The document discusses nested PCR, which is a modification of polymerase chain reaction (PCR) that improves specificity. It involves two rounds of PCR where the product of the first reaction is used as a template for the second reaction with a nested primer set. This increases specificity by reducing non-specific binding. Some key advantages are improved accuracy and sensitivity for low abundance targets or difficult templates. However, it is more time-consuming and costly than standard PCR due to the extra reagents and steps required. Nested PCR has applications in microbial detection, genetic analysis, and other areas where high specificity is needed.

1. PRESENTATION ON
NESTED PCR

2. hello!
I am HRITIKA SINHA
ROLL NO - 1815054028
BRANCH - BIOTECHNOLOGY
SEMESTER – 5TH
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3. CONTENTS
• INTRODUCTION OF PCR
• TYPES OF PCR
• NESTED PCR
• PICTORIAL REPRESENTATION OF PROCESS
• PROTOCOL FOR NESTED PCR
• ADVANTAGES OF NESTED PCR
• DISADVANTAGES OF NESTED PCR
• APPLICATIONS
• REFERENCES
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4. 1.
POLYMERASE CHAIN
REACTION
Let’s start with the first set of slides
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5. “✘PCR is a revolutionary method developed by Kary Mullis in
the 1980s.
✘PCR is based on using the ability of DNA polymerase to
synthesize new strand of DNA complementary to the
offered template strand.
✘Because DNA polymerase can add a nucleotide only onto a
preexisting 3'-OH group, it needs a primer to which it can
add the first nucleotide.
✘This requirement makes it possible to delineate a specific
region of template sequence that the researcher wants to
amplify.
✘At the end of the PCR reaction, the specific sequence will
be accumulated in billions of copies (amplicons).
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6. TYPES OF PCR
✘ NESTED PCR
✘ INVERSE PCR
✘ AFLP-PCR
✘ ALLELE SPECIFIC PCR
✘ ASSEMBLY PCR
✘ ASYMMETRIC PCR
✘ HOT START PCR
✘ COLONY PCR
✘ SINGLE CELL PCR
✘ REAL TIME PCR / qPCR
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2.

7. NESTED PCR
It is a modification of polymerase chain reaction intended to reduce non-
specific binding in products due to the amplification of unexpected primer
binding sites.
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8. ✘ Used to increase the specificity of DNA amplification.
✘ Two sets of primers are used in two successive reactions.
✘ In the first PCR, one pair of primers is used to generate DNA products, which will be the
target for the second reaction.
✘ Using one ('hemi-nesting') or two different primers whose binding sites are located (nested)
within the first set, thus increasing specificity.
✘ Nested PCR is a simple and easy modification of conventional PCR which actually increases
the specificity of any reaction.
✘ The nested PCR is the best choice in the microbial identification and 16s RNA analysis.
What are nested pcr
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9. SETS OF PRIMERS
✘ The outer set of primer:
The outer primers are primers
that are upstream to the inner
set of primers. The outer primers
are bind to the outside to the
flanking region of out target DNA.
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 lead to false-
positive results.
✘ The inner set of primer:
Even if the non-specific DNA sequences
can be amplified in the first round of PCR,
that non-specific DNA will not be
amplified in the second set of
amplification.
The second set of primer is specific to
the inner sequence (amplicon of the first
round of PCR).
It is very unlikely that the inner set of
primers binds to other than its specific
site because the amplicon from the first
round of PCR is the template for the
second round of amplification.
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10. 10
PICTORIAL REPRESENTATION OF THE PROCESS

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Protocol for the nested PCR:
✘ In the year 1993, Kamolvarin and co-workers described the method for use of two sets of primers
for increasing sensitivity and specificity of the PCR.
✘ The nested PCR reaction is complete into two steps, a first round of amplification with the outer
forward and reverse primers. The protocol is as described,
Component Concentration Quantity
Master mix 1X 12µL
PCR reaction buffer 1X 5µL
Outer Forward primer 10pM 1µL
Outer Reverse primer 10pM 1µL
Template DNA 30ng 3µL
Water 3µL
Total ——————————- 25µL

12. 12
PCR Steps
Initial
Denaturation Denaturation Annealing Extension
Final
extension
Temperature 90 ̊C-95 ̊C 90 ̊C-95 ̊C 55 ̊C-60 ̊C 72 ̊C 72 ̊C
Time 3min 1min 50sec 1min 7 min
——————– ——————- 25 cycles ————— ———————
• After the reaction preparation, put the PCR as shown into the table below,
• After the completion of the first round of amplification, take the tubes and prepare the
reaction for the second round of amplification.
Now add 1µL inner forward primer and 1µL inner reverse primer to the PCR reaction
tubes of the first round of amplification.
Or
We can use another method in which, 3µL of PCR product is taken from the first
amplification and use it as a template, prepare the reaction as followed.

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Component Concentration Quantity
Master mix 1X 12µL
PCR reaction buffer 1X 5µL
Inner Forward primer 10pM 1µL
Inner Reverse primer 10pM 1µL
The amplicon from the first PCR (as a
template DNA) —————————————- 3µL
Water 3µL
Total —————————————- 25µL
PCR Steps
Initial
Denaturation Denaturation Annealing Extension Final extension
Temperature 90 ̊C-95 ̊C 90 ̊C-95 ̊C 55 ̊C-60 ̊C 72 ̊C 72 ̊C
Time 3min 1min 50sec 1min 7 min
——————– ——————- 35 cycles ————— ———————
• Place it back into the PCR machine.
 Instead of 25 cycles, set the PCR at 35 cycles. Higher amplification is achieved by
increasing the cycles in the second round of PCR. we can amplify more amount of gene of
our interest.

14. ADVANTAGES OF NESTED PCR
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 It is beneficial in studies such as phylogenetic analysis and
genetic polymorphism.
 The main advantage of the present method is that it gives
100% accuracy, specificity and sensitivity.
 For the impossible templates where the GC content might be
high or chance of non-specific banding is higher, nested PCR
offers the best results.
 It is also useful in the amplification of genes with the low
abundance.
 Further, nested PCR is the best choice for carcinoma and viral
infection studies.

15. DISADVANTAGES OF NESTED PCR
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The method is time-consuming.
Required more reagents such as an extra set of
primer and one extra round of agarose gel
electrophoresis. Which means the method is quite
costly.
The chance of contamination is also higher.

16. APPLICATIONS
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 To improve the sensitivity of the assay, nested PCR has been used in many PCR
assays. It is particularly useful for suboptimal nucleic acid samples, such as
those extracted from formalin-fixed, paraffin-embedded tissue.
 Nested PCRs have proven valuable for the detection of microorganisms when
they are present in very low quantities. For example:
 detection of Rickettsia, Bartonella, and similar organisms in blood (bacteremia)
and tissues,
 detection of herpesvirus and enterovirus in the CSF, and
 detection of M. tuberculosis in sputum sample.

17. thanks!
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18. REFERENCES
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✘ https://geneticeducation.co.in/what-is-nested-pcr/
✘ https://www.google.com/search?q=PIC+OF+NESTED+PCR
✘ https://en.wikipedia.org/wiki/Nested_polymerase_chain_reaction
✘ https://microbeonline.com/nested-pcr-principle-applications/