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BIOTECHNOLOGY
Sanger Sequencing
BIOTECH 101
BACKGROUNDER
Page 1 of 1
SANGER SEQUENCING
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
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
Figure 1A (left): Standard gel electrophoresis.
Figure 1B (right): Sequencing using
fluorophore to each nucleotide, sequencing can be performed
fluorophores.
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!
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