Sanger Method

"Sequencing ladder", gel radioautograph

The Sanger method - DNA sequencing ( nucleotide sequencing ) is also known as the chain termination method. This sequencing method was first proposed by Frederick Sanger in 1977 ^[1] , for which he was awarded the Nobel Prize in Chemistry in 1980. This method has been most common for 40 years.

Content

Principle Method

In the classical variant of the Sanger method, one of the chains of the analyzed DNA acts as a matrix for the synthesis of a complementary chain by the enzyme DNA polymerase . The reaction with the same matrix is carried out in four different test tubes, each of which contains:

the primer is a small single-stranded DNA molecule complementary to the beginning of the region to be sequenced. The primer is necessary because DNA polymerases cannot start the synthesis of the chain “from scratch”; they only attach the next nucleotide to the already existing 3′- hydroxyl (OH) group of the previous one. Primer, therefore, is a "seed" in DNA synthesis;
four standard deoxynucleotides (dATP, dGTP, dCTP and dTTP);
a small amount (at a concentration of 1 to 100) of one of the radioactively labeled deoxynucleotides (dideoxynucleotide) (for example, [ ³² P] -dATP), which is incorporated into the DNA during synthesis and allows you to subsequently visualize the products of the reaction;

Dideoxyribonucleotides (ddATP, ddGTP, ddCTP, or ddTTP) lack a 3'-hydroxyl group, therefore, after their incorporation into the chain, further synthesis terminates. Thus, in each test tube, a set of DNA fragments of different lengths is formed, which end with the same nucleotide (in accordance with the added dideoxynucleotide). After completion of the reaction, the contents of the tubes are separated by polyacrylamide gel electrophoresis under denaturing conditions and autoradiography of the gels is performed. The products of the four reactions form a "sequencing ladder", which allows you to "read" the nucleotide sequence of the DNA fragment ^[2] ^[1] .

The Sanger method also allows one to determine the nucleotide sequence of RNA , but it must first be “rewritten” in the form of DNA using reverse transcription .

Current State

Graphic presentation of DNA sequencing results

To date, DNA sequencing according to Sanger is fully automated and is carried out on special instruments, sequencers. The use of dideoxynucleotides with fluorescent labels with different emission wavelengths allows the reaction to be carried out in the same tube. The reaction mixture is separated by capillary electrophoresis in solution, DNA fragments leaving the capillary column are recorded by a fluorescence detector. The results are analyzed by computer and presented in the form of a sequence of multi-colored peaks corresponding to four nucleotides. Sequencers of this type can “read” at once sequences of 500-1000 nucleotides in length. For comparison, the pyrosequencing method, developed in 1996, makes it possible to determine the sequence of a significantly smaller number of nucleotides in one stage. Automation significantly accelerated the sequencing process and allowed the sequencing of entire genomes , including the human genome ^[2] .

Notes

↑ ¹ ² Sanger F., Nicklen S., Coulson AR DNA sequencing with chain-terminating inhibitors. // Proc Natl Acad Sci US A .. - 1977. - Vol. 74 , no. 12 - p . 5463-5467 . - PMID 271968 .
² ¹ ² Blackburn MG Chapter 5: Nucleic Acids in Biotechnology // Nucleic Acids in Chemistry And Biology . - Great Britain: Royal Society of Chemistry, 2006. - P. 168. - ISBN 0854046542 .

Literature

Sanger F., Niclein S., Coulson AR DNA sequencing with chain-terminating inhibitors // Proc Natl Acad Sci USA. - 1977. - T. 74 . - p . 5463-5467 .
Sanger F., Coulson AR: Rapid syntesis with DNA polymerase // J Mol Biol. - 1975. - T. 94 . - p . 444-448 .