Nested PCR is a modification of conventional PCR that uses two sets of primers to improve sensitivity and specificity. It involves two rounds of amplification, where the first round uses outer primers that bind outside the target DNA to amplify a larger fragment. The second round uses inner primers that bind within the first amplified fragment to specifically amplify the target DNA. This blocks non-specific amplification. Nested PCR allows for the accurate detection of pathogens or genes present at low levels. While more sensitive and specific than conventional PCR, it is also more time-consuming and prone to contamination due to the use of two primer sets.

1. SUBMITTED TO
DR.ATIF KAMRAN
MS (SELF-SUPPORTING)
SUBMITTED BY
FIAZA IDREES (MSBT28F20)
MARIA JABEEN (MSBT29F20)
FATIMA KHALID (MSBT30F20)
AMINA ALI (MSBT31F20)
TOPIC: NESTED PCR
SUBJECT
PCR AND ITS IMPLICATIONS IN PLANTS

2. NESTED PCR
 RATIONALE AND PURPOSES:
 Nested PCR is a modification in Conventional PCR that was designed to
improve sensitivity and specificity. The main difference from conventional
PCR is that two set of primer pairs are used in it which blocks the non-specific
amplicons.

3. PROCESS
 Two sets of primers are used to
achieve high sensitivity in the
nested PCR. Here both primers
have different and unique
properties. The first set of primer
binds outside of our target DNA
and amplifies larger fragment, this
set of primer is referred to as a
outer primer. Another set of primer
binds specifically at the inner of site
target site and in the second round
of amplification, it amplifies only the
target DNA, this set of primer is
referred to as nested or inner
primer.
SET OF PRIMERS

4. PROCESS OF AMPLIFICATION
. In the first round of PCR, It is possible that this
primer can bind to the site other than the target site
and amplifies it. Multiple DNA bands might be
observed and they have nonspecific sequences.

5. APPLICATIONS
 For the detection of Bartonella, Rickettsia, and
many organisms in tissues and blood
(bacteremia).
 For the detection of enterovirus and herpesvirus
in the CSF (Cerebrospinal Fluid).
 For the detection of Tuberculosis in a sputum
sample.
Commercial application
• The Bio Fire Film Array from bio
Meraux is a commercially available system
that employs nested, multiplex, and single
plex PCR reactions for the detection of a
variety of pathogens.

6. NESTED PCR
 It is beneficial in studies such as
genetic polymorphism.
 The main advantage of the present
method is that it gives maximum
accuracy, specificity and
sensitivity..
 It is also useful in the amplification
of genes with the low abundance.
 Nested PCR is the best choice for
carcinoma and viral infection
studies.
 For gene amplification, it is a
useful process with minimal
abundance.
 It is also a useful process in
reducing the non-specific
amplification of the sequence of
interest.
 It is considered a quite time-
consuming process.
 The risks of contamination
during the performance of
this process are high
because of two sets of
primers are used. The cost
will increase dramatically if
assays are repeated when
contamination occurs.
 It is a quite costly method as
it needs more reagents like
extra primer-set and extra
rounds of agarose gel
electrophoresis.
Advantages Disadvantages

7. REFRENCES
 Chang-Hui Shen (2019). Amplification of Nucleic
Acids. Diagnostic Molecular Biology.
 Deepachandi, B., Weerasinghe, S., Soysa, P. et al
(2019). A highly sensitive modified nested PCR to
enhance case detection in leishmaniasis. BMC
Infect Dis 19, 623 doi:10.1186/s12879-019-4180-3
 Souza G., Almeida A., Farias A., Leal N., Abath F.
(2007). Development and Evaluation of a Single
Tube Nested PCR Based Approach (STNPCR) for
the Diagnosis of Plague. In: Perry R.D., Fetherston
J.D. (eds) The Genus Yersinia. Advances in
Experimental Medicine and Biology, vol 603.
Springer, New York, NY