BIOL 200 Lecture Notes - Lecture 18: Ethidium Bromide, Open Reading Frame, Multiple Cloning Site
13 Oct 2016
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Lecture 21: molecular techniques: pcr and dna sequencing. Size: easiest way to separate dna; the only charged group on dna is phosphate; southern blotting + hybridization works only with denatured, ssdna, not dsdna. Cloned dna molecules are sequenced rapidly by the dideoxy chain-termination method. Complete characterization of any cloned dna fragment requires determination of its nucleotide sequence. Basic idea: synthesize from the dna fragment to be sequenced a set of daughter strands that are labeled at one end and differ in length by 1 nucleotide. Separation of truncated daughter strands by gel electrophoresis can then establish nucleotide sequence of original dna fragment. Dideoxyribonucleoside triphosphates ddntps lack a 3" hydroxyl group; thus ddntps can be incorporated into a growing dna chain by dna polymerase, but cannot form a phosphodiester bond with incoming dntp. Add ddntp to mixture of polymerase action, in addition to normal nucleotides; ddntp concentration very low compared to dntp; synthetic oligodeoxynucleotide used as primer for the polymerization rxn.
