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Molecular Biology

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    1. 1 Molecular Biology Materials & Methods 5: PCR and DNA Markers

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    3. 3 PCR: Polymerase Chain Reaction Amplify DNA by in-vitro (in plastico) synthesis Key requirements: enzyme: Taq DNA polymerase, not denatured at high temps used to denature DNA primers: short (~ 20 b) oligonucleotides bind to denatured DNA, required to start DNA synthesis

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    18. 18 PCR: polymerase chain reaction Advantages: powerful: works with miniscule amounts of DNA (even single cells) highly specific targets many primers work with broad range of targets (plants, animals, fungi) adapted to simplify DNA sequencing Disadvantages: difficulty / expense of primer development troubleshooting when reactions fail

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    22. 22 Thermosequencing: dideoxy sequencing + PCR Recall fundamental step of Sanger sequencing is enzymatic replication of DNA PCR using only one primer, plus different fluorescent nucleotides for each of 4 ddNTP reactions, produces copies of one strand Time-consuming steps of digestion & cloning in M13 can be avoided

    23. 23 PCR anthem from Bio-Rad http://www.cnpg.com/video/redirect.aspx?redirectid=65

    24. 24 RFLPs: Restriction Fragment Length Polymorphisms Restriction enzymes digest DNA by cutting only at specific sequences (typically 6 bp palindromes) Named because they restrict the entry of foreign (e.g. viral) DNA into bacteria, by shredding it

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    31. 31 VNTRs DNA fingerprints A kind of RFLP: combines restriction digest & probing Detects variable number of tandem repeats polymorphism

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    37. 37 VNTR fingerprints Advantages: Low development cost: probes work on most taxa (plants and animals) Highly polymorphic Disadvantages: Bands anonymous: cant determine how many loci or alleles are present Mostly forensic applications Comparisons among a small & defined set of related individuals

    38. 38 RAPDs: Randomly Amplified Polymorphic DNA PCR using short (10 b) primers Short primers bind to many sites scattered throughout the genome because the

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    45. 45 RAPDs: Randomly Amplified Polymorphic DNAs Advantages: Modest development cost Low application cost High polymorphism Modest database for comparison Disadvantages: Bands dominant & anonymous: cant recognize alleles for a locus Generally not comparable among labs

    46. 46 AFLPs: Amplification Fragment Length Polymorphisms Combines RFLPs + PCR

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    53. 53 AFLPs: Amplification Fragment Length Polymorphisms Advantages: Moderate development cost Highly polymorphic Disadvantages Anonymous bands (cant tell loci from alleles) Dominant (complex & uncertain genetics) Rather expensive (multiple restrictions plus PCR plus capillary electrophoresis)

    54. 54 Microsatellites / SSRs: simple sequence repeats PCR-based assay for very small VNTRs Amplify using one of: radiolabeled nucleotides fluorophore-labeled nucleotides fluorophore-labeled primers

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    61. 61 SSR Advantages Defined loci Defined alleles capillary eph gives < 0.5 b resolution Robust amplification targets Codominant !! Highly polymorphic

    62. 62 SSR Disadvantages Primer development takes lots of work digest, clone, probe, sequence, design primer Primers not broadly applicable across taxa usually work within genera (plants) Fluorescent primers are expensive

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