Chain Termination Method
Sanger sequencing is a method of DNA sequencing that involves electrophoresis and is based on the random incorporation of chain-terminating dideoxynucleotides by DNA polymerase during in vitro DNA replication. After first being developed by Frederick Sanger and colleagues in 1977, it became the most widely used sequencing method for approximately 40 years. It was first commercialized by Applied Biosystems in 1986. More recently, higher volume Sanger sequencing has been replaced by next generation sequencing methods, especially for large-scale, automated genome analyses. However, the Sanger method remains in wide use for smaller-scale projects and for validation of deep sequencing results. It still has the advantage over short-read sequencing technologies (like Illumina) in that it can produce DNA sequence reads of > 500 nucleotides and maintains a very low error rate with accuracies around 99.99%. Sanger sequencing is still actively being used in efforts for public health initiat ...
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Dideoxynucleotide
Dideoxynucleotides are chain-elongating inhibitors of DNA polymerase, used in the Sanger method for DNA sequencing. They are also known as 2',3' because both the 2' and 3' positions on the ribose lack hydroxyl groups, and are abbreviated as ''ddNTPs'' (ddGTP, ddATP, ddTTP and ddCTP). Role in the Sanger method The Sanger method is used to amplify a target segment of DNA, so that the DNA sequence can be determined precisely. The incorporation of ddNTPs in the reaction valves are simply used to terminate the synthesis of a growing DNA strand, resulting in partially replicated DNA fragments. This is because DNA polymerase requires the 3' OH group of the growing chain and the 5' phosphate group of the incoming dNTP to create a phosphodiester bond. Sometimes the DNA polymerase will incorporate a ddNTP and the absence of the 3' OH group will interrupt the condensation reaction between the 5' phosphate (following the cleavage of pyrophospate) of the incoming nucleotide with the 3' ...
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