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Current Sequencing Technologies

The two basic sequencing approaches, Maxam-Gilbert and Sanger, differ primarily in the way the nested DNA fragments are produced. Both methods work because gel electro-phoresis produces very high resolution separations of DNA molecules; even fragments that differ in size by only a single nucleotide can be resolved. Almost all steps in these sequencing methods are now automated. Maxam-Gilbert sequencing (also called the chemical degradation method) uses chemicals to cleave DNA at specific bases, resulting in fragments of different lengths. A refinement to the Maxam-Gilbert method known as multiplex sequencing enables investigators to analyze about 40 clones on a single DNA sequencing gel. Sanger sequencing (also called the chain termination or dideoxy method) involves using an enzymatic procedure to synthesize DNA chains of varying length in four different reactions, stopping the DNA replication at positions occupied by one of the four bases, and then determining the resulting fragment lengths (Fig. 12).

These first-generation gel-based sequencing technologies are now being used to sequence small regions of interest in the human genome. Although investigators could use existing technology to sequence whole chromo-somes, time and cost considerations make large-scale sequencing projects of this nature impractical. The smallest human chromosome (Y) contains 50 Mb; the largest (chromosome 1) has 250 Mb. The largest continuous DNA sequence obtained thus far, however, is approximately 350,000 bp, and the best available equipment can sequence only 50,000 to 100,000 bases per year at an approximate cost of $1 to $2 per base. At that rate, an unaccept-able 30,000 work-years and at least $3 billion would be required for sequenc-ing alone.
Fig. 12. DNA Sequencing. Dideoxy sequencing (also called chain-termination or Sanger method) uses an enzymatic procedure to synthesize DNA chains of varying lengths, stopping DNA replication at one of the four bases and then determining the resulting fragment lengths. Each sequencing reaction tube (T, C, G, and A) in the diagram contains
  • a DNA template, a primer sequence, and a DNA polymerase to initiate synthesis of a new strand of DNA at the point where the primer is hybridized to the template;
  • the four deoxynucleotide triphosphates (dATP, dTTP, dCTP, and dGTP) to extend the DNA strand;
  • one labeled deoxynucleotide triphosphate (using a radioactive element or dye); and
  • one di deoxynucleotide triphosphate, which terminates the growing chain wherever it is incorporated. Tube A has di dATP, tube C has di dCTP, etc.

For example, in the A reaction tube the ratio of the dATP to di dATP is adjusted so that each tube will have a collection of DNA fragments with a di dATP incorporated for each adenine position on the template DNA fragments. The fragments of varying length are then separated by electrophoresis (1) and the positions of the nucleotides analyzed to determine sequence. The fragments are separated on the basis of size, with  the shorter fragments moving faster and appearing at the bottom of the gel. Sequence is read from bottom to top (2). (Source: see Fig. 11.)

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