- All commonly used methods of nucleotide sequencing have these features in common:
- A population of oligo- and polynucleotides is analyzed.
- All analyzed oligo- or polynucleotides have one end in common (the 5' end, left in the diagram).
- The other end (in this case, the 3' end) is base-specific.
- The oligo- and polynucleotide population is separated according to chain length by polyacrylamide gel electrophoresis. Such electrophoresis can take place in slab gels or in capillaries.
- In the enzymatic method (Sanger):
- The common end is provided by an oligonucleotide primer complementary to a sequence in the template DNA.
- The oligonucleotides are elongated by a DNA polymerase using 2' deoxynucleoside triphosphate (dNTP) substrates.
- The base-specific end is obtained by doping the polymerase reaction with a 2', 3' dideoxyNTP.
- Synthesis products can be distinguished from template and other DNAs by incorporation of radioactive or fluorescent labels.
- In the chemical method (Maxam & Gilbert):
- The common end is usually generated by restriction endonuclease digestion.
- The end is labeled, often with radioactivity.
- The base-specific ends are generated by chemical cleavage reactions. They are not carried out to completion, allowing individual molecules to be cleaved at different distances from the end
- In one commonly used variation, the DNA polymerase used is that from a thermostable organism so that multiple cycles of DNA synthesis, melting, and primer annealing can be used. A "Shockwave" animation of this procedure, called thermocycle sequencing is available.
- In the Sanger method, the label can be incorporated into the primer or attached to the dideoxyNTP that terminates chain elongation.
- When labels are attached to the terminating dideoxyNTP, different labels can be attached to each of the four dideoxyNTPs. In that case, one reaction, incorporating all four dideoxyNTP's can be run. Distinction of which nucleotide terminated a chain of a particular length is then based on the spectral properties of the chain. This technology is patented, so that equipment from only one company allows one-tube reactions. However, others have been able to sequence one template in both directions, using differently tagged primers.
- Lengths of reading of sequence from one primer are typically between 300 and 500 nt. Some protocols allow reading up to about 1400 nt.
- In one company's application, the fluorescent dyes absorb infrared radiation.
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This is page 427 of Molecular Genetics by Ulrich Melcher, ©1997, 1998, 1999, 2000
E-mail inquiries to U. Melcher------------Last Updated: 6 September, 2000