Maxam & Gilbert Sequencing
There are four chemical cleavage reactions at the core of the Maxam and Gilbert sequencing system. The figure below left shows an example from these reactions, the reaction cleaving specifically at guanine. The other three reactions cleave at G+A, C+T, or C. Guanine and cytosine, therefore, give bands in 2 lanes, adenine and thymine in only one. An example of the gel pattern produced is presented below right.
![]() |
![]() |
Maxam and Gilbert DNA sequencing reaction specific for Guanidine residues. The Guanine base is first modified with Dimethyl Sulfate (DMS), which makes the chain susceptible to cleavage by piperidine, destroying the Guanidine residue and releasing a labeled fragment for electrophoresis. | In a Maxam and Gilbert gel, the identity of guanine or cytosine in the sequence can be assigned most easily because two of the four reaction sets cleave at those bases alone. Adenine or thymine are slightly more difficult, being represented by those bands in the G+A or C+T lanes which do not appear, respectively, in the G or C lanes. |
The DNA to be sequenced must first be end labeled, at one end only. This is accomplished by kinase treatment with 32P ATP, which labels both ends, followed by restriction digestion and isolation of the two labeled fragments. Alternatively, digestion of a plasmid containing a clone of the DNA of interest with an appropriate enzyme can yield a unique labeling site. Plasmid vectors containing the rare site for Tth111I, which leaves a single 5' base overlap, have been generated for this purpose. Cleavage with Tth111I leaves a G at one end and a C at the other in these vectors. By filling in the gap with Klenow polymerase fragment in the presence of dGTP or dCTP, one end or the other can be labeled specifically. Labeled DNA is first precipitated to remove any salts which might interfere in the cleavage reactions. It is then modified, cleaved and run on a denaturing gel for analysis. NB: THE HYDRAZINE AND DMS USED IN THESE PROTOCOLS ARE TOXIC AND VOLATILE. KEEP TUBES SEALED AND WORK IN A HOOD.
Maxam and Gilbert Sequencing Reactions
|
NEXT TOPIC: Sanger Sequencing