Heteroduplex Mobility Assay
gbbmvea917 at univ-angers.fr
Wed Feb 28 08:29:36 EST 1996
From: Philippe Simoneau at univ-angers.fr
Need help to improve the resolution of my HMA.
I use this test to rapidly screen polymorphism in the ITS region. When
heteroduplexes are formed by mixing amplified sequences from moderately
distant species the HMA work perfectly but when I use sequences from
closely related species (i.e sequences that have 97-98% identities)
I am not able to discriminate between homo- and heteroduplexes.
I know that detecting single base mutations using HMA has been reported.
I should therefore be able to separate the 2 DNA populations.
My protocol is:
-Heteroduplexes formation: mix equal vol of two separately amplified
products and 1/10 vol of annealing buffer (1M NaCl, 100mMtris, 20mMEDTA)
then heat at 98°C for 5 min. and cool on ice for 1 hour then add non
denaturing loading buffer
-running conditions: run in PA gel 5% (30:0.8 acryl:bis)in TBE (1or0.5X)
at 250V for 2hours (gels 12X12 cm & 1mm thick).
I've tried including 1M urea in the gel without success.
Does anyone have a solution?
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