DNA Sequencing and Analysis
DNA Sequencing
- DNA sequencing is the process by which the order of base pairs is determined.
- Frederick Sanger was the first to map the entire genome of an organism; PhiX174, a bacteriophage, using radioactive tracers and gel electrophoresis.
- Since then the process has become automated, and in the 70s, Sanger was able to process up to 800 bases at a time. Fluorescent tags are now used instead of radioactive ones.
- The human genome project was set up in 1990, as an international project to map the entire human genome. In 2003, 2 years ahead of schedule and under budget, the entire human genome had been sequenced.
- Sequencing requires the DNA to be split into fragments, and then each fragment is subsequently sequenced.
- Length of the fragment is determined by where a terminator base is used, which stops the polymerisation of any other bases.
- The terminator bases have fluorescent tags for ease of use.
- The capillary method of DNA sequencing is detailed below.
Next-Generation Sequence
- Instead of gel or capillaries, sequencing now occurs on a slide called a flow cell, allowing millions of DNA fragments to be replicated in-situ in PCR.
- As many DNA clusters are sequenced at once, the process is known a 'massively parallel sequencing'.
- Massively Parallel Sequencing is being constantly refined, and integrated with state-of-the-art computers.
- High throughput methods are extremely fast and efficient, sequencing bacterium genomes in less than 24 hours!