In article <Bz1w8o.ExA at ncifcrf.gov>, oberste at fcs260c.ncifcrf.gov (Oberste) writes:
> In article <1g71l0INN4h8 at huon.itd.adelaide.edu.au> ajeffrie at waite.adelaide.edu.au (Alex Jeffries) writes:
>>>> Fact1: Virus "species" are best described as a population of minor
>>sequence varients. The term quasispecies is usually used here.
>>>> Fact2: PCR amplification has an error rate associated with it. The rate
>>is dependent on the type of polymerase used and other factors such as Mg-2+
>>concentration and the number of cycles.
>>>> Question1: When sequencing a virus, where the clones have been
>>generated by PCR, how many instances of a particular sequence would have to be
>>seen before people here would be happy that they have the "correct" sequence and
>>not a PCR artifact.
>>>> Since a PCR-induced base change is propagated through subsequent cycles,
> it is important to collect and compare sequence from independent PCR
> amplifications. It would be extremely unlikely to see the same artefact
> in independent amplifications.
>> A problem that we have been thinking about is that, in the absence of cloning,
> mutations may be masked or seen as ambiguities, since the starting virus
> population is heterogeneous. If we go with cloning before sequencing, there
> are extra steps and lots of clones to screen if we are looking for multiple
> variants in the same population. Anyone have any novel approaches to these
> problems?
>> -Steve
>>> --
> Steve Oberste Internet: oberste at ncifcrf.gov> LCMS, PRI, NCI-FCRDC
> PO Box B "Never put off until tomorrow that which
> Frederick, MD 21702-1201 you can do the day after tomorrow"
You might be interested in direct sequencing, that way you don't have to worry
so much about Taq- artefacts, just sequence the bulk quasispecies
PCR-fragment. If the misincorportion happens in the first cycle of PCR, then
it's intensity on the sequencing film will be only one fourth of the other
bands, IF you are using manganese together with T7 pol. After the first PCR
cycle, 4 strands of DNA will be present. Only after the second cycle, one of
one of these will have established the mutation on both strands. Have a look at
the following ref for a method:
Salminen M. Rapid and simple solid phase direct sequencing of in vivo HIV-1
quasispecies. AIDS Research and Human Retroviruses 1992;8:1733-42.
------------
Orig. templ.: ------------
PCR 1 ------------ ----------
-----x------ ----------
PCR 2 -----x----- --------- -------- --------
-----x----- --------- -------- --------
an so on...
You see my point? If there is a TRUE ambiguity of equal proportions of mixed
bases in the original template, this will be seen as equal intensities of bands
in the gel.
Of course, even the above 1/4th band intensity is very unlikely, as you seldom
start with one copy of template. So, forget about hundreds of parallel clones,
go for direct sequencing!!!
Mika Salminen
msalminen at nphi.fi (Internet)
msalminen at finnphi (Bitnet)
P.S. If you are interested, I can give you my very well working trimmed dirseq
protocol.
