DNA Sequencing with the Maxam-Gilbert Method

For example, given an initial fragment, labeled at the 5 -end, that has the sequence 32P-GCTGCTAGGTGCCGAGC, partial cleavage at (and removal of) the G residues will yield the following detectable fragments  

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

Both the G and C reactions are specific for these bases. The two other reactions needed for sequencing result in cleavage at both G and A residues, and at both C and T residues, respectively. 

The G + A Reaction In this reaction, acid is used to weaken the glycosidic bond by protonating (rather than methylating as in the G reaction) at the N7 position of both A and G. This protonated form is susceptible to piperidine displacement, which then occurs as shown in Reaction 10.3. 

The C +T Reaction The C + T reaction is performed under identical conditions to the C reaction, except that the 1 M NaCl used in the C reaction is absent. Under these conditions, cleavage will occur at both C and T residues. For example, the fragments expected for the cleavage of the 5 -labelled fragment 32P-ACTGTAGC in each reaction are shown in Table 10.4. 

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