Sanger's DNA Squencing

 

 

 

In DNA sequencing the nucleotide sequence of DNA is determined. In sequencing, appropriate treatments are used to generate DNA fragments that end at the four bases: adenine (A), guanine (G), cytosine (C) and thymine (T). Then the fragments are subjected to polyacrylamide gel electrophoresis so that molecules with one nucleotide difference in length are separated on the gel.

 

"G" tube: all four dNTP's, ddGTP and DNA polymerase

"A" tube: all four dNTP's, ddATP and DNA polymerase

"T" tube: all four dNTP's, ddTTP and DNA polymerase

"C" tube: all four dNTP's, ddCTP and DNA polymerase

 

The ‘Sanger’, ‘dideoxy’, or ‘chain-termination’ method of DNA sequencing relies on the enzymatic synthesis of DNA in vitro in the presence of chain-terminating inhibitors. In the enzymatic Sanger dideoxy procedure the sequence is determined by making a copy of the single-stranded DNA, using DNA polymerase. This enzyme uses deoxyribonucleoside triphosphates (dNTPs) as substrates and adds them to a primer. The primer is hydrogen bonded to the 3' end of the DNA to be sequenced. The DNA with the primer is divided into four separate reaction mixtures. Each reaction mixture contains all four dNTPs and in addition, one of the four dideoxy analogs (dideoxyribonucleoside triphosphates ddNTPs) of the deoxyribonucleoside triphosphates. Because in the dideoxy sugar the 3'-hydroxyl has been replaced by hydrogen, continued extension of the chain cannot occur. The dideoxy analog thus acts as specific chain-termination reagent. Fragments of variable length are obtained depending on the ddNTP in the mixture. The formed nucleic acid fragments are visualizes by using either a labelled (radioactive or fluorescent) primer or dNTPs.