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DNA sequencing technique - Sanger sequencing
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Sanger Sequencing Notes
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Sanger SequencingWhat Is Sanger Sequencing?
Sanger sequencing is the process of selective incorporation of chain-
terminating dideoxynucleotides by DNA polymerase during in vitro DNA replication.
Sanger sequencing, also known as the “chain termination method,” was developed by the English
biochemist Frederick Sanger and his colleagues in 1977. This method is designed for determining the
sequence of nucleotide bases in a piece of DNA (commonly less than 1,000 bp in length). Sanger
sequencing with 99.99% base accuracy is considered the “gold standard” for validating DNA
sequences, including those already sequenced through next-generation sequencing (NGS). Sanger
sequencing was used in the Human Genome Project to determine the sequences of relatively small
fragments of human DNA (900 bp or less). These fragments were used to assemble larger DNA
fragments and, eventually, entire chromosomes.
How Does Sanger Sequencing Work?
In Sanger sequencing, a DNA primer complementary to the template DNA (the DNA to be
sequenced) is used to be a starting point for DNA synthesis. In the presence of the four
deoxynucleotide triphosphates (dNTPs: A, G, C, and T), the polymerase extends the primer by adding
the complementary dNTP to the template DNA strand. To determine which nucleotide is
incorporated into the chain of nucleotides, four dideoxynucleotide triphosphates (ddNTPs: ddATP,
ddGTP, ddCTP, and ddTTP) labeled with a distinct fluorescent dye are used to terminate the
synthesis reaction. Compared to dNTPs, ddNTPs has an oxygen atom removed from the
ribonucleotide, hence cannot form a link with the next nucleotide. Following synthesis, the reaction
products are loaded into four lanes of a single gel depending on the diverse chain-terminating
nucleotide and subjected to gel electrophoresis. According to their sizes, the sequence of the DNA is
thus determined.
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