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Sanger sequencing, also known as chain-termination sequencing or dideoxy sequencing has been the powerhouse of DNA sequencing since its invention in the 1970s. The process is based on the detection of labelled chain-terminating nucleotides that are incorporated by a DNA polymerase during the replication of a template.

The method has been extensively used to advance the field of functional and comparative genomics, evolutionary genetics and complex disease research. Notably, the dideoxy method was employed in sequencing the first human genome in 2002. Because of its suitability for routine validation of cloning experiments and PCR fragments, Sanger sequencing remains a popular technique in many laboratories across the world.

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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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1. DNA Sequence for Chain Termination PCR

2. Size Separation by Gel Electrophoresis

3. Gel Analysis & Determination of DNA Sequence

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