It is also possible to undertake nucleotide sequencing from double-stranded
molecules such as plasmid cloning vectors and PCR amplicons directly. The double-
stranded DNA must be denatured prior to annealing with primer. In the case of
plasmids an alkaline denaturation step is sufficient; however, for amplicons this is
more problematic and a focus of much research. Unlike plasmids amplicons are short
and reanneal rapidly, therefore preventing the reannealing process or biasing the
amplification towards one strand by using a primer ratio of 100 : 1 overcomes this
problem to a certain extent. Denaturants such as formamide or DMSO have also been
used with some success in preventing the reannealing of PCR strands following their
separation.
It is possible to physically separate and retain one PCR strand by incorporating a
molecule such as biotin into one of the primers. Following PCR one strand with an
affinity molecule may be removed by affinity chromatography with strepavidin,
leaving the complementary PCR strand. This affinity purification provides single-
stranded DNA derived from the PCR amplicon and although it is somewhat time-
consuming does provide high-quality single-stranded DNA for sequencing.5.11.4 PCR cycle sequencing
One of the most useful methods of sequencing PCR amplicons is termedPCR cycle
sequencing. This is not strictly a PCR since it involves linear amplification with a
single primer. Approximately 20 cycles of denaturation, annealing and extension take
place. Radiolabelled or fluorescent-labelled dideoxynucleotides are then introduced in
the final stages of the reaction to generate the chain-terminated extension products
(Fig. 5.39). Automated direct PCR sequencing is increasingly being refined allowing
greater lengths of DNA to be analysed in one sequencing run and provides a very
rapid means of analysing DNA sequences.5.11.5 Automated fluorescent DNA sequencing
Advances in fluorescent dye terminator and labelling chemistry have led to the
development of high-throughput automated sequencing techniques. Essentially most
systems involve the use of dideoxynucleotides labelled with different fluorochromes.
Thus the label is incorporated into the ddNTP and this is used to carry out chain
termination as in the standard reaction indicated in Section 5.11.1. The advantage of
this modification is that since a different label is incorporated with each ddNTP it is
unnecessary to perform four separate reactions. Therefore the four chain-terminated
products are run on the same track of a denaturing electrophoresis gel. Each product
with its base-specific dye is excited by a laser and the dye then emits light at its
characteristic wavelength. A diffraction grating separates the emissions which are
detected by a charge-coupled device (CCD) and the sequence is interpreted by a
computer. The advantages of the technique include real-time detection of the sequence.
In addition the lengths of sequence that may be analysed are in excess of 500 bp
(Fig. 5.40). Capillary electrophoresis is increasingly being used for the detection of191 5.11 Nucleotide sequencing of DNA