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.

In a Maxam and Gilbert gel, the

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.
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

1. Precipitate the substrate: To the 32P labeled DNA, add 0.1 vol.
3M Sodium Acetate and 1 vol. Isopropanol. Precipitate at -70°C for 10
minutes, and centrifuge at max RPM in a microcentrifuge for 5 minutes
to collect the DNA. Wash the pellet twice with 1 ml cold 70% ethanol to
remove all salt. Redissolve the DNA in 45 µl of sterile water. Count one
microliter of the solution in scintillation cocktail to confirm >5x103 cpm
total counts.
2. Aliquot 10 µl of the DNA solution into each of 4 tubes. Label the
tubes C, G, C+T, G+A.
3. Reactions:
• C: Add 10µl 2.5M NaCl and mix well. Add 30µl of
Hydrazine (toxic!) and incubate at 25°C for 7-9 minutes.
• G: Add 200µl of: 50mM sodium cacodylate, pH 8, 1mM
EDTA. Mix well and add 1µl Dimethyl Sulfate (DMS)
(Toxic!) and incubate at 25°C for 4-5 minutes.
• C+T: Add 10µl H2O and mix well. Add 30µl Hydrazine
and incubate at 25°C for 7-9 minutes.
• G+A: Add 25µl of formic acid, mix well and incubate at
25°C for 4-5 minutes.
4. Stop the reactions:

Stop buffers:
• G reaction: Add 50µl of:1.5M sodium acetate pH 7, 1M
mercaptoethanol, 100µg/ml tRNA.
• All other reactions: Add 200µl of 0.3M sodium acetate, pH
7, 0.1mM EDTA, 25µg/ml tRNA.
 Ethanol precipitation:
Add 750µl of Ethanol, and transfer reactions to a -70°C bath for 5
minutes. Collect DNA by microcentrifugation for 5 minutes.
Discard the supernatants as appropriate for DMS or Hydrazine
waste. Rinse the pellets twice with 70% ethanol. Redissolve the
pellets in 300µl of water, add 30µl of 3M sodium acetate and 1ml
of ethanol. Pellet DNA and wash twice with 70% ethanol. Allow
the pellets to air dry.

5. Piperidine cleavage reactions:

Resuspend pellets in 75µl of 10% piperidine, and transfer to screw top
tubes. It is essential that the tubes used for the piperidine reaction seal
well in order to ensure that the reaction goes to completion. Incubate the
tubes at 90°C for 30 minutes. Cool the tubes, centrifuge briefly to collect
the condensate, and evaporate to dryness in a speedvac. Redissolve the
pellet in 40µl of water and dry again. Repeat the rehydration and drying
once more to ensure that all of the piperidine has been removed. The
samples are now ready for denaturing PAGE.