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Bio 321 F13 lecture 6 for post

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Unformatted text preview: dividuals digest with restric1on enzyme(s) run on gel blot on nitrocellulose and hybridize bound DNA to a radioac1ve probe corresponding to a specific fragment containing a variable restric1on site expose to autoradiographic film •  •  •  •  •  •  •  •  •  5 individuals (assume haploids or homozygous diploids) EcoR1 HindIII 1 EcoR1 1 1 kb EcoR1 HindIII 3 kb EcoR1 2 EcoR1 4kb -3kb -- EcoR1 3 EcoR1 HindIII EcoR1 1kb -- 4 EcoR1 5 EcoR1 isolate genomic DNA from individuals PCR amplify fragment of interest containing variable restric1on site digest with variable restric1on enzyme only run on gel EcoR1 HindIII EcoR1 2 3 4 5 RFLPs are co-dominant markers =can tell all three genotypes RAPD markers •  Random Amplifica1on of Polymorphic DNA –  PCR with 1 primer of arbitrary sequence –  a series of fragments are produced, most invariable, some variable between individuals •  these are inherited like alleles, either band, or no band –  Have problems with reproducability – no longer used much http://www.informatics.jax.org/silver/figures/figure8-9.shtml RAPD markers are are dominant markers two alleles: band produced no band produced =heterozygote for the band looks the same as homozygote (although quantification is possible in some cases) AFLP markers •  Amplified Fragment Length Polymorphism ①  isolate genomic DNA and digest with restric1on enzyme ②  ligate (a)ach) “adaptors” onto “s1cky ends” of restric1on- digested DNA fragments ③  perform PCR with primers complementary to adaptor sequences plus 1 or a few nucleo1des into the restric1on fragments •  results in PCR amplifica1on of a subset of the fragments in the sample ④  run products on gel – many bands are variable between individuals – some are inherited as band or no band markers •  gels (or sets of capillaries) are analyzed by computer from e.g. F2 popula1on with known parents and those segrega1ng as alleles are iden1fied and assembled into linkage maps example AFLP outputs gel with many individuals capillary of one individual http://en.wikipedia.org/wiki/File:Electropherogram_trace.jpg http://www.qiagen.com/products/ genomicdnastabilizationpurification/dneasyplantsystem/ dneasy96plantkit.aspx AFLP markers are dominant markers two alleles: band produced no band produced =heterozygote for the band looks the same as homozygote (although quantification is possible in some cases) microsatellite markers •  simple sequence repeats (usually) in noncoding DNA –...
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