1 molecular technologies discovery of dna structure/function (1953) molecular biology powerful...
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Molecular Technologies
Discovery of DNA structure/function (1953)
Molecular Biology
Powerful Techniques - Gene Analysis & Manipulation
Widely Applicable
Ecology, Behavior, Medicine, Agriculture
Pharmaceuticals, Horticulture, Animal Breeding, etc....
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Denaturation of DNA
High temperature, high pH - break H bonds
hyperchromatic effect
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Denaturation of DNA
Relative G:C content
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Renaturation of DNA
Homology affects renaturaton (hybridization)
Stringency [Salt] Temperature
High Low High
Low High Low
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Renaturation of DNA
Concentration affects rate of hybridization
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Renaturation of DNA
Concentration affects rate of hybridization
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Nucleic Acid Probes
DNA/RNA probes
Target sequences
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Phylogenetic Relationships
Amount bound ~ homology
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In situ Hybridization
FISH
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In situ Hybridization
Chromosome Painting
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In situ Hybridization
Detecting Chromosomal Rearrangements
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One specific sequence at each spot
Hybridize with labeled RNAfrom different cells or individuals (red or green)
Detect binding and interpret results
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Microarrays (Gene Chips)
Demonstration: http://www.bio.davidson.edu/Courses/genomics/chip/chip.html http://learn.genetics.utah.edu/content/labs/microarray/
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Microarrays (Gene Chips)
mRNA isolated from cells (cancer/normal; aerobic/anaerobic)
Converted to cDNA Fluorescent labels
http://www.bio.davidson.edu/Courses/genomics/chip/chip.html
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Microarrays (Gene Chips)
Mixture of labeled cDNA hybridized to microarray
Specific binding Scan and evaluate expression
http://www.bio.davidson.edu/Courses/genomics/chip/chip.html
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Microarrays (Gene Chips)
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Microarrays (Gene Chips)
Applications:
Evaluate gene expressionDevelopment, Cancer, Disease progression
Determine presence of specific mutations Genotyping, SNP association studies
Detect infectious genomes
Virus, Bacteria, etc.
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Restriction Endonucleases
Bacterial immune system
Recognize and cleave foreign DNA
Names
EcoRI - E. coli strain RI
HinDIII - H. influenzae strain DIII
Each recognizes and cuts a specific sequence of DNA
Specific molecular scissors
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Restriction Endonucleases
Palindromic Sequence
‘AND MADAM DNA’
5’>3’ = 3’>5’
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Restriction Endonucleases
Ends:
Blunt
or
Sticky
(complementary)
3’ or 5’ overhangs
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Restriction Endonucleases
Recognition Methylation
Enzyme Site Sensitive ?
AluI 5’...AG CT...3’
No3’...TC GA...5’
BamHI 5’...G GATCC...3’
No3’...CCTAG G...5’
BspEI 5’...T CCGGA...3’
Yes 3’...AGGCC T...5’
KpnI 5’...GGTAC C...3’
No3’...C CATGG...5’
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Recombinant DNA
RE fragments from two sources joined by DNA ligase
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Cloning DNA
DNA inserted into vector for growth in another cell
Vector enables growth in host cell (ori or ARS)
Selective markers (antibiotic resistance, nutritional requirement)
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Plasmid Vector
Bacterial Vector
pUC19
pBluescript
LacZ at MCS
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Eukaryotic Vectors
Yeast Artificial Chromosome
Shuttle Vectors
CEN; ARS plus ORI; markers
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Gene Cloning
Source of DNA Insert
Genomic DNA - dsDNA - coding and noncoding
cDNA - RNA > DNA - coding only
One specific gene
or
Comprehensive collection -
Genomic
Library, cDNA Library
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Genomic Library
Clone collection of
overlapping fragments (contigs)
Ave. fragment size
256 bp = (44)
4096 bp = (46)
Comprehensive library
for 3 x 109 bp genome
4096 bp fragments
> 732,000 clones
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Genomic Library
Probability of having at least one copy of any DNA
sequence in a genomic library
N = ln(1-P) N = number of clones needed ln(1-f) P = probability desired
f = ave. size fragment
cloned/genome size
Ex. Human genome 106 kb; if fragments average 15 kb,
More than 920,000 clones are needed
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cDNA Library (Expression Library)
cDNA - complementary DNA
copy mRNA - reverse transcriptase, polydT primer
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Transferring Recombinant DNA into Host Cells
Transformation
Each colony contains vector, Only some have insert
Clones - each with specific segment of genome
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Identifying Transformants Containing Gene Inserts
Blue/White Screen - pBluescript
Ampicillin resistant
lacZ gene-galactosidaseIPTGXgal (white) > blue
White coloniesall contain insert in MCS
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Screening Clones for Specific Gene Sequences
Detection of specific sequence - probe
Colonies transferred to membraneDNA released, hybridization with probe
Identify clone with desired gene
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Screening cDNA Libraries for Specific Gene Products
Detection of protein produced by cells with desired gene
Identify clone expressing gene
Specific Antibody
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Screening Clones for Ability to Rescue Mutants
DNA genomic library in shuttle vectorused to transform eukaryotic cells with defective gene
Only those with good copy of the gene grow
Permissive (30OC) Restrictive (37OC)
Complementation
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Screening Clones for Ability to Rescue Mutants
Cloning of yeast ARG1 gene
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Restriction Mapping
Relative positions of RE sites along DNA - molecular markers
670 1,500 500 250
BstEB
bp distances
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Restriction Mapping
Specificity of enzymes - reproducible fragments produced
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Determining Sizes of Restriction Fragments
Gel Electrophoresis - Agarose (200 bp+), Polyacrylamide
Determine distance migrated *Extrapolate size
*
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Determining Sizes of Restriction Fragments
Visualize DNA bands - Ethidium Bromide, UV transilluminator
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Generating Restriction Maps
Complete Digestion with HindIII and/or EcoRI
kb
kbkb
kb
kb
Answer:
4 H 7 E 9
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Generating Restriction Maps
Partial Digestion with PstI
Possible partial digestion fragments (*complete)
19, 15, 14, 12, 10, 8*, 7, 5*, 4*, 2* kb
Is a 6 kb fragment possible? A 13 kb fragment?
