Dear evoldir members:

I have several answers for my question, so thanks a lot for your time folks!

To be more precise about the low height peak in one of the locus of
microsatellite I am dealing with, I should say that the low peaks were at a
particular locus, no multiplex PCR was done, and the fluorochrome used for
the marked primer was FAM.

Anyway, here you have some of the comments:

Have you tried increasing the concentration of flourochrome tagged pcr
product that is run on the sequencer? This is the simplest way of increasing
peak height. Hope this helps

Cheers

Alex

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do you this problem for each of you samples? Or only in some individuals,
especially if they are form the same localities? It could be that there is a
mutation in the primer region causing a lower yield of the PCR reaction.
This could be shown by sequencing with primers outside you current fragment.

You could try to run the PCR one or a few cycles longer to increase yield
(and hope that you don't get too much unspecific products after that...).

Do you dilute the PCR product before loading it into the sequencer? Dilute
less, and see what happens.

Is the occurs in a multiplex reaction with other primers? Maybe these other
mSATs outperform you locus under consideration. You can tune the multiplex
PCR reaction by varying the amount of primer for each locus. E.g. for the
'problematic' one you can increase the amount of primer; for ones that work
really weel, you can decrease it.

Good luck,
Cheers,

Robert

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Are you using primers developed for your species? If you are using
heterologous primers (developed for another species), you might be
having the problem of a NULL allele.  That happens when there are
mutations on the annealing site of your species so that the primer anneals
imperfectly. Depending on the number and position of the mismatches, the
PCR is potentially less efficient. The mutation on the annealing site
could be specific to one allele.  Thus this particular allele amplify
"sub-optimally" (low peak) or it doesn't amplify at all.

You could run CERVUS (
http://www.fieldgenetics.com/pages/aboutCervus_Overview.jsp)
to test for null alleles in your loci.

Best,
Paulo.

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I had a similar problem with one of my loci too.  Is this in a multiplex?  I
found when I was genotyping with my loci in a multiplex that one of the loci
only produced very low peak heights when all the others were much higher.
You could go through the hassle of redesigning primers etc, but if they are
all working, the simplest solution may be to increase the concentration of
the primer that's producing the lower peak heights.

Good luck!

     Javier

jsmf77@gmail.com