DNA Sequencing

A. Sequencing by chemical degradation

• base-specific cleavage of DNA by dimethyl sulfate (DMS) and piperidine
• Four different chemicals are used in four reactions, one for each base.
  
• Each reaction produces a set of DNA fragments of different sizes. The sizes of the fragments
• A double-stranded or singlestranded fragment of DNA to be sequenced is processed to obtain a single strand labeled with a radioactive isotope at the 5! end (1).
• This DNA strand is treated with one of the four chemicals for one of the four reactions.
  

Dimethyl sulfate attaches a methyl group to the purine ring of G nucleotides. The amount of DMS used is limited so that on average just one G nucleotide per strand is methylated, not the others (shown here in four different positions of G). When a second chemical, piperidine, is added, the nucleotide purine ring is removed and the DNA molecule is cleaved at the phosphodiester bond just upstream of the site without the base. The overall procedure results in a set of labeled fragments of defined sizes according to the positions of G in the DNA sample being sequenced.. The four reaction mixtures, one for each of the bases, are run in separate lanes of a polyacrylamide gel electrophoresis. Each of the four lanes represents one of the four bases G, A, T, or C. T

B. Sequencing by chain termination

References:

Brown, T.A.: Genomes. Bios Scientific Publ., Oxford, 1999.
Rosenthal, N.: Fine structure of a gene—DNA sequencing. New Eng. J. Med. 332:589–591, 1995.
Strachan, T., Read, A.P.: Human Molecular Genetics. 2nd ed. Bios scientific Publishers
http://en.wikipedia.org/wiki/DNA_sequencing
http://www.springerprotocols.com/Abstract/doi/10.1385/0-89603-248-5:261