PCR Problem

Grant Morahan morahan at wehi.edu.au
Mon Jul 17 22:13:55 EST 1995


   In reply to your post about PCR problems, it sounds to me like your
problem is that one of your strains has a mismatch with the 3' end of one
of your oligos. If that is the case, you need to do one of 2 things: use
another marker; or derive a new oligo from the sequence of the MIT
microsatellite you are interested in (assuming that there will be no
further changes). Obviously the former is preferable.

   Also, if you are doing LOH analyses, you should include F1 (nontumour)
DNA - in addition to DNA from each parental strain - in each run. That
will provide a check for preferential amplification of 1 allele in the
presence of the other, whioch can happen sometimes.

    Regards, Grant

In article <3tt7k3$o0i at news.unimelb.EDU.AU>, julia
<juliam at ariel.ucs.unimelb.edu.au> wrote:

> Hi
> My name is Julia.  I am undertaking my PhD at the Peter Maccallum 
> Cancer Institute in Melbourne Australia.  I am using PCR to amplify
>  mouse DNA using a variety of SSLP's obtained from the Whitehead
>  Institute.  I am writing for help because all of a sudden my PCR has
>  gone from working beautifully to half working to not working at all. 
Here is my procedure.  Please if anyone can give me any clues that would
be great.  
> Because I am dealing with many tumour DNA samples at once,  the 
> procedure I use is to make up a PCR mix.  This contains everything
>  except the DNA.  That is the primers, both unlabelled and labelled,
>  MgCl2, dNTP's, taq polymerase, polymerase buffer and H2O.  I add the
>  DNA to the PCR mix.  I then place the samples into the PCR machine 
> and do a 5 min. denature at 94C before starting the amplification 
> cycles.  Usually what I see on a PAGE/formamide gel are 2 bands per
>  DNA sample since the DNA was isolated from heterozygous mice. 
>  Occassionally I will see loss of heterozygosity which is what I am
> looking for.  Now what I am seeing on these gels is one band only. 
>  Bizarre! It appears that the primers are preferentially annealing to 
> and amplifying one allele.  However,  when I run the 2 genomic DNA 
> controls I get amplification of one of them, the same one that
> amplified in the tumour  DNA samples but there is no amplification of
>  the other DNA control. What am I to make of this? Has something
>  happened to the DNA? Should I try a hot start? I await your reply. 
>  Thanks.

Grant Morahan, Ph.D.
The Walter and Eliza Hall Institute of Medical Research
Parkville, Victoria

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