Detecting two alleles

[Chris Boyd]

Ian A. York (iayork at panix.com) wrote:
: I have some data which is consistent with my mutant cell expressing a
: dominant negative allele, though there are also many other possible
: explanations.  I've cloned and sequenced the relevant gene, and it has no
: abnormalities.  But of course, I may only be seeing a normal allele in the
: sequencing.  So what I want to know is:  How far do you have to go to be
: comfortable in saying that there is only one allele expressed?
(snip)

I think you have a difficult problem, with no obvious quick solutions.
I don't know if it's been tried, but what about this approach, turning
what John Ladasky cited as a possible problem into a virtue:

If you can identify any sequence variation (DNA polymorphism(s))
between the two alleles from the wt cell line progenitor of your mutant
(by sequencing a range of RT-PCR products) then you can design primer
pairs that are virtually allele-specific by arranging for one of the
primers in each pair to vary at its 3' end according to the
polymorphism(s). Allele-specific semi-quantitative RT-PCR on the mutant
cell-line will then help you decide if there is gross imbalance in
allele expression (as long as the expression levels ratio does not
exceed the threshold  -- 1000:1, we're told -- at which amplification
of the highly-expressed allele by mispriming starts to become a
significant issue).

With luck, you will nevertheless be able to amplify both alleles and
look for mutations by sequencing the respective products, but you'd
need a lot more luck to find enough sufficiently well spaced
polymorphisms to allow a complete examination of both alleles by
RT-PCR -- unless you're an expert in long range PCR.

Best wishes,
-- 
Chris Boyd                      | from (but not \  MRC Human Genetics Unit
Christopher.Boyd at hgu.mrc.ac.uk  | on behalf of) /      Crewe Rd, Edinburgh
http://www.hgu.mrc.ac.uk/Users/Christopher.Boyd          EH4 2XU, SCOTLAND