Why are dideoxynucleotides used in DNA sequencing

Sanger sequencing

after Frederick Sanger (1918 to 2013), British biochemist and double Nobel Prize winner
Synonym: dideoxy method, classical DNA sequencing, chain termination method
English: Sanger sequencing

1 definition

The Sanger sequencing or. Sanger's dideoxy method is a method of DNA sequencing. It is mainly used in genetics and biochemistry and enables the base sequence to be determined in a particular DNA molecule.[1]

2 background

Two methods of DNA sequencing have developed independently of one another: the chemical method according to Maxam and Gilbert and the said dideoxy method according to Sanger. The second was technically developed in such a way that it has established itself and proven itself worldwide because it is faster and easier to automate.

Sanger sequencing is based on in vitro replication of the DNA molecule to be examined. The prerequisite for this, however, is that a sequence region of the molecule is already known.[2]

3 procedures

The dideoxy method is based on a labeled oligodeoxynucleotide primer that hybridizes to the already known sequence region of the molecule. A DNA polymerase catalyzes the synthesis of the complementary DNA strand in four parallel approaches. These four approaches are basically identical - however, in addition to the dNTPs, they also have a different base as a 2'-3'-dideoxynucleotide (ddNTP) in low concentration. It is known that chain extension can only take place at the 3'-OH end of a nucleotide. Because of this, the synthesis will come to a standstill as soon as a ddNTP has been incorporated. The respective ddNTP competes with the corresponding dNTP, which is why the chain termination takes place at different points at random. This is also the reason why a series of synthesis products of different lengths is obtained.[2]

The sequencing process can basically be divided into three steps:

3.1 Denaturation

Since double-stranded DNA is initially present, the hydrogen bonds between the opposing strands must be broken by denaturation (at about 94 ° C., 1 minute) in order to obtain two single strands each.

3.2 Annealing

The annealing process takes 15 seconds at 50 ° C. Here (in contrast to the polymerase chain reaction) only one primer is used, so either one forward primer or a reverse primer binds.

3.3 Extension

For the extension reaction, the temperature for the Taq polymerase must be increased again (60 ° C, 4 minutes). In addition to the polymerase, a mixture of four dNTPs and four ddNTPs is also used. Here the ddNTPs are equipped with fluorescence markers (so-called fluorescence-marked terminators) so that they appear in different colors (red, black, green, blue).

4 evaluation

After an average of 30 cycles, the process can be stopped so that it can then be separated and read using gel electrophoresis.[1]

5 sources

  1. 1,01,1"Basic knowledge of human genetics" - Christian P. Schaaf, J. Zschocke, Springer-Verlag, 2nd edition
  2. 2,02,1"Dual Biochemistry Series" - Joachim Rassow et. al., Thieme-Verlag, 3rd edition