Detection of CA repeat polymorphisms.
"Dr." Shoumo Bhattacharya B1 061
sbhattac at mrc-crc.ac.uk
Thu Oct 24 15:34:33 EST 1991
In <9110240858.AA21285 at genbank.bio.net> Roger C Green writes:
I am beginning to use polymorphic dinucleotide repeats (CA
repeats) as genetic markers. I understand there are some technical
problems which may occur. I am confused by the proliferation of
methods for detection of the PCR products, e.g.--
1. Ethidium staining of ds DNA on agarose gels
2. Using labelled dNTP or labelled primers followed by use of
denaturing (urea +/- formamide) gels, then ARG.
3. Running ss DNA on non-denaturing gels which will separate
the CA strand from the TG strand.
I would appreciate any technical tips that would assist in
the detection of polymorphic CA repeat sequences.
The following may be useful:
1. The polymorphisms are best detected using a labelled primer: this
labels only one strand getting rid of irritating doublets and non-specific
amplified product is not visualised. Search the EMBL database with your primer
sequence to ensure specificity. Most polymorphic repetitive sequences are close to
Alu repeats, this makes finding a specific primer difficult. Obviously, label the primer
that is more likely to be unique.
A very small amount of 32P labelled primer e.g. 2-3 ng
is needed per 25 ul reaction but must be to a high specific activity. We add unlabelled primers
as well eg. 20 - 60 ng of each primer.
2. The products are best visualised on 4 -6% sequencing (6M urea- 4-6%PAGE) gels. In case of
'laddered' products reduce the no. of PCR cycles from 30 to 25 or to 20. We have
found that Stratagene's PerfectMatch helps.
Hope this is useful.
MRC Clinical Research Centre
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