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Generating Restriction Maps
Partial Digestion with SalI
Which fragmentsare adjacent?
4.2 = 3.5 = 8.2 =
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Using Restriction Maps to Analyze Constructs
Characterize insert in recombinant DNA of clone
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Using Restriction Maps to Analyze Constructs
Orientation of insert can be determined
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Analysis of Genomic Sequences
Human genome - cut with RE (6 bp site) - ~7.3 X 105 fragments
How can you find and identifya specific fragment?
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Southern Blot Analysis
Southern Blot
DNA transfer -
detect fragment
specific probe
Northern BlotRNA
Western BlotProtein
Transfer tonylon membrane
Hybridize with probe
Visualizeautoradiographychemiluminescence
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RFLP Analysis
Restriction Fragment Length Polymorphisms (RFLP)
Basis for polymorphic DNA sequences
1. Change in RE cleavage site
Gel Pattern
A
a
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RFLP Analysis
Basis for polymorphic DNA sequences
2. Change in number of repeats
VNTR (variable numbers tandem repeats: 10 - 1000
bp)
STRP (simple tandem repeats: CAGCAGCAG)
Southern blot with probe to repeat
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SNP Analysis using Southern Blot
Single Nucleotide Polymorphisms that affect restriction sites
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Mutation Detection using Southern Blot
Detection of Sickle-cell gene by DdeI fragment
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Association of RFLP with Disease Allele
If RFLP pattern and disease are inherited together,
the RFLP site (probe) and the disease gene are linked.
If < 1% recombination, they are within 1 million bp of each other.
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Association of RFLP with Disease Allele
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DNA Fingerprinting
Alex Jeffreys - Southern Blot, probes to hypervariable regions
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DNA Fingerprinting
Paternity test Forensic
test
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Polymerase Chain Reaction - PCR
Amplification of specific region of DNA - specific primers
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Polymerase Chain Reaction - PCR
Thermal cycler - 25+ cycles - 3.4 x 107 copies of template
95OC 50 - 60OC 72OCdenature anneal extension
Major productvisible on gel
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Polymerase Chain Reaction - PCR
Using PCR to determine which STR alleles are present
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Polymerase Chain Reaction - PCR
Using PCR to study SNPs that affect RE sites
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DNA Profiling using PCR
Different primer set for each allele used in multiplex PCR
Products size-separated by capillary gel electrophoresis, HPLC
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DNA Profiling for FBI
STR profile -
13 STR loci,
X/Y markers,
2 other loci
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Determining Probability of Match in DNA Profiling
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DNA Sequencing
DNA template - labeled primer (or nucleotides), DNA polymerase, dNTPs, dideoxynucleotides (ddNTP)
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DNA Sequencing
Polyacrylamide gel electrophoresis, autoradiography
Sequence deduced from bands on autoradiogram
5’ A- G-C-C-T-A-G-A-C-T 3’
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DNA Sequencing
Capillary gel electrophoresis, fluorescent ddNTPs
http://www.wellcome.ac.uk/News/2009/Features/WTX056032.htm
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Gene Therapy
Somatic Cell Therapy - feasible for single gene disorders
Somatic cells isolated from individual with defect
Transformed with cloned wild-type gene
virus, direct injection
Cells reintroduced into individual
bone marrow, blood stream
Ideally, cells survive and make gene product
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Gene Therapy
Transgenic cell - contains artificially introduced gene
Transgene - gene used
Examples of successful gene therapy
SCID (severe combined immunodeficiency) adenosine deaminase (ADA)
periodic infusions of transformed T cells
Sickle-cell anemia - bone marrow - permanent cure
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Biotechnology - Transgenic Animals
Expression in mammary tissue - -Lactoglobulin promoter
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Biotechnology - Transgenic Animals
GloFish - Available at That Fish Place
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Biotechnology - Transgenic Plants
Agrobacterium tumefaciens - Ti plasmid
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Biotechnology - Transgenic Plants
Roundup™ Readytobacco
Modified bacterialgene for EPSPS
- herbicide resistance
(5-enolpyruvylshikimate-3-phosphate synthase)
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Applications of Genetic Technology
Isolation and characterization of genes and genomes
Potential for gene therapy and molecular diagnosis
Agriculture: plants - insect resistance, enhanced nutrition/flavor animals - increased production
Environmental: oil-eating bacteria
Manufacture of human gene products:human insulin, growth hormone, clotting factor, etc.
Genome projects - tremendous potential for rapid development