PCR ghost bands

Tracy Aquilla aquilla at salus.med.uvm.edu
Fri Jun 24 17:09:56 EST 1994


In Article <2uca3i$lp9 at apakabar.cc.columbia.edu>,
pcj1 at konichiwa.cc.columbia.edu (Pierre Jelenc) wrote:
>
>Very often (3/4 of the time), I see a PCR product shorter than the correct
>one. This extra band is sometimes much stronger than the correct one, and
>the difference in size between correct and incorrect is pretty much random
>from primer pair to primer pair. I.e. it is not a fixed number of
>nucleotides, nor is it proportional to the size of the correct fragment. 
>
>This extra band is _always_ shorter, typically by 10-30 nucleotides, and 
>it is absolutely reproducible with a given primer pair, regardless of PCR 
>conditions (Mg concentration, annealing temperature, elongation time, 
>number of cycles between 20 and 40, etc). It is readily visible in 
>denaturing acrylamide gels, and seems to be there in concentrated agarose 
>gels as well, although the lower resolution in the latter makes it 
>difficult to be sure.
>
>It does not appear to be a gel artifact, because I have run side by side 
>reactions with and without extra bands, nor to be simply due to the 
>reaction conditions, because I run several reactions from the same premix 
>of reagents, added to the template and primers that have been premelted 
>and annealed under wax.
>
>I am at a complete loss.
>
>Pierre
>
>Pierre Jelenc                        pcj1 at columbia.edu 
>                                    Columbia University, New York

Hi Pierre,
    I suspect that your 'ghost bands' are due to one of your primers
annealing at more than one site (ie: internal or external) on the template
DNA. If your primers have a high GC content, they may be false-priming with
a relatively high percentage of mismatch. How much did you try to raise the
annealing temp? You might try making it even higher, a few degrees at a
time, until the ghost (or all) bands go away. Southern analysis will tell
you if the extra bands contain a portion of the region you want to amplify.
Sequencing the extra bands will give more precise information. This is
especially common if there are many repeated sequences in your template DNA,
and the problem can be even worse if you are using genomic DNA as the
template.  You can check this by doing a dot-plot of the template vs.
itself. You could also search both template strands for the sense and
antisense sequences of the primers, allowing for some significant
mismatches. There are also PC programs that will tell you what sizes of
amplification products to expect when using a given template and primer pair
(ie: Amplify for the Mac), and these can often identify the extra bands you
are seeing on the gel. Hope this helps. Good luck.
                                                Tracy
 
Tracy Aquilla, Post-doctoral Research Associate
Department of Molecular Physiology and Biophysics
University of Vermont, College of Medicine
aquilla at salus.med.uvm.edu



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