Detecting two alleles

Ian A. York iayork at
Mon Nov 23 10:17:27 EST 1998

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?

I've sequenced bulk PCR products, and three individual clones.  The PCR
doesn't give me super-quality sequence, so there are a couple of slightly
fuzzy areas, and I'm also not convinced I would see an ambiguity as
genuine rather than PCR/sequencing effect.   

With the clones, there are a couple of difference which are fairly clearly
PCR artifacts (non-coding change, inconsistent with the PCR sequencing).
So both the clone approach and the bulk PCR approach have their

I assume that the proper way to do this is to sequence multiple
independent clones until I can say there are no consistent changes.  Since
I don't want to spend infinite amounts of money or take infinite periods
of time, I want this to be the minimum number of clones.

What, then, is the minimum number of clones I should sequence?

Ian A. York        iayork at
"I'm more not Ian York than you'll ever not be!" --James "Kibo" Parry

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