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Introduction to Genetic Analysis (Introduction to Genetic Analysis (Griffiths))

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Chapter 20: Gene Isolation and Manipulation Add. Info. For Chapter 1, 4 I) Probing to find specific nucleic acid in mixture A) Gel electrophoresis-fragments of DNA move at speeds inversely dependent on size B) Blotting 1) Southern—denature DNA, stick to membrane 2) Northern—detect specific RNA, determine whether specific gene is transcribed in a tissue or under certain environmental conditions C) Exploits ability of nucleic acids with complementary nucleotide sequences to find and bind to each other II) Finding specific clones by functional complementation aka mutant rescue A) Introducing functional DNA back into mutants to restore function B) Through clones in bacterial or phage library III) Positional cloning—method for finding a specific clone that makes use of info about gene’s position on its chromosome. Need: A) Some genetic landmarks to set boundaries of where gene may be (i.e RFLPs, SNPS, molecular polymorphisms) B) Ability to investigate continuous segment of DNA by extending b/t the delimiting
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Unformatted text preview: genetic landmarks 1) Genome sequence—genes in this block of DNA in model organisms 2) Chromosome walk—for some species to find and order the clones falling b/t genetic landmarks a) Use sequence of nearby landmark as probe to ID second set of clones that overlaps marker clone containing landmark but extends into two directions b) Series of steps from one adjacent clone to next IV) Polymerase chain reaction—to amplify DNA in vitro A) If we know sequence of at least some parts of gene B) Uses multiple copies of primers (15-20 bases long) that bind to different end of gene to be amplified C) Primers bind to opposite DNA strands with their 3’ ends pointing at each other, location of primers determines specificity of DNA segment that is amplified D) Taq Polymerase adds bases to primers E) Polymerization process shuttles back and forth b/t them F) Forms exponentially long # of double-stranded DNA molecules...
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