PCR primers

Greg Tobin tobin at fcs260c.ncifcrf.gov
Wed Aug 16 14:00:05 EST 1995


In article <25822 at sci.med.aids> tmiller at newssun.med.miami.edu (Todd Miller - Pharmacology) writes:
>	I've noticed that the PCR, applied to HIV, almost always
>uses the SK primers that amplify part of the gag sequence.  Does
>anyone know of studies that compared the results of this primer
>pair to other primer pairs also specific for HIV, but in other
>parts of the genome?  Are there factors that would limit the
>degree of correlation between the results of different primer
>pairs?  How close would you expect different primer pairs to be
>as far as providing an answer to 1)infected or not infected and
>2)quantitative measurements of viral load?
>
>

I believe that the original paper describing the SK primers presentes 
several sets of primers that amplified various segments of the genome.
BTW, SK refers to the initials of the person who made the primers rather
than Sac1-Kpn1 in pBS vectors.

The choice of PCR primers depends upon the purpose of the study.
If you just want to know if a sample is positive or negative, you
should choose primers from a conserved region of the viral genome.
These primers should work well on most HIV-1 templates and provide
some semblence of quantitation when used correctly.  If you would like
to determine if a template is from a particular Clad (clade?) or strain
of HIV-1, you would choose primers from less conserved regions.

Primer choice for routine, conserved amplification starts with 
identification of conserved target sites.  After several sets of primers
are made, they are tested for sensitivity and specificity.  Although 
computer alignments are needed for the first step, I (and others) have
found that a certain amount of trial and error is needed to get
good, reproducible results.  While better primer pairs may exist,
the SK sets have been used with good results by many so they continue
to be used.  

	Thanks for any help.  This question was too tough to
>extract out of the literature, so I'm hoping someone has come
>across this, perhaps even WRT other viruses.
>
>Todd Miller
>

-- 
Greg Tobin, Ph.D.                        tobin at lcms-1.ncifcrf.gov




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