tadem repeats PCR
czhu at changbioscience.com
Wed Jul 30 01:26:17 EST 2003
I've been testing a software for PCR amplification through repeat
regions (http://www.changbioscience.com/primo/primohl.html). The
software will pick primers with minimum binding to the repeats, but
you are raising a different question: product that is not fully
extended serve as primer in the next round. In theory, you can avoid
this by making sure the specific primers are always in excess (higher
primer concentration and lower initial template concentration) and
products are fully extended (more enzyme, longer extension time?).
May I ask for the template and primer sequences?
khmerb at stanford.ed wrote in message news:<20030720224119.3228.qmail at ww02.hostica.com>...
> I have been trying to do PCR off of a template that contains 8
tandem repeats, ~220bp in length. I have problems with enzyme
slippage along my template and any product that is not fully extended
in one round can tehn serve as a primer in the next round and can
non-specifically anneal anwhere along the repetitive template. I have
experimented with various polymerases and see that some work better
than others as well as more starting material and lower numbers of
cycles but I still see products that have anywhere from 1-8 repeats.
any advice or references of people doing this would be really helpful.
I have looked at some references by people looking at satellite DNA,
but they seem to use primers specific to the repeats in order to
amplify outside regions instead of amplifying through the repetitive
region. Thanks in advance for any help!
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