Sanger Sequencing

Education Services / L’équipe des services d’éducation
14 September 2012

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Sequencing DNA is a way to determine the order of the four nucleotides along a strand of DNA. Sequencing DNA has become vital to the fields of basic research, biotechnology, forensics and medical diagnostics. In the late 1970’s, biology saw the first two methods to sequence DNA. One method, Maxam-Gilbert sequencing, uses chemicals to break up DNA in order to determine its sequence. Frederick Sanger developed the second method for which he and Maxam and Gilbert were awarded the Nobel Prize. The Sanger method is based on the idea that by making copies of DNA strands and monitoring what nucleotides are added, one by one, the sequence of nucleotides is found. Both methods revolutionized biology; however, Sanger sequencing has become the method of choice, and is now routine in most biological laboratories and is the focus of this article.

The Sanger method: How it works 

Figure 1A (left)
standard gel electrophoresis.
Figure 1B (right)
Sequencing using fluorophores.
To start, you need a piece of DNA which you want to sequence. Next, you add a DNA priming sequence, the four nucleotides and an enzyme called DNA polymerase which incorporates new nucleotide bases making a new piece of DNA which is a copy of the original piece. In Sanger’s original method, four different sequencing reactions are performed. Each reaction contains a different modified nucleotide that once incorporated results in DNA chain termination, which leads to the identity of the final base.  These samples are then subjected to gel electrophoresis, which is a method to separate the new DNA pieces on a gel base using an electric current. The DNA pieces can then be seen using x-rays or UV light. To read the gel, you start at the bottom and look at the bands (black dash marks) to determine the sequencing of the DNA fragment.  Each lane (column) of the gel has a different base at the end, and so each band on the gel represents a piece of DNA with that base at the end (Figure 1A). 

  

Further advances in this method have incorporated the use of fluorophores, which are small chemical compounds that give off coloured light. By adding a different coloured fluorophore to each nucleotide, sequencing can be performed in a single reaction mixture with a single gel lane to resolve the DNA pieces, using the colour of the band to indicate the base at the end of the DNA fragment (Figure 1B).  This innovation in automation (computers and machines do the work) has enabled many more pieces of DNA to be sequenced. Sanger sequencing has enabled scientists to sequencing a diverse range of organisms, from bacteria to humans. Recently, DNA sequencing technologies on a previously unheard of scale have been developed. These machines are capable of sequencing a person’s DNA (all 3 billion bases) in a few days, compared with the years it took previously! 

Education Services / L’équipe des services d’éducation

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