Principle of the plus and minus method

Principle of the plus and minus method (i) Primed synthesis 

Electrophoresis of 8 mixtures from plus and minus systems 

The autoradiograph of the -A channel will consist of a set of bands, each of which corresponds to an extension product up to, but not including, the next A residue in the sequence. Thus the positions of the A residues are located. In a similar way the positions of the other residues are located from the sequencing channels and, in principle, the sequence of the DNA is read off from the autoradiograph. Usually, however, this system alone is not sufficient to establish the sequence and a second line of attack, the plus system is used to confirm and complement the data from the minus system. 

Detailed protocol for the plus and minus primed synthesis method 

Stop reaction by blowing into lOyu.1 formamide-dye mixture. 

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Fig. 2.2. Principle of the plus and minus method for sequencing DNA (from Sanger and Coulson, 1975). For description see text.

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