Nucleotide sequencing Maxam-Gilbert method

Sequence analysis of nucleotides by the Sanger or Maxam-Gilbert method... 

The final point to note is that sequencing by the primed synthesis methods will not reveal the presence of methylated or other modified nucleotides in the original DNA sequence. This is also true of the Maxam-Gilbert method where the DNA to be sequenced has been cloned into a plasmid vector. 

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.

Fig. 3. Nucleic acid sequencing. Scheme showing the chemical cleavage method of Maxam and Gilbert applied to the determination of the nucleotide sequence in a hypothetical length of DNA. a = original DNA, i.e. the uncleaved but P-labeled DNA being sequenced, b > location of each nucleotide in the DNA being sequenced, numbering from the 5 -end.

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. 

Figure 4 The principles of the methods of nick translation left) and of Maxam and Gilbert (right), for determination of the sequence of first 48 nucleotides in the hypothetical Hae III firagment of sequence...

The sequencing of DNA can be accomplished by using both chemical (introduced by Gilbert with his student Maxam) and enzymatic methods, especially the Sanger dideoxy-nucleotide protocol. In both approaches, and in analogy to protein analysis (Section 26-5), the unwieldy DNA chain is first cleaved at specific points by enzymes called restriction endonucleases. 

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