DNA Sequencing
In section 6.5, the forensic use of mtDNA is described. As the differences between the
mtDNA of individuals are mainly point mutations, they are usually revealed by
applying the technique of DNA sequencing. This is another major technique of
molecular genetics with forensic applications. DNA sequencing allows the precise
base-sequence (Section 6.2.1) of a section of DNA to be determined. Mutations and
different alleles are initially characterised using DNA sequencing. It is capable of
revealing all types of mutation.
The method of DNA sequencing, illustrated in Figure A, commonly used (the dideoxy
or Sanger-Coulson method) relies on DNA synthesis using the enzyme DNA
polymerase which adds nucleotides (DNA pre-cursors, Figure 6.2a) to the primer.
DNA synthesised is complementary to the DNA of interest. Figure A shows the
primer bound in front of the sequence to be determined.
Here, the sequence is shown for clarity, normally it would be unknown. The reaction
contains the precursors of DNA, nucleotides from which the new DNA will be
synthesised. In addition to the normal deoxynucleotides (dNTPs) in the reaction are
modified versions called dideoxynucleotides (ddNTPs) which are labelled with
fluorescent tags. The DNA polymerase enzyme adds nucleotides one at a time
starting from the primer. The nucleotide added is complementary to that on the strand
being sequenced. In Figure 6.9, the first nucleotide to be added is complementary to
the base C, i.e. G. If a normal dNTP containing G is added to the growing DNA chain
then the extension can continue after this. If however a ddNTP containing G is added
it stops further DNA synthesis on that molecule and it labels the DNA strand with a
particular fluorescent colour tag. Each different ddNTP is labelled with a different
colour according to which base (A, C, G, or T) it carries. Hence, a molecule which
ends in an A will fluoresce, say, red; a molecule ending with a G will fluoresce, say,
green etc. Every time a nucleotide is added to the end of the growing DNA chain it
can be a dNTP or a ddNTP. If it is a ddNTP the reaction stops and the DNA is
labelled with the fluorescent tag of the colour of the last base added. The products of
the sequencing reaction are a set of DNA molecules which differ in length by single
bases and which are fluorescently labeled.
The products of the reaction are separated by gel electrophoresis, a laser beam causes
the molecules to fluoresce, and they are detected and presented as an
electropherogram. Each peak represents a molecule of a particular size, the next peak
is one base longer, and the colour of the peak indicates the base at the end of the
molecule. From this the sequence of the target DNA can simply be read.
This is a complex but important technique, it is not used routinely in forensic science
but its main use is for mitochondrial DNA analysis (Section 6.5).
The Animation Library at the Dolan Learning Centre at the Cold Spring Harbour
Laboratory at http://www.dnalc.org/resources/BiologyAnimationLibrary.htm contains
an animation called DNA Sequencing – this is an excellent animation showing the
Sanger –Coulson method.