Why are ddNTPs used in Sanger sequencing

Sanger sequencing

Sanger sequencing, Dideoxy method, Chain termination method, a method of sequencing DNA (nucleic acid sequencing). The S. is now more widespread than the Maxam-Gilbert method because the necessary reagents are now readily available and the method has been automated.

In S., a DNA polymerase catalyzes the synthesis of complementary copies of the single-stranded DNA that is to be sequenced. The nucleotide sequence of the complementary copy is determined directly and the sequence of the original DNA derived on the basis of the base pairing. This means that the DNA to be sequenced is used as a template for making complementary copies. For this purpose it is inserted into a suitable vector (phage or plasmid) and cloned. To initiate the replication process, the DNA polymerase requires a primer (a short DNA segment) that forms stable base pairs to a complementary nucleotide sequence of the phage or plasmid DNA. Synthetic primers are approximately 20 nucleotides in length and are commercially available. When the template-primer duplex is incubated with the four deoxyribonucleoside 5'-triphosphates (dATP, dGTP, dCTP, dTTP) in the presence of the appropriate DNA polymerase, the primer is continuously extended by adding deoxyribonucleoside 5'-monophosphate residues to the terminal 3'-hydroxyl group (5'®3 'direction) are added in an order given by the template and the laws of base pairing. Each new phosphodiester bond is formed by condensation of the α-phosphate residue on the 5'-hydroxyl group of the incoming deoxyribonucleotide with the 3'-hydroxyl group of the terminal deoxyribonucleotide of the extending chain. The elongation therefore depends on the presence of a 3'-hydroxyl group on the terminal deoxyribonucleotide of the chain that is being elongated.

To determine the sequence, four incubations are carried out, each of which contains the template-primer duplex, the appropriate DNA polymerase, equal amounts of the four deoxyribonucleoside-5'-phosphates, of which at least one is marked in the α-phosphate residue, for example with 32P, and one Contains a small amount of the 2 ', 3'-dideoxy derivative of one of the four deoxyribonucleotide 5'-phosphates (ddATP, ddGTP, ddCTP or ddTTP). If the analogous 2 ', 3'-dideoxy derivative is incorporated into the growing polynucleotide chain instead of a 2'-deoxynucleotide, the chain extension stops because a 3'-hydroxyl group is missing. The amount of the 2 ', 3'-dideoxynucleotide present in each incubation mixture is small relative to each of the 2'-deoxyribonucleotides so that a set of 32P-labeled chains of different lengths is created. If ddGTP is used and the template DNA the sequence (3')TCTGACGGTAGC(5') owns, the following sentence is attached 32P-labeled chains released by the enzyme: primer(5')AddG(3'), Primer(5')AGACTddG(3') and primer(5')AGACTGCCATCddG(3'). This and the other three sets of nucleotide chains generated using the other 2 ', 3'-dideoxyribonucleotides are separated in parallel lanes of a polyacrylamide sequencing gel. The banding pattern can then be determined, for example, by autoradiography. The figure shows the application of this method to the template DNA mentioned above (3')TCTGACGGTAGC(5').

The basic method was varied and improved in several ways, but the basic concept remained the same. The improvements concerned the following five areas: 1) the applicability and convenience of the vectors into which the DNA to be sequenced is incorporated, 2) the way in which the 2 ', 3'-dideoxyribonucleotide terminated chains are labeled, 3) the use of 2'-deoxyribonucleoside-5'-triphosphate analogs to eliminate the so-called "compression" of the bands on the sequencing gel, 4) the DNA polymerases used and 5) the possibility of sequencing both double-stranded and single-stranded DNA templates.

Specific vectors so developed are phages (e.g. M13 and its constructs such as M13mp19), plasmids (e.g. the pUC series) and phagemids (e.g. pBluescript II KS +/-) with high copy number, resistance to specific antibiotics, multiple restriction endonuclease cleavage sites and known coding sequences into which the DNA to be sequenced can be inserted precisely.

To mark the DNA, first [35S] dATPαS instead of [32P] dATP used. With this connection one forms 35S atom has a double bond to the P atom of the α-phosphate residue in the 2'-deoxyribonucleotide instead of the O - as in the dATP. If a dAMP residue is incorporated into the growing DNA chain with the help of DNA polymerase, this is due to the formation of an atypical phosphodiester bond [R-O-P (35S) (OH) -O-R '] with 35S marked. Since the β-particles produced by 35S emitted have about 10 times less energy than that of 32P, there is less radiological destruction of the labeled DNA chains and the bands on the autoradiograms of the sequencing gels are narrower and less diffuse.