DNA sequencing is the determination of the precise sequence of nucleotides in a sample of DNA.
The most popular method for doing this is called the dideoxy method or Sanger method (named after its inventor, Frederick Sanger, who was awarded the 1980 Nobel prize in chemistry [his second] for this achievment).
DNA is synthesized from four deoxynucleotide triphosphates. The top formula shows one of them: deoxythymidine triphosphate (dTTP). Each new nucleotide is added to the 3′ -OH group of the last nucleotide added.
|Link to discussion of DNA synthesis.|
The dideoxy method gets its name from the critical role played by synthetic nucleotides that lack the -OH at the 3′ carbon atom (red arrow). A dideoxynucleotide (dideoxythymidine triphosphate — ddTTP — is the one shown here) can be added to the growing DNA strand but when it is, chain elongation stops because there is no 3′ -OH for the next nucleotide to be attached to. For this reason, the dideoxy method is also called the chain termination method.
|The bottom formula shows the structure of azidothymidine (AZT), a drug used to treat AIDS. AZT (which is also called zidovudine) is taken up by cells where it is converted into the triphosphate. The reverse transcriptase of the human immunodeficiency virus (HIV) prefers AZT triphosphate to the normal nucleotide (dTTP). Because AZT has no 3′ -OH group, DNA synthesis by reverse transcriptase halts when AZT triphosphate is incorporated in the growing DNA strand. Fortunately, the DNA polymerases of the host cell prefer dTTP, so side effects from the drug are not so severe as might have been predicted.|
The DNA to be sequenced is prepared as a single strand.
This template DNA is supplied with
- a mixture of all four normal (deoxy) nucleotides in ample quantities
- a mixture of all four dideoxynucleotides, each present in limiting quantities and each labeled with a "tag" that fluoresces a different color:
- DNA polymerase I
Because all four normal nucleotides are present, chain elongation proceeds normally until, by chance, DNA polymerase inserts a dideoxy nucleotide (shown as colored letters) instead of the normal deoxynucleotide (shown as vertical lines). If the ratio of normal nucleotide to the dideoxy versions is high enough, some DNA strands will succeed in adding several hundred nucleotides before insertion of the dideoxy version halts the process.
At the end of the incubation period, the fragments are separated by length from longest to shortest. The resolution is so good that a difference of one nucleotide is enough to separate that strand from the next shorter and next longer strand. Each of the four dideoxynucleotides fluoresces a different color when illuminated by a laser beam and an automatic scanner provides a printout of the sequence.
Here is a representative example of a DNA sequence (455 nucleotides of the lysU gene of E. coli) which was generated by an automated sequencing device.
(The image is courtesy of Pharmacia Biotech Inc., Piscataway, NJ.)
|Animation of the procedure|
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9 November 2015