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Principle of Sanger Sequencing

 

Research in molecular biology, DNA sequence analysis is further study and improvement of the basis of the target gene. Sequencing technologies currently used mainly in Sanger (1977) invented the double DNA chain termination method, Sanger sequencing method is currently widely applied. Sanger method is based on the nucleotide start at a fixed point, random in a particular base at the end, and in the back of each base fluorescent marker, resulting in A, T, C, G the end of the four groups a series of nucleotides of different lengths, and then denatured in urea PAGE gel electrophoresis for detection, resulting in visible DNA base sequence. The principle is the Sanger sequencing, each reaction containing all four deoxynucleotide triphosphate (dNTP) to the expansion, and a limited mix of different dideoxy nucleoside triphosphate (ddNTP) to the end. Extension of the ddNTP lacks the 3'-OH required for groups, so that the extended oligonucleotide selectively in G, A, T or C at the end, end point from the corresponding dideoxy reaction may be. Each of the relative concentrations of dNTPs and ddNTPs can be adjusted so that the reaction of a few to a thousand or more a head, a difference of one base a series of clips. They have a common starting point, but in a different termination nucleotide, it can be separated by high-resolution denaturing gel electrophoresis, fragments of different sizes, the gel can be treated with X-ray film autoradiography or non-isotopic markers detection.

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