Detecting two alleles

John Ladasky jladasky at pmgm.Stanford.EDU
Mon Nov 23 14:40:41 EST 1998


In article <73bua7$3pn at panix3.panix.com>, 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?
>
>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
>complications.
>
>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?

Hi, Ian.

	I'll weigh in with my opinion -- there is *no* practical limit to
the number of clones you should sequence.  I'll give you examples from 
our work here in the Parham lab with class I MHC.  We read both alleles
of each class I gene from RT-PCR products cloned into plasmids, with the
goal of getting each allele in triplicate.  Ideally, this takes only six
clones per locus, but more practically, we sequence twelve.  (Having an
automated fluorsecent sequencer all to ourselves sure helps.)

	Sometimes, twelve clones are not enough.  In fact, with a few of
the samples, all twelve of the clones are from a single allele.  This 
may be due to the fact that some alleles express little mRNA (often dis-
countable by looking at a protein gel), or that the allele has an unde-
tected polymorphism that sits right under a PCR primer.  Taking more
clones can help -- we've occasionally had to go up to 48 clones in order
to read a troublesome allele.  You could also try moving your primers.

	If you're having trouble reading sequences and you're not sure
of a mutation call, you could sequence the gene from the parent cell 
line as a control.

	Can you test your hypothesis in a completely different way?  Do
you know on what chromosome your putative dominant negative gene is
located?  I know it's a project, but you could make somatic cell hybrids,
and isolate several lines containing your chromosome of interest.  If
you have a dominant negative mutant, half of your cells that contain
the chromosome on which your gene is located should express the mutant
phenotype, and the other half should not.

>-- 
>Ian A. York        iayork at panix.com      http://www.panix.com/~iayork
>"I'm more not Ian York than you'll ever not be!" --James "Kibo" Parry

	A Kibo quote?  Your brush with fame, eh? :^)

-- 
Rainforest laid low.
"Wake up and smell the ozone,"
Says man with chainsaw.					- John Ladasky



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