lecture11 - 1. DNA Sequencing 2 Methods (developed in 1977;...

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DNA Sequencing 2 Methods (developed in 1977; newer versions adapted) Both rely on analysis of radioactive products on denaturing polyacrylamide gels 1) Chemical Cleavage a/k/a • Depends on chemical reactions that specifically cleave at different residues (A,T, G, or C) 2) Chain termination a/k/a dideoxynucleotide sequencing a/k/a Sanger method • Depends on base-specific interruption of DNA synthesis by incorporation of nucleotide analogues that serve as chain terminators 1.
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Maxam-Gilbert Sequencing 2 basic types of reactions to target purines vs. pyrimidines 1) Depurination Dimethyl sulfate methylates purines • Methylated purines are susceptible to depurination • At positions of depurination, polynucleotide chain can be cleaved by hydrolysis in hot 0.1 N alkali • Can select for G vs. A by using different pHs for depurination 2) Hydrazinolysis of pyrimidines Hydrazine used to cleave pyrimidines from backbone Piperidine is added to cleave the backbone at these positions • Different conditions favor C vs. T 2.
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This note was uploaded on 04/06/2010 for the course BCH BCH 453 taught by Professor Heminway during the Spring '10 term at N.C. State.

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lecture11 - 1. DNA Sequencing 2 Methods (developed in 1977;...

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