Daniel GAUTHERET wrote:
>> I bet this issue has already been debated, but I can't
> find any relevant data in the litterature. So here it is:
> Direct sequencing of PCR products often yields sequences
> with many inaccuracies (Ns). Can we say that the position
> of these undefined nucleotides is related to actual genetic
> variation at these particular sites?
>> Thank you.
>> Daniel Gautheret
> CNRS - IGS - 31 ch. Joseph Aiguier - 13 402
> Marseille Cedex 20 - France
> Tel: (33) (0)4 91 16 45 48 - Fax: (33) (0)4 91 16 45 49
The answer depends on the situation. people have used
PCR of heterogeneous templates, followed by direct sequencing
of the products, to get data on the variable sites in the
heterogeneous templates. See for example Leitner et al
BioTechniques 15: 120-126 (1993) and Leitner et al Virology
209: 136-146 (1995) which used the technique to study HIV
sequence diversity in AIDS patients.
Others in AIDS research have used this technique to
study the ratio of drug-resistant to drug-sensitive virions
in the blood during anti-retroviral treatment.
There is a big difference between carefully controlled
conditions in which such heterogeneity of template can
be measured, and just assuming that any site that cannot
be read by an automated sequencer was heterogeneous in the
template. In both automated sequencing and in conventional
35-S or 32-P labelled dideoxy sequencing, there are very often
sites which are difficult to call even when the template
for sequencing was homogeneous, grown from a single cloned
plasmid in E. coli. PCR only makes things worse.
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