I am doing research on the occurrence and polymorphism of microsatellites in co
nifers. I haven't found a lot of polymorphisms in GT/CA repeats or CT/GA. Howev
er, I have an AT/AT microsatellite that shows a high rate of variability. There
are a few problems with it though.
1) I originally isolated it as a CA-repeat that was followed by a stretch of TA
s. I amplified it from the genomic DNA and found that all the products I obtain
ed are shorter than the original. Then I cloned the PCR amplified fragments and
sequenced them. To my surprise, the CA/GT microsatellite was not present. What
was left was a stretch of TAs. Am I amplifying a microsatellite family and is
the CA/GT + TA member that I cloned only a minority???? I am not so sure since
I haven't obtained more than 2 alleles from a single tree up to now.
2) When I PCR the plasmids containing the different AT stretches, I obtain two
or more distinct bands as a result. Instability of the TA repeat in the plasmid
/bacterial host is almost ruled out. I get a nice sequencing gel, upon isolatio
n of plasmid from single colonies I always get two identical bands in several i
solates, I get distinct bands and not smears. I assume that something is going
on during the PCR amplification of the TA repeats. It cannot be a random proces
s, because I get distinct bands, not a smear. What can it be????? Has anyone se
en such results before?
Any ideas on what might be going on are welcome. If you need more info, please
E-mail meor if it is relevant to the whole net, post it.
Dept of Forest Environment Science, Univ of Maine
5755 Nutting Hall, Orono, ME 04473
207 581 2819
IO01072 at maine.maine.edu