Maxam-Gilbert method: DNA cloning and polymerization reactions made the sanger method less popular than maxam-gilbert DNA sequencing method. This method was discovered by Allan Maxam and Walter Gilbert in 1977 which is based on chemical modification and subsequent cleavage. In this method, a 3' or 5' radiolabeled DNA is treated with a base specific chemicals which randomly cleaves the DNA at their specific target nucleotide. These fragments are analyzed on a high resolution polyacrylamide gel and a autoradiogram is developed (Figure 27.3). The fragment with terminal radiolabel appears as band in the gel. The chemical reactions are performed in two steps;

Base Specific Reaction: Different base specific reagents are used to modify the target nucleotide.

Reaction 1: Dimethylsulfate (DMS) modifies the N7 of guanine and then opens the ring between C8 and N9.

Reaction 2: Formic acid acts on purine nucleotide (G+A) by attacking on glycosidic bond.

Reaction 3: Hydrazine breaks the ring of pyridine (T+C).

Reaction 4: Where as in the presence of salt (NaCl), it breaks the ring of cytosine.

Cleavage reaction : After the base specific reactions, piperidine is added which will replace the modified base and catalyzes the cleavage of phsophodiester bond next to the modified nucleotide.

Interpretation of the band in autoradiogram: The fragment in G lane is read as “G” where as fragment present in G+A but absent in G is read as “A”. Similarly fragment in C is read as “C” where as fragment present in T+C but absent in C is read as “T”. To get the DNA sequence, the band with the lowest molecular weight is read followed by next band in the four lane. For example in the given figure 27.3, in G lane the band is of lowest molecular weight followed by band in A lane etc.