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??


Brendan Scott BSc. PGDip(Hons) UQld
Human Genetics Group
Division of Molecular Medicine
John Curtin School of Medical Research
Australian National University
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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