Heteroduplex Mobility Assay
mc at u.washington.edu
Thu Feb 29 20:52:08 EST 1996
In article <4h1lc0$72m at news.univ-angers.fr>, gbbmv
<gbbmvea917 at univ-angers.fr> wrote:
> 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?
We do a great deal of HMAs in our lab. Perhaps the following suggestions
will be of some help to you.
1) Single base _frame shifts_ (insertions or deletions) are resolvable,
but NOT single base substitutions (unless the following is true).
2) Base substitutions are resolvable if they are in (or, of course,
greater than) the range of 1-2% of the total.
3) The protocol you are following sounds fine, however, I wonder if you
place the samples on ice immediately from the 98oC bath? A very rapid
temperature drop is critical for the formation of heteroduplexes.
4) Your annealing buffer is fine, I wouldn't change it; we use that buffer
5) Perhaps you can try longer sequences or sequences from regions of the
ITS that are not so well conserved.
"The two most common things in the universe are hydrogen and stupidity" Harlan Ellison
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