RNA PCR primers

Robert Horton horton at biosci.cbs.umn.edu
Mon Sep 18 13:01:26 EST 1995


My dear virtual colleagues:

Sami Kohan <skohan at ucla.edu> wrote:

> :Why is it standard to use DNA oligonucleotides in PCR when in
> :vivo, when DNA is replicating, the polymerase use RNA primers?

To which I offered the following explanation:

> Using RNA primers would require an RNA-dependant DNA polymerase
> activity for use in PCR.

E-mail responses have convinced me that I should expound on this.

In PCR, the molecules that act as primers in one round must act as
template in subsequent rounds. Bear with me as I try this in ASCII
art:

(I'll draw the primer molecule with plus signs (+) to differentiate
it.)

++++++++>  primer strand
<-------------------------------------------------  template strand


Polymerase extends primer to form a molecule that looks like this:
++++++++>---------------------------------------->

The primer is actually incorporated into this molecule. If the primer
is RNA, then the strand just formed has RNA in it. Now, when the
primer at the other end is extended, you get a molecule like this:

++++++++>----------------------------------------->
        <------------------------------------------

It can't fill in the rest of the sequence unless it can use the
primer as a template. With RNA primers, this would require a
polymerase with reverse transcriptase activity. If this last bit of
the molecule where the primer binds is not filled in, you cannot have
exponential amplification. Hence, no PCR.

You can make RNA primers synthetically (they are expensive). You can
also make them by in vitro transcription from an engineered DNA
template. You could probably even find a way to get around the
problem of RNA being less stable than DNA when heated. You just can't
use them in PCR without reverse transcriptase activity.


Robert M. Horton (PhD!) /\ "Crash programs fail because of the theory that
U of M Dermatology Dept || with nine women pregnant you get a baby a month"
Box 98 UMHC, 4-154 PWB /||\ -W. von Braun.   Disclaimer: "Bob who?"
Minneapolis, MN 55455   ^^   horton at biosci.cbs.umn.edu       (612)625-8941





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