>Variable PCR results - Ideas and Suggestions?
Mr. T. Weissensteiner
tweissen at hgmp.mrc.ac.uk
Fri Oct 24 11:53:47 EST 1997
Doug Jones (jonesd at telenet.net) wrote:
>>I have been having a problem with an RT-PCR for murine IL-6. After
>> optimization, which went well, I have been unable to obtain consistent
>> results, even in the same run.
>> The PCR is done in a mix using 1.5 mM MgCl, 50mM KCl, 10mM Tris, 0.1%
>> Triton X-100, .1% NP-40, .1% Gelatin, 0.25 mM each dNTP's, 5 U Taq
>> (Promega) and primers in a volume of 50 microliters. The cycle is 1
>> min at 95, 1 minute at 55, and 1.5 minutes at 72 degrees, for 35
>> cycles in a Stratagene RoboCycler.
>> The problem is that there is run-to-run inconsistency, with bands
>> either appearing or not in the spleen cDNA, and within run
>> inconsistency [...]
To the many good points that have been made I would like to add
1. Bob Steinberg wrote:
> [ ...] this sounds like sample well variability within the thermal
> cycler, either because the wells are not uniform or because the
> tubes are not making equal contact with the wells
This can be a problem especially with small sample volumes and with
PCR tubes whose tips do not fit the heat block wells as well as the
sides. Try adding oil into the heat block wells (but leave the outer
wells empty, as the oil will expand when heated, and may spill into
the cycler !)
2. Alexander Dobrovic and Duncan Clark noticed the unusually high
concentrations of Taq and dNTPs.
I remember a paper in the former "PCR Methods and Applications" claiming
that increasing both Taq and dNTPs substantially increased the PCR product
yield. I doubt, however, that this will work for all templates.
Any comments ?
3. Warren Gallin wrote
> You have too much dNTP. Anything over 200 micromolar inhibits the
> enzyme. This is not simply due to complexing Mg, it can not be
> rescued by adding more MgCl2
Is your amplfied IL6 fragment "GC-rich" (> 70%) ? Such amplicons
can be very sensitive to cation concentrations and dNTP come as salts.
Adding more salt (MgCl2), of course, won't help.
In this case, however, you should try adding some dsDNA destabilizing
additive, such as glycerol, or betaine.
Moreover, this is a good alternative to a high denaturation
temperature, as it will be much kinder to the Taq polymerase.
4.) "Allelic dropout" can cause the random failure of PCR reactions with low
Here are some examples from the extensive and growing list of
publications on PCR failure. They contain more details on the above points
(and if nothing else, show at least that Doug is not alone with
his problem :-) ).
PS Walsh, HA Erlich & R. Higuchi : Preferential amplifiaction of
alleles: mechanisms and solultions
PCR Methods and Applications (1992) 1, 241 - 250
GL Mutter & KA Boynton : PCR bias in amplification of androgen
receptor alleles, a trinucleotide repeat marker used in clonality studies.
Nucleic Acids Research (1995) 23, 1411 - 1418
Weissensteiner,T, Lanchbury,J.S. (1995) How to control preferential amplification in PCR typing: Lessons from an assay for alleles of the HLA-B locus. Europ. J. Immunogenet. 22, 100
Weissensteiner,T, Lanchbury,J.S. (1996) Strategy for controlling
preferential amplification and avoiding false negatives in PCR typing
Biotechniques 21, 1102 - 1108
Pai,C.Y., Chou,S.L., Tang,T.K., Wei,Y.H., Yang,C.H. (1996) Prevention
of false results from preferential amplification of VNTR alleles.
J. Formosan Med. Assoc. 95, 69 - 72
Rosner,A., Maslenin,L., Spiegel,S. (1997) The use of short and long PCR
products for improved detection of prunus necrotic ringspot virus in
woody plants. J. Virol. Meth. 67, 135-141
Weissensteiner, T. / Taberlet,P. (1997) Guidelines for microsatellite
allele typing. Nucleic Acids Research 25(3), Scientific Correspondence
Jenner Institute for Vaccine Research
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