Nucleic Acid Tracing

As DNA & RNA are polymers comprised of a very limited number of distinct monomers, biomedical tracing of these molecules principally consists of introducing modified base monomers into the structure of the nucleic acid of interest. Modification may also occur by adding molecular marker groups with chemistries similar to what we have described for proteins & other molecules. Radioactivity, chromogens, fluorogens, particles, & density labels have all been used to tag nucleic acids. Antibodies also exist that can distinguish the 2D & 3D features of specific nucleic acids or sequences.

NA applets: http://www.oligo.net/Enzymology of DNA replication:Nucleic Acid Labeling\CUHKBio2310TutorialFile1Expt3NABasics.pdfwww.ufrgs.br/depbiot/blaber/section1/section1.htm

Background:Background:http://www.gene-quantification.info/

Background & General Information

Biochemistry of polymerases & ligases for NA manipulation:www.chups.jussieu.fr/polys/biochimie/BGbioch/POLY.Part.I.html

Bioinformatics Info: www.dur.ac.uk/stat.web/Bioinformatics/Projects/hint2.htmwww.dur.ac.uk/stat.web/Bioinformatics/DNA_corner.htmhttp://www.dur.ac.uk/biological.sciences/Bioinformatics/DNA_corner.htm

bio.fsu.edu/~stevet/BioInfoSurvey.BCH5425.05.ppt#256,1,The “Nuts and Bolts” of ‘doing’ bioinformatics with the Wisconsin Package at FSU

DNA labeling & other NA protocols:http://info.med.yale.edu/genetics/ward/tavi/n_label.html

Nick-translation defined:Nick-translation defined:http://www.fmv.ulg.ac.be/genmol/MODGEN/ChapterIII/SR5_2A.htm

Nick-translation Labeling

http://www.bio.davidson.edu/courses/genomics/method/randompriming.html

http://www.chups.jussieu.fr/polys/biochimie/BGbioch/POLY.Chp.9.12.html

Random Priming Labelling

Polymerase Chain

Reaction

One of the most common means of amplifying, modifying, or tagging nuclei acids. Amplification may reach millions of copies after 20-30 cycles.

PCR Parameters

NA length, redundancy, GC/AT content

Polymerase features (Taq, Vent, …)

[Mg++]

[Primers]

[dNTPs, NTPs]

Time & temperature of denaturation

Time & temperature of primer annealling

Time & temperature of extension

Presence of additives (intended, impurities)

Number of cycles

Nature of labeled dNTPs, NTPs

Variants of PCR

RT-PCR (Reverse Transcriptase-PCR)

RT-PCR (Real-Time PCR)

using hairpin primers

using intercalation labels

Universal primers

Rolling circle amplification

LCR (Ligase Chain Reaction)

http://www.faculty.virginia.edu/landers/Images/pcr2.gif

Landers et al. at Virginia are working to put PCR in a micro-fluidics system that uses IR heating.

Real Time PCR

Methods notes that include competitive quantitative PCR:http://ccm.ucdavis.edu/cpl/Tech%20updates/TechUpdates.htm

Real Time sites: http://dna-9.int-med.uiowa.edu/realtime.htm

http://www.promega.com/paguide/paguide_us.pdf (Also includes many,many more protocols; 299 p, 24MB)

Taqman Approach to PCR

Perkin Elmer Taqman PCR: http://cgr.otago.ac.nz/SLIDES/TAQMAN/INDEX.HTM

Reverse Transcriptase PCR

http://www.promega.com/guides/

http://www.westburg.eu/en/site/life-sciences/pcr-rt-pcr

Rolling Circle Amplification

http://courses.agri.huji.ac.il/71953/ic7.htm

Alia L. Merla, Amersham presented an alternative to DNA plasmid amplification, 7/16/03, Seminar at UCLA,

http://genoseq.ucla.edu/action/view/Seminars_and_news

http://www1.gelifesciences.com/APTRIX/upp01077.nsf/Content/sample_preparation~product_selection_category~rolling_circle_amplification/$file/flash.htm

LCR

T4 Ligase:

http://dwb.unl.edu/Teacher/NSF/C08/C08Links/www.worthington-biochem.com/manual/D/DNAT4L.htmlhttp://www.biochem.uwo.ca/community/molbio/ligate.html

Southern & Northern BlotsSouthern Protocol plus:Southern Protocol plus:http://www.ableweb.org/volumes/vol-12/1-karche/1-karche.htmhttp://www.healthsystem.virginia.edu/internet/transgenic-mouse/southern.cfm

http://center.intron.co.kr/method/protocol_2.asp

http://www.bio.davidson.edu/courses/genomics/method/Northernblot.html

Apoptosis Assays:http://sciencepark.mdanderson.org/fcores/histology/apop.htmlhttp://www.compucyte.com/apoptosis.htmhttp://www.ikp.unibe.ch/lab2/REG/index.htmhttp://www.phnxflow.com/apo.overview.html

TUNEL Assays

Protocols:www.animal.ufl.edu/hansen/protocols/tunel.htmhttp://sciencepark.mdanderson.org/flow/files/TUNEL.html

Detection of apoptotic cells in the mouse testis using the TUNEL assay (methyl green counterstaining). http://sciencepark.mdanderson.org/fcores/histology/histology_picts/staining/apoptosis_assays/TunelTesticle_10xMethgre-04.jpg

