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DNA Sequencing |
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The Sanger method being used today was pioneered by Fred Sanger in the 1970s. It is also known as the Dideoxy method, because a small amount of dideoxynucleotides are mixed with normal deoxynucleotides during sequencing. The dideoxynucleotides lack both 2' and 3' hydroxyl groups, while the deoxynucleotides lack only the 2' hydroxyl group. As discussed in Chapter 3, the 3' hydroxyl group is essential for forming the phosphodiester bond that connects two nucleotides. Therefore, the dideoxynucleotide will be the terminator of a polynucleotide chain since it lacks the essential 3' hydroxyl group. The Sanger method may sequence a DNA fragment containing up to 500 nucleotides. For large scale sequencing (such as the entire human genome), a strategy known as the shotgun sequencing is commonly used. In this approach, the DNA molecule of interest is randomly chopped into numerous small pieces. After these small pieces are sequenced, the whole sequence is assembled by their common overlaps. Review Articles: Automation for Genomics, Part Two: Sequencers, Microarrays, and Future Trends - Genome Research, 2000. Pyrosequencing
Sheds Light on DNA Sequencing - From Genome Research, 2001.
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