Template-PCR Geno-Screening problem

Brendan Scott Brendan.Scott at anu.edu.au
Wed Nov 7 02:24:00 EST 2001


Hi!  Does anyone have any suggestions to explain a problem I am having with
a genotype screening PCR?

Basicly I have two different screening methods for genotypes of two
different genes.  One is a 2 primer PCR that amplifies alternate products
of c.140bp and/or 170 bps in size.  The other is a 3 primer PCR (two
alternate forward primers and one reverse primer) amplifying alternate
products c.1400bps and/or 1800bps in size.  There are virtually no other
differences.  I am otherwise using the same templates, the same reagents
(except very slightly different buffers), and I set up the reactions the
same way - though with slightly different concentrations established from
initial optimisation.

The 140-170 PCR is working fine.  But the 1400-1800 is erratic.  Some
samples will work fine for the 1400-1800, but mostly one or some (or all)
of the replicates will fail;  frequently amplification is poor for some
templates, but not for others, despite the template concentration being the
same (this includes my positive controls).  I keep tinkering around the
edges with it, readjusting primer concentration and template concentration
until my optimisation reactions work perfectly - but when I go and do the
large-scale screening PCRs using the new conditions, I get erratic results
again.  Both PCRs are producing spurious bands in addition to the useful
products, mostly (I assume) from amplifying the endogenous DNA from the
home-grown polymerase mix we use, but this hasn't interfered at all with
the results from the 140-170 PCR.

Any ideas appreciated greatly!  If its the different product sizes, for
example, why??

Cheers,
Scotty

++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++
Brendan Scott BSc. PGDip(Hons) UQld
Human Genetics Group
Division of Molecular Medicine
John Curtin School of Medical Research
Australian National University
CANBERRA ACT 0200
Australia
Ph: 02 6125 0417/3725  Fax:  02 6125 4712
0418 871 514
** From 1st January, 2001, ANU phone prefixes change from 6249 to 6125
++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++


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