Sequencing of PCR Products
Two basic methods are available for DNA sequencing, the Maxam-Gilbert and the Sanger method. The chemical method (Maxam and Gilbert, 1977) is based on base-specific chemical modification of the DNA and subsequent nicking of the sugar-phosphate bonds of the DNA at the modified bases. The enzymatic method (Sanger et al., 1987) works by elongating a primer on a single-stranded DNA template by incorporating deoxynucleotides (dNTPs) with a polymerase enzyme and simultaneously terminating the chains by base-specific dideoxynucleotides (ddNTPs). Both methods use incomplete reactions (chemical or enzymatic, respectively) in order to generate fragment populations that give a sequence ladder after separation by denaturing electrophoresis. The sequence ladders are visualized by radioactive or nonradioactive means.
KeywordsDirect Sequencing Sequence Ladder Thermostable Polymerase Automatic Fluorescent Sequencing
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