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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