PCR Techniques
By
Ahmad Mansour Mohamed Alzohairy
Department of Genetics, Zagazig
University,Zagazig, Egypt
2015
Nested PCR
Definition of Nested
:PCR
Nested PCR is a variation of the
polymerase chain reaction
(PCR), in that two pairs (instead
of one pair) of PCR primers are
used to amplify a fragment.
The Nested PCR Reaction
- Nested PCR requires two sets of
primers which are used to amplify a
specific DNA fragment using two
separate runs of PCR.
- The first pair of PCR primers amplify a
fragment similar to a standard PCR.
However, a second pair of primers called
nested primers (as they lie / are nested within
the first fragment) bind inside the first PCR
product fragment to allow amplification of a
second PCR product which is shorter than the
first one.
The usefulness of nested PCR is two-fold:
- First specificity, because two different sets
of primers are used, specificity is increased
and the amplification of contaminants
decreased because if the wrong PCR
fragment was amplified, the probability is
quite low that the region would be amplified a
second time by the second set of primers.
Thus, Nested PCR is a very specific PCR amplification.
- Second, nested PCR can be very,
very sensitive, even more sensitive
than “simple” PCR, depending
upon the number of amplification
cycles used in each PCR step.
It’s that sensitivity that allows nested
PCR to amplify very tiny amounts
of target sequence