Sanger sequencing

Sanger sequencing, also known as the chain termination method, is a method of DNA sequencing first commercialized by Frederick Sanger and his colleagues in 1977. This method is based on the selective incorporation of chain-terminating dideoxynucleotides by DNA polymerase during in vitro DNA replication.

History

The Sanger sequencing method was developed in 1977 by two-time Nobel laureate Frederick Sanger and his colleagues. This method quickly replaced the then-used plus and minus method and became the most widely used DNA sequencing method for the next 40 years.

Method

The Sanger sequencing method involves four separate DNA synthesis reactions. Each reaction includes a DNA template, a DNA primer, the four deoxyribonucleotide (dNTP) bases, DNA polymerase, and a small amount of one of the four dideoxynucleotide (ddNTP) bases. The ddNTPs cause DNA synthesis to stop, as they lack the 3'-OH group required for the formation of a phosphodiester bond between two nucleotides, causing DNA polymerase to cease extension of DNA.

Applications

Sanger sequencing is used in a wide range of applications, including gene sequencing, mutation detection, and genetic disease diagnosis. It is also used in research for studying genetic disorders, cancer, and infectious diseases.

Limitations

While Sanger sequencing is a reliable and accurate method for DNA sequencing, it has several limitations. These include the inability to sequence very long DNA molecules, the requirement for a large amount of purified DNA, and the relatively high cost and time-consuming nature of the method.

See also

• DNA sequencing
• Frederick Sanger
• Dideoxynucleotide
• DNA polymerase