Sanger-Coulson method

The Maxam-Gilbert method doesn t require synthesis of a primer and it sometimes works well for sequences that are difficult to obtain with the Sanger -Coulson procedure. The two methods may both be used to provide additional certainty about a sequence. The Maxam-Gilbert method is very convenient for sequencing small oligonucleotides which often react poorly with the polymerase used for the chain termination method. The method usually requires that a restriction map be prepared. 

Sanger, F., and A. R. Coulson, A rapid method for determining sequences in DNA by primed synthesis with DNA polymerase. J. Mol. Biol. 94 444-448, 1975. 

In 1970, Maxam and Gilbert and, in parallel, Sanger and Coulson, invented their method of DNA sequencing, of which, in principle, the latter is still in use today. 

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... 

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

In the ribosubstitution method these problems are circumvented by the addition of one or more ribonucleotides between the DNA primer and the radioactive cDNA. This site is susceptible to cleavage with ribonuclease or alkali. This method can also be used in conjunction with other primed synthesis methods for DNA sequencing (Barnes, 1978 Sanger, Nicklen and Coulson, 1977). 

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... 

The thin-layer gel system used for resolving the different fragments produced by the chemical degradation method is essentially that of Sanger, Nicklen and Coulson (1977) as described in Chapter 4. An 8% polyacrylamide gel is usually used though for special purposes a 10 or 12% gel can give an improved resolution of the smallest fragments. For the standard 8% gel the polymerization mixture contains 7.6% (wt/vol) acrylamide, 0.4% (wt/vol) bisacrylamide, 50% (wt/vol) urea, 100 mM Tris-borate, pH 8.3, 2mM EDTA, 0.07% (wt/vol) ammonium persulphate and TEMED catalyst. This solution is poured or injected into a 0.4 x 200 x 400 mm mold to form a gel slab. 

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