Maxam-Gilbert

The base-specific chemical cleavage (or Maxam-Gilbert) method starts with a single-stranded DNA that is labeled at one end with radioactive (Double-stranded DNA can be used if only one strand is labeled at only one of its ends.) The DNA strand is then randomly cleaved by reactions that specifically fragment its sugar-phosphate backbone only where certain bases have been chemically removed. There is no unique reaction for each of the four bases. However,... 

FIGURE 12.4 Maxam-Gilbert sequencing of DNA cleavage at purines uses dimethyl sulfate, followed by strand scission with piperidine. 

Note that the key to Maxam-Gilbert sequencing is to modify a base chemically so that it is removed from its sugar. Then piperidine excises the sugar from its 5 - and 3 -links in a /3-elimination reaction. The conditions of chemical cleavage described in Figures 12.4 and 12.5 are generally adjusted so that,... 

In principle, the Maxam-Gilbert method can provide the total sequence of a dsDNA molecule just by determining the purine positions on one strand and then the purines on the complementary strand. Complementary base-pairing rules then reveal the pyrimidines along each strand, T complementary to where A is, C complementary to where G occurs. (The analogous approach of locating the pyrimidines on each strand would also provide sufficient information to write the total sequence.)... 

Fig. 3 Autoradiograms of the denaturing sequencing gel for photoreactions of duplexes 9/10, 11/12, 13/14, and 15/16. Lanes 1-4, ODN 9 lane 5, ODN 11 lane 6, ODN 13 lanes 7 and 8, ODN 15 ODNs in lanes 3-7 were photoirradiated all ODNs except in lane 3 were heated with piperidine lanes 1 and 8, Maxam-Gilbert G+A sequencing reactions for ODNs 9 and 15, respectively. Partial base sequences of oligomers were shown on the side. dCNBPU was located opposite to the A (shown with a box)...

Fig. 8 Autoradiograms of a denaturing polyacrylamide gel for photooxidation of duplex 35/36 in the presence of BamH I. ODNs 35 (a) and 36 (b) were separately 5 -32P-end labeled and hybridized to a non-labeled complementary strand. Lane 1, Maxam-Gilbert G+A sequencing reactions lane 2, in the absence of BamH I lanes 3-5, BamH I. ODNs in lanes 2-4 were irradiated at 312 nm. All samples except in lane 2 were heated with piperidine. The BamH I site and dCNBPU (X) are shown in bold face. For clarity, the autoradiogram for ODN 36 is shown upside-down...

Determination of DNA Sequence Information. Almost all DNA sequence is determined by enzymatic methods (12) which exploit the properties of the enzyme DNA polymerase. Whereas a chemical method for DNA sequencing exists, its use has been supplanted for the most part in the initial determination of a sequence. Chemical or Maxam-Gilbert sequencing (13) is more often used for mapping functional sites on DNA fragments of known sequence. 

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. 

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

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