Quantitative PCR

William Alexander ALEXANDERW at cber.cber.fda.gov
Thu Aug 31 06:14:39 EST 1995


In Article <41vo24$ok2 at miasun.med.miami.edu>, tmiller at newssun.med.miami.edu
(Todd Miller - Pharmacology) wrote:
>In article <thomas.kreuzer.62.0012F0A2 at kfunigraz.ac.at>,
>Thomas Kreuzer <thomas.kreuzer at kfunigraz.ac.at> wrote:
>>On Thu. 24 Aug Frank Chen wrote as a reply to a question concerning 
>>quantitative RT-PCR:
>>
>>" No matter how you do it and how good your controls are , your results from 
>>quantitative PCR will be questionable."
>>
>>Since I am trying to put up a quantitative RT-PCR - method myself (and being 
>>quite new to the method of PCR in common) I would be interested in the major 
>>reservations against the principle of quantitative PCR and especially 
>>quantitative RT-PCR. What exactly makes itYs results questionable and why 
>>canYt these problems be avoided ?
>>It would be a great help if you could also give me some advice about 
>>literature dealing with these problems.
>>
>I would highly recommend reading an article by Luc Raeymaekers in
>_Analytical Biochemistry_ (I think it is volume 214, 1993, but you
>may have to check your favorite citation source).  He goes through
>all the mathematical theory of quantitative competitive PCR and comes
>to the conclusion that there are many papers published that have
>ignored some basic theory and predictions, which makes the data
>very suspicious.  Basically, the assumption that your competitor and
>your target have equal amplification efficiencies is almost never
>borne out, and this results in huge errors by the time you go through
>all the cycles.  
>
>Quantitative PCR works only for HIV and AIDS patients...  ;-)
>
>Todd Miller, PhD
>University of Miami School of Medicine
>tmiller at newssun.med.miami.edu
>
> 
>>Thanks in advance,
>>
>>Tom Kreuzer
>
>
Todd Miller, Did you read Isaac Kim's Post?  He is correct (great arguments
and the experiments were done) and so is Tracy.  Quantitative PCR does work,
you should not use a "house-keeping" gene (GAPDH) as your competitor and the
source of the nucleic acids has nothing to do with this topic.  

Todd said:
Basically, the assumption that your competitor and
>your target have equal amplification efficiencies is almost never
>borne out, and this results in huge errors by the time you go through
>all the cycles.  

The whole idea is to design the competitor so that it does amplify at the
same efficiency as the target.   (See Celi et al., "A rapid and versatile
method to synthesize internal standards for competitive PCR" Nucleic Acids
Research, 1993, Vol. 21, No.4, 1047.)  Yes, Todd is also correct, if the two
do not amplify at the same rate, the paper should be ignored.  

There are at least two simple ways to design good competitors:
1.  Use the "same" product as your competitor by adding or removing a
restriction site so that the two products can be distinguished an a gel
after restriction.  (This extra restriction step can be a pain.)

2.  Use a competitor that is slightly different in size to the original
target so that the only sequence difference is this extra Nucleic acid
fragment. 
This is the case where Todd gets caught up in mathematics.  The enzymes used
for the amplification are extremely fast (do you need references?).  
Therefore, the size difference can be designed so that it is detectable on a
gel but does not change the efficiency of the amplification of the target
and the competitor.

Two US Patents (there may be more) have been issued in this area.
1. Wang et al., Patent # 5,219,727, Date of Patent:  June 15, 1993, Filed:
Sep. 28, 1989; "Quantitation of Nucleic acids using the polymerase chain
reaction"


2. Bunn et al., Patent # 5,213,961, Date of Patent:  May 25, 1993, Filed:
Aug. 31, 1989; "Accurate quantitation of RNA and DNA by competitive
polymerase chain reaction"

These patents are great scientific papers and should be available around the
country (Frank Chen, U of MD College Park should have them in their lib.). 
As you can see, they were both filed in 1989 and issued in 1993. They are
both too "old" to be included in the full text biotech patents available on
the WWW.  (See:
http://www.inform.umd.edu:8080/EdRes/Topic/AgrEnv/Biotech/Biotechnology_Pate
nts-full_text)

I just don't think this area is so new anymore.

Bill Alexander
alexanderw at cber.cber.fda.gov



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