Sequencing


DNA sequencing refers to the process by which the sequence of bases in a DNA molecule is elucidated

The most widely used method of DNA sequencing is the Sanger method, which involves using chain-terminating dideoxynucleotides


The Sanger Method

The Sanger method involves four PCR reactions:

  • Each reaction contains the four normal nucleotides plus one dideoxynucleotide stock
  • Dideoxynucleotides are modified nucleotides that prevent the addition of subsequent nucleotides and hence end a nucleotide chain when incorporated
  • As a typical PCR reaction generates over 1 billion DNA molecules, each of the four PCR reactions will generate all of the possible terminating fragments for that particular base
  • Dideoxynucleotides are fluorescently labelled and so, when the four PCR samples are run through gel electrophoresis, the sequence of the fragments can be detected by a laser and represented via a chromatogram


DNA Sequencing via the Sanger Method


Human Genome Project

The Human Genome Project (HGP) was an international cooperative venture established to sequence the 3 billion base pair (~25,000 genes) in the human genome 

The outcomes of this project include:

  • Mapping:  We now know the number, location and basic sequence of human genes
  • Screening:  This has allowed for the production of specific gene probes to detect sufferers and carriers of genetic disease conditions
  • Medicine:  With the discovery of new proteins and their functions, we can develop improved treatments (pharmacogenetics and rational drug design)
  • Ancestry:  It will give us improved insight into the origins, evolution and historical migratory patterns of humans 


With the completion of the Human Genome Project in 2003, researcher have begun to sequence the genomes of several non-human organisms