02. Maxam-Gilbert Chemical Sequence Method

Before the popular Sanger sequencing came about, there were two DNA sequencing methods introduced by Alan Maxam and Walter Gilbert in 1973 and 1976.

The first is known as the wandering-spot analysis, which reported sequence of a *whopping* 24 base pairs.

The second, more effective yet still limiting method used chemical sequencing. This means it used chemical processes to terminate DNA strands. These fragment DNA pieces were then run through a gel to resolve the sequence order.

Procedure

  1. Denature a double-stranded DNA to single-stranded by increasing temperature.
  2. Radioactively label one 5' end of the DNA fragment to be sequenced by a kinase reaction using gamma-32P.
  3. Cleave DNA strand at specific positions using chemical reactions. For example, we can use one of two chemicals followed by piperdine. Dimethyl sulphate selectively attacks purine (A and G), while hydrazine selectively attacks pyrimidines (C and T). The chemical treatments outlined in Maxam-Gilbert's paper cleaved at G, A+G, C and C+T. A+G means that it cleaves at A, but occasionally at G as well.
  4. Now in four reaction tubes, we will have several differently sized DNA strands.
  5. Fragments are electrophoresed in high-resolution acrylamide gels for size separation.
  6. These gels are placed under X-ray film, which then yields a series of dark bands which show the location of radiolabeled DNA molecules. The fragments are ordered by size and so we can deduce the sequence of the DNA molecule.
Maxam Gilbert Sequence Illustration

Pros

Maxam-Gilbert sequencing was at one point more popular than the Sanger method. Purified DNA could be used directly, while the Sanger method required that each read start be cloned for production of single-stranded DNA.

Cons

Cons included difficulties scaling up, and the handling of X-rays and radiolabeling, which were harmful to technicians.

References

Gilbert, W., and A. Maxam. "The Nucleotide Sequence of the Lac Operator." Proceedings of the National Academy of Sciences 70.12 (1973): 3581-584. Web.

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