PCR is a technique used to amplify specific DNA sequences. It involves using DNA polymerase to replicate target DNA sequences across multiple temperature cycles. Key points: 1. PCR amplifies specific DNA segments through repeated cycles of heating and cooling. This allows millions of copies of the target DNA to be generated. 2. It was invented by Kary Mullis in 1983 and revolutionized molecular biology. Mullis shared the 1993 Nobel Prize in Chemistry for his invention. 3. PCR has many applications including pathogen detection, genetic testing, forensics, and molecular cloning. It is a core technique in molecular biology and genetics research.

1. “Polymerase chain reaction (PCR)"
By
B. Bhaskar

2. Introduction
• Polymerase Chain Reaction (PCR) is a powerful method for amplifying
particular segments of DNA, distinct from cloning and propagation within
the host cell.
• PCR procedure is carried out entirely biochemically, that is, in vitro. PCR
was invented by Kary Mullis in 1983 (American biochemist).
• He shared the Nobel Prize in chemistry with Michael Smith in 1993.
• PCR or Polymerase Chain Reaction is a technique used in molecular
biology to create several copies of a certain DNA segment.
• PCR has made it possible to generate millions of copies of a small segment
of DNA.
• What is PCR used for in pathology?
• PCR has become established as a routine method in pathology diagnostics
and has been adapted for the detection of infectious agents, mutations,
translocations and gene amplifications, and for molecular forensic
applications including identity testing.
• 1) The amplification of gene fragments as fast alternative of cloning. 2)
The modification of DNA fragments. 3) The sensitive detection of
pathogenic microorganisms, if desired followed by an accurate
genotyping. 4) DNA analysis of arachaeological specimens.

3. Principle of PCR
• Principle of PCR
• PCR uses the enzyme DNA polymerase that directs the synthesis of DNA from
deoxynucleotide substrates on a single-stranded DNA template.
• DNA polymerase adds nucleotides to the 3` end of a custom-designed oligonucleotide when
it is annealed to a longer template DNA.
• Thus, if a synthetic oligonucleotide is annealed to a single-stranded template that contains a
region complementary to the oligonucleotide, DNA polymerase can use the oligonucleotide
as a primer and elongate its 3` end to generate an extended region of double stranded DNA.
• 1. Denaturation
• The DNA template is heated to 94° C. This breaks the weak hydrogen bonds that hold DNA
strands together in a helix, allowing the strands to separate creating single stranded DNA.
• 2. Annealing
• The mixture is cooled to anywhere from 50-70° C. This allows the primers to bind (anneal)
to their complementary sequence in the template DNA.
• 3. Extension
• The reaction is then heated to 72° C, the optimal temperature for