real time pcr

Susanne Rohrer "srohrer(removethis)" at
Sun Jun 25 08:26:29 EST 2000

T V Naga Rama Chander wrote:

> dear friendz,
> can anyone explain me what is real time pcr?

This means analysis of PCR kinetics by detecting PCR products as they
accumulate.You can plot the increase in fluorescence of a dye that is
incorporated in the PCR mix versus cycle number. In the end you can
quantitate the product.
You can also do it with a traget-specific probe. The probe is an
oligonucleotide with both a reporter fluorescent dye and a quencher dye
attached. While the probe is intact, the fluorescence is quenched until
the target is amplified.
using mutation-specific probes it is possible to detect point mutations
and so on. Go to the Roche homepage etc., other suppliers of those
expensive Machines to find out more.

> what is meant by nested pcr?

Nested PCR is used to make sure (surer?! it's happened that you still
get sth wrong) you amplify what you really want. You make 2 primer pairs
of which the second is "inside" or "nested" in the first. A second round
of PCR with the second primer pair increases specificity.
Like this:

<---------- 1stPCR
<----------------                  2nd PCR

Susanne Rohrer
Institute of Medical Microbiology
University of Zurich

srohrer at immv dot unizh dot ch

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