Plus and minus method

The forerunner of the primed synthesis methods was the plus and minus method developed by Sanger and Coulson in 1975 and this marked the first real breakthrough in the search for new and efficient ways of sequencing DNA. This method is the subject of Chapter 2. By itself this procedure does have distinct limitations and additional back-up procedures had to be employed to confirm regions of the sequences deduced. One of these, the depurination method originally introduced by Burton and Petersen, proved a particularly useful adjunct and a description of this method is included as applied to the analysis of pyrimidine clusters in the... 

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

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

Additional methods useful in conjunction with plus and minus method... 

The plus and minus method represented a major breakthrough in the development of primed synthesis methods for the rapid determination of DNA sequences. 

Taken all in all the plus and minus method is a powerful technique. However, it does have its limitations and the more recent developments using the dideoxynucleoside triphosphates as specific chain inhibitors have much improved both the speed and accuracy of the method and will usually be the method of choice for primed synthesis sequencing. This is described in the next chapter. 

This recently described procedure is essentially a marrying of the Maat and Smith (1978) nick translation method and the plus and minus method of Sanger and Coulson (1975). The reason for this development was to circumvent one recurrent problem of the nick translation methods, namely the uneven distribution of band intensities obtained particularly when a row of repeated nucleotides... 

Maxam and Gilbert Method. In 1977, Allan Maxam and Walter Gilbert developed a sequencing method that replaced the plus and minus method. This method was similar, as it required a radioactive label, gel electrophoresis for fragment separation, and the use of X-ray autoradiography for product visualization and inference of the sequence. However, the method differed in that it allowed for the direct analysis of purified double-stranded DNA and used another way of creating products ending in a specific nucleotide. 

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