Hetero-mobility Assay

Ranga Uday Kumar uday at jncasr.ac.in
Mon Aug 14 09:48:53 EST 2000


Dear Netters,

    We have been trying to standardize the hetero-mobility assay (HMA)
in our lab to identify HIV sub-types. We have been having some problems.
We follow the standard protocol as recommended by the HMA kit supplied
by the NIH HIV reagents sharing program. 5% polyacrylamide gel (29:1
ratio) is used to identify homo- and hetero-duplexes and the single
stranded DNA. The problem is that the heteroduplexes don't enter the gel
if the viral strains are phylogenetically distant (for instance
sub-types B and C). With in subtype-C (comparison among C1, C2, C3 and
C4) the hetero-duplexes enter the gel but to defferent extent.
Hetero-duplexes between C1 and C3 freely enter the gel while those
between C1 and C4 hardly do so and stay very close to the well. We
checked the concentrations of our reagents, it is indeed 5% acrylamide
gel. We polymerise the gels over-night. All the reagents are mol-bio
quality from Pharmacea and made fresh. Our power pack is local. By the
time we stop the gel homo-duplexes are close to the bottom and single
stranded DNA is half way. The gel is run for approximately 4 hrs at 250
volts at room temp. The gel is not over-heated during the run. Does any
one faced similar problem? The gel dimension are as reported by standard
protocols.
    Any suggestions and comments are appreciated.
    Is it possible (will it help?) to run a 4% gel? Or gel of 39:1 of
acrylamide to bis?
    On reviewing the literature, we do not see any consistancy the way
different species run on gels reported from different labs. In some
reports you see that the hetero-duplexes run between homo-duplexes and
single stranded DNA, while in others (using the same viral sub-types)
hetero-duplexes run after the single stranded DNA. What makes the
difference?
--
________________________________________________________
Ranga Uday Kumar Ph.D.
Faculty Fellow
Molecular Biology and Genetics Unit
Jawaharlal Nehru Center for Advanced Scientific Research
Jakkur Post, Bangalore 560 064
India
Tel: +91 (80) 846 2750 Ext. 241
Fax: +91 (80) 846 2766
________________________________________________________



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