DNA Microarray

Point mutation detection with DNA microchips:http://www.clinchem.org/cgi/reprint/46/10/1555.pdf

Reagents & methods:www.cambridgebluegnome.com/Products/www.bioxing.com/Products/bxdesigner.htm

Statistical methods:http://www.garnetthenley.com/Descriptive.pdf

Tutorials: Tutorials: http://www.transcriptome.ens.fr/sgdb/presentation/principle.phphttp://www.ebi.ac.uk/ebisearch/search.ebi?db=allebi&query=Microarray

DNA Sequencing

Maxam-Gilbert• Amplify NA or fragments using vectors or PCR • Radiolabel 5’ end• Fragment DNA using base selective chemical agents• Separate products on PAGE or capillary electrophoresis• Read autoradiograms

Manual protocol; 100’s of bp/day; low # bp/read; >>$250/Mb

Sanger• Amplify NA or fragments using vectors or PCR • Radio- or dye-labelled ddNTPs + polymerase used in a final labeling replication/amplification• Separate products on PAGE or capillary electrophoresis• Read autoradiograms or laser excited electroperograms produced by PMT or CCD

Manual, Fast = ABI sequencer; kb/day; 400-800 bp/read; ~$234/Mb

http://www.campus.skelleftea.se/biomine/molecular/index_14.htm

Maxam-Gilbert Sequencing

Sanger Sequencing

http://www3.appliedbiosystems.com/AB_Home/applicationstechnologies/DNASequencingbyCapillaryElectrophoresis/index.htm

Next/Second Generation Pyrosequencing: 454 Life Sciences

• Emulsion PCR of NA fragments• Picotiter well serial reaction with dNTPs• Luciferase coupling of luciferin to ATP from PPi via sulfurylase exchange of S on APS• CCD detection of light pulse amplitude α # N’s added

100 Mb/run (7h); 250 bp/read; $84/Mb

http://www.454.com/enabling-technology/the-technology.asp

Genome Sequencer 20 System 2006 Roche Diagnostics GmbH.

Genome Sequencer 20 System 2006 Roche Diagnostics GmbH.

Next/Second Generation (cont.)Bridge-amplification & fluorescent dyes: Illumina

•Adapter ligation, surface attachment, bridge amplification•Denature, cluster•Single F-dNTP extension•Image clusters•Deblock & remove fluorophore•Repeat labeling & detection

1300 Mb/run (4d); 32 - 40 bp/read; $6/Mb

http://www.illumina.com/pages.ilmn?ID=203

http://seqanswers.com/forums/showthread.php?t=21

http://seqanswers.com/forums/showthread.php?t=21

IlluminaSteps 1-6

IlluminaSteps 7-12

http://seqanswers.com/forums/showthread.php?t=21

Next/Second Generation (cont.) Ligation-based, dual nucleotide: SOLiD

• Ligate adapters & hybridize to beads w/ complementary adapters• Amplify w/ emulsion PCR• Tether beads to a surface• Hybridize anchoring adapters & 1st of F-8mer-known-diN probes• Ligate probes, wash, read signal• Remove F- & 3 3’ N’s, wash• Repeat hybridization, ligation & reading• Software calls sequences from color patterns

3000 Mb/run (5d); 25 - 35 bp/read; $6/Mb

http://marketing.appliedbiosystems.com/images/Product/Solid_Knowledge/flash/102207/solid.html

Third Generation MethodsHelicosTechnique appears similar to Illumina but is done on single DNA strands w/o prior amplification

7.5 GB/run (14d); > 25 b/read; $2.40/MB

http://www.helicosbio.com/Technology/tabid/62/Default.aspx

Single Molecule Real TimeTethered polymerase adds F-dNTP then cleaves F to add next F’-dNTP; CCD images single polymerase rxns

Nanopore SequencingDetection of exonuclease product dNDPs via modified electrical flow in a nanopore tethered to the enzyme

http://www.pacificbiosciences.com/index.php?q=technology-introduction

http://www.nanoporetech.com/sequences

http://www.pacificbiosciences.com/template/image_popup.php?img=tech/zmw_dna_poly_phos_nucl_lg.jpg&imgTitle=ZMW%20with%20DNA%20polymerase%20and%20phospholink%20nucleotides&background=#000

Methods: http://www.mcb.uct.ac.za/Sequencing%20Service%20web/index.htmhttp://www.epibio.com/litindex.asp?method=alpha

Background: http://www.roswellpark.org/document_3636_639.html

New Sequencing Approaches

http://www.liv.ac.uk/agf/454sequencing.html

454 Life Sciences, Pyrosequencing

http://www.dkfz.de/gpcf/242.html

http://jeb.biologists.org/cgi/content-nw/full/210/9/1518/FIG2

http://www.genengnews.com/sequencing/supp_02.aspx

Original Description

https://www.roche-applied-science.com/servlet/RCConfigureUser?URL=StoreFramesetView&storeId=10202&catalogId=10202&langId=-1&countryId=us

More Methods Manuals:http://www.dwalab.ca/labman/index.htmlhttp://www.uhmc.sunysb.edu/bioscience/methods/methods.htmhttp://www.methods.info/

Additional Molecular Methods (especially Drosophila):http://www.dhgp.org/current/index.html