"good" PCR primers ?!

Bill O'Reitz ewok at access.digex.net
Thu Sep 23 08:17:17 EST 1993


I've recently been examining the program "Amplify 1.2", a shareware PCR
utility written by Bill Engels for the Macintosh.  This software allows
one to probe the performance of various primer sequences in carrying out
PCR on target DNA sequences.  Variant primer sequences are scored on
their "primability" (ie. sequence similarity to various sites in the
target sequence) and on "stability" (related to the total strength due
to the variable number of hydrogen bonds for varying G/C vs A/T compos-
ition of the primer).  These quantities are used to determine how "good"
a primer may be.

My question: what is meant by "good" here?

My thinking is that if a primer is good, it will prime the reaction early
and often, so that product is formed at a higher rate (assuming primer-
target duplex formation is a rate limiting factor in the subsequent
polymerization) than if the primer is "bad".

Is this a reasonable supposition?  What if the reaction is rate limited by
the amount of polymerase - do "good" and "bad" exist as useful descriptions
in the above sense?  And what if each round is given a very long time, so
that the reaction runs to exhaustion within each round.  What is the
meaning of good and bad here?

Now, in the first case (priming is rate limiting), how does a measure of
"goodness" show up in the total amount of product; doesn't it appear as
a factor in the exponent, so that aften _n rounds, a primer of measure
of goodness _g1 will have produced 2^{_a*_g1*_n} product (where _a
contains any other rate constants).  In this way shouldn't the relative
growth of product using two primers of goodness measure _g1 and _g2 be
2^{_a*_n*(_g1-_g2)}?

Finally, assuming I'm not missing something or everything in the above,
how does temperature fit into the goodness measure (ie. stability)?  Is
it reasonable to factor this into the _g values, or should this be
abosrbed into _a?

Sorry for the make-it-up-as-you-go-along notation; as a neophyte in this
area I lack the formal education that would allow me to phrase these
questions in the normal nomenclature and symbology.

Thanks....

Bill O'Reitz



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