Maxam and Gilbert method

Figure 13.19 The Maxam and Gilbert method of DNA sequencing. The labelled DNA strand is divided into four aliquots. Each is treated to cleave the strand next to a different base resulting in a mixture of different length nucleotides. These nucleotides are separated by polyacrylamide gel electrophoresis and the DNA sequence read from the gel.

In the Maxam and Gilbert method (M2) the starting point is a defined DNA restriction fragment. The DNA strand to be sequenced must be radioactively labeled at one end with a 32P-labeled phosphate group. This DNA can be single stranded or of duplex form. The base-specific cleavages depend upon the following points ... 

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. 

In the Maxam and Gilbert method, the double-stranded DNA is radioactively labeled, cutwith restriction enzymes, and denatured. Subsequently, the DNA is treated chemically in four separate reactions, which cut DNA at different nucleotides. The first reaction, called the A+G reaction, cuts the nucleotides adenine (A) and guanine (G). The second reaction, called the G reaction, cuts at G. The third and fourth reactions are similar, but involve cytosine (G) and thymine (T) in the G+T and the G reaction. The products of these four reactions are run through gel electrophoresis in four adjacent wells and analyzed for the sequence. The G reactions determine the placement of G, the A+G reactions determine the location of A, and so forth. 

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. 

Detailed procedures for 5 - or 3 -end labelling DNA fragments, following the method of Maxam and Gilbert (1980) are given in Chapter 5. Essentially similar protocols are given by Wu et al. (1976) and Roychoudhury and Wu (1980). 

Primed synthesis methods require that either the primer or the template DNA is in single-stranded form. Sometimes it is possible to physically separate the complementary strands from a duplex DNA but this is not always a straightforward procedure. The methods developed by Maxam and Gilbert (1977) (Chapter 5 of this volume), while eminently suitable for separating single... 

Maxam and Gilbert (1980) recommend the following procedure for small segments of gel. For longer pieces of gel the method described under Procedure A may be used ... 

Two basic protocols for DNA sequencing are commonly adapted in laboratories the chemical cleavage method (Maxam and Gilbert, 1977) and the enzymatic chain termination or dideoxy method (Sanger et al, 1977). Of the two approaches, the Sanger dideoxy... 

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