Maxam-Gilbert sequencing

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,... 

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 pathway for strand cleavage using the Maxam-Gilbert sequencing method (1) with a thymidine residue and the cleavage method of the current invention are illustrated in Eqs 2 and 3, respectively. 

Modified Maxam-Gilbert Sequencing Method According to the Current Invention... 

What is the main difference between the Sanger dideoxy method and the Maxam-Gilbert sequencing method ... 

TABLE 10.4. Labeled Fragments Expected from the 32P-ACTGTAGC Cleavage Using the Maxam-Gilbert Sequencing Method... 

Figure 10.10. Autoradiogram of DNA sequencing gel obtained after Maxam-Gilbert sequencing reactions of 32P-ACTGTAGC.

Figure Z Polyacrylamide gel electrophoretic analysis of strand I after treatment of the 22-bp 5 -end labeled duplex I/n (or ODN I, lane 14) with activated Mn-TMPyP. Reaction mixtures contained 1 pM of ODN. Lanes 1,2,3 and 4, Maxam-Gilbert sequencing reactions (A + G, G + Q G and C respectively). Lane 5 to 11 40 mM Tris/HCl pH 8 buffer and 100 mM NaQ lane 5, duplex control lane 6, 0.5 pM Mn-TMPyP followed by alkali + heating treatment lane 7, 0.5 pM Mn-TMPyP + 40 pM KHSO5 Mn-TMPyP + 40 pM KHSO5 followed by alkali + heating...

Depurination Cleavage of the glycosidic bond between C-1 of deoxyribose and a purine base in DNA. Used in Maxam-Gilbert sequence analysis. 

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