you can
- ProbePrimer-based Amplification Technologies
- CARTRIDGE dependent Applications eg via WAVE Systems
- Slide 27
- Slide 28
- Slide 29
- Chromatograms and pedigree of patients 3 and 16 A DHPLC analy
- Example of Oligo Analysis by DHPLC
- Slide 32
- Thanks for Your Attentions Reference
- Thanks for Your Attentions
-
Chromatograms and pedigree of patients 3 and 16 A DHPLC analysis in normal control patients 3 and 16 Both patients show abnormal DHPLC peak patterns with two peaks in comparison to normal control B Sequencing data in patient 3 and his family members C Sequencing data in patient 16 and his family members
Example of Oligo Analysis by DHPLC
Thanks for Your AttentionsReference
Thanks for Your Attentions
ReferenceMethods in Molecular Medicine Vol 108
Hypertension Methods and Protocols Edited by J P Fennell and A H Baker copy Humana Press Inc Totowa NJ Chapter 13
- DHPLC Principles An Introduction to Denaturing High Performanc
- Nucleic Acids
- Nucleic Acids OMICS
- Genomics Variations as MutationPolymorphism
- The Principles for Gene Variation Analysis
- Scanning Procedures
- Scanning Procedures (2)
- Screening Procedures
- Screening Procedures (2)
- Heteroduplex analysis (HET) Keen J et al (1991) Rapid dete
- Technological Improvement of HET to eg DHPLC
- Technological Improvement of HET to eg DHPLC (2)
- Slide 13
- DHPLC Graphical Scheme
- Software 1 HSM (Hitachi) and WAVEMAKER 41 (Transgenomic) soft
- Slide 16
- Slide 17
- Nucleic Acid Amplifications eg PCR
- Equipments
- Solutions
- Slide 21
- Results
- Applications
- With the WAVE System you can
- ProbePrimer-based Amplification Technologies
- CARTRIDGE dependent Applications eg via WAVE Systems
- Slide 27
- Slide 28
- Slide 29
- Chromatograms and pedigree of patients 3 and 16 A DHPLC analy
- Example of Oligo Analysis by DHPLC
- Slide 32
- Thanks for Your Attentions Reference
- Thanks for Your Attentions
-
Example of Oligo Analysis by DHPLC
Thanks for Your AttentionsReference
Thanks for Your Attentions
ReferenceMethods in Molecular Medicine Vol 108
Hypertension Methods and Protocols Edited by J P Fennell and A H Baker copy Humana Press Inc Totowa NJ Chapter 13
- DHPLC Principles An Introduction to Denaturing High Performanc
- Nucleic Acids
- Nucleic Acids OMICS
- Genomics Variations as MutationPolymorphism
- The Principles for Gene Variation Analysis
- Scanning Procedures
- Scanning Procedures (2)
- Screening Procedures
- Screening Procedures (2)
- Heteroduplex analysis (HET) Keen J et al (1991) Rapid dete
- Technological Improvement of HET to eg DHPLC
- Technological Improvement of HET to eg DHPLC (2)
- Slide 13
- DHPLC Graphical Scheme
- Software 1 HSM (Hitachi) and WAVEMAKER 41 (Transgenomic) soft
- Slide 16
- Slide 17
- Nucleic Acid Amplifications eg PCR
- Equipments
- Solutions
- Slide 21
- Results
- Applications
- With the WAVE System you can
- ProbePrimer-based Amplification Technologies
- CARTRIDGE dependent Applications eg via WAVE Systems
- Slide 27
- Slide 28
- Slide 29
- Chromatograms and pedigree of patients 3 and 16 A DHPLC analy
- Example of Oligo Analysis by DHPLC
- Slide 32
- Thanks for Your Attentions Reference
- Thanks for Your Attentions
-
Thanks for Your AttentionsReference
Thanks for Your Attentions
ReferenceMethods in Molecular Medicine Vol 108
Hypertension Methods and Protocols Edited by J P Fennell and A H Baker copy Humana Press Inc Totowa NJ Chapter 13
- DHPLC Principles An Introduction to Denaturing High Performanc
- Nucleic Acids
- Nucleic Acids OMICS
- Genomics Variations as MutationPolymorphism
- The Principles for Gene Variation Analysis
- Scanning Procedures
- Scanning Procedures (2)
- Screening Procedures
- Screening Procedures (2)
- Heteroduplex analysis (HET) Keen J et al (1991) Rapid dete
- Technological Improvement of HET to eg DHPLC
- Technological Improvement of HET to eg DHPLC (2)
- Slide 13
- DHPLC Graphical Scheme
- Software 1 HSM (Hitachi) and WAVEMAKER 41 (Transgenomic) soft
- Slide 16
- Slide 17
- Nucleic Acid Amplifications eg PCR
- Equipments
- Solutions
- Slide 21
- Results
- Applications
- With the WAVE System you can
- ProbePrimer-based Amplification Technologies
- CARTRIDGE dependent Applications eg via WAVE Systems
- Slide 27
- Slide 28
- Slide 29
- Chromatograms and pedigree of patients 3 and 16 A DHPLC analy
- Example of Oligo Analysis by DHPLC
- Slide 32
- Thanks for Your Attentions Reference
- Thanks for Your Attentions
-
Thanks for Your Attentions
ReferenceMethods in Molecular Medicine Vol 108
Hypertension Methods and Protocols Edited by J P Fennell and A H Baker copy Humana Press Inc Totowa NJ Chapter 13
- DHPLC Principles An Introduction to Denaturing High Performanc
- Nucleic Acids
- Nucleic Acids OMICS
- Genomics Variations as MutationPolymorphism
- The Principles for Gene Variation Analysis
- Scanning Procedures
- Scanning Procedures (2)
- Screening Procedures
- Screening Procedures (2)
- Heteroduplex analysis (HET) Keen J et al (1991) Rapid dete
- Technological Improvement of HET to eg DHPLC
- Technological Improvement of HET to eg DHPLC (2)
- Slide 13
- DHPLC Graphical Scheme
- Software 1 HSM (Hitachi) and WAVEMAKER 41 (Transgenomic) soft
- Slide 16
- Slide 17
- Nucleic Acid Amplifications eg PCR
- Equipments
- Solutions
- Slide 21
- Results
- Applications
- With the WAVE System you can
- ProbePrimer-based Amplification Technologies
- CARTRIDGE dependent Applications eg via WAVE Systems
- Slide 27
- Slide 28
- Slide 29
- Chromatograms and pedigree of patients 3 and 16 A DHPLC analy
- Example of Oligo Analysis by DHPLC
- Slide 32
- Thanks for Your Attentions Reference
- Thanks for Your Attentions
-