SSCP

bagley at FAUNA.UCDAVIS.EDU bagley at FAUNA.UCDAVIS.EDU
Sat Jan 13 14:51:24 EST 1996


In article <scottf19-1301960117150001 at dial108.pcix.com>, scottf19 at nfi.com (Scot Federman) writes:
>In article <1996Jan11.114105.1 at leif>, ynakao at kean.ucs.mun.ca wrote:
>
>> Hello, Netters,
>> 
>>     Would you please answer my stupid question? In PCR-SSCP, there are 3 - 
>> 6 positive band on the film. Which are the real positive? I think there 
>> must be two bands, one from sense and another from anti-sense if there is 
>> no mutation. From where do other bands come from???
>> 
>> Thanks in advance,
>> 
>> Yoshi
>
>Yoshi,
>
>      I had the same question when I was running PCR-SSCP.  One possible
>explanation I came up with was that they are an artifact of PCR with Taq. 
>T-overhangs created on the 3' end are not created with 100% efficiency,
>and I thought that this might be an explanation.  However, this can only
>account for up to 4 bands, and not 6.  I don't know what else can be the
>cause.  Maybe someone else knows where these mystery bands come from?
>
Well I don't know for sure where the bands come from either, but here is some
more data.  The intensity of the different  bands changes with formamide
concentration.  Adding 0.6X formamide  to the PCR sample enhances the higher
set of bands.  Use of 2-3X formamide seems to bring up 4 bands in homozygotes
and use of higher amounts of formamide seems to enhance the lower bands.
Could the excess bands you see be due to renaturation?  I am thinking that the
pattern  I see is  due to renaturation of excess primer under low formamide
conditions.  Thus, I would expect to see 4 bands in homos and 8 bands in hets
at intermediate formamide  concentrations.  You could get an additional band if
double-stranded template remains.  You'd get less if some bands overlap.  I
personally use 0.6X formamide, giving me a strong signal from the higher set of
bands and a very weak signal from the lower set.

Anybody buy this theory?
Mark.





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