Multi primers PCR

anon anon
Wed Dec 7 11:47:30 EST 1994


In article <1994Dec6.161419.1 at leif>, mmai at kean.ucs.mun.ca wrote:

> Hi. Does anyone have the experience to do multi primers PCR? It means that
> more than one gene fragments are amplified with two or more pairs of primers.
> If it works this technique will be very good the check low abundance mRNA
> with a control PCR band. Thanx in advance.
> 
> 
> --mming
> Terry Fox Cancer Res Lbs
> Faculty of Medicine
> Memorial university
> St. John's NF A1B 3V6

Yes, I routinely use 2 to 3 sets of primers, all of which flank introns in
their respective genes, in multiplex reverse transcription PCR. I use a
Mac based program called Amplify to ensure that the oligos do not
cross-prime or cross-dimerize. All of the oligos have similar Tm's (within
4 degrees of each other is fine) and I try to group the products in
similar, non-overlapping sizes (i.e. 300, 350, 400 bp). The relative
abundances of the primer pairs and the Mg concentration are very important
variables which you will have to optimize for each multiplex. When I use
actin as an internal control versus a rare message, I use 5x higher primer
conc. for the rare message as for actin. The optimal Mg will depend on the
total primer concentration of the reaction.

The biggest problem which I have encountered is in adjusting the primer
relative abundances. Too much of an internal control product can swamp the
reaction, making the control output nonlinear, and therefore
unquantifiable, and sometimes preventing me from getting the product of
interest. Still, duplex is pretty straightforward; triplex is often more
than 50% harder to make reliable.

Cheers (and good luck),

Andy Shenk
Dept. of Biological Chemistry
U. of California
Irvine, CA 92717
MASHENK at UCI.EDU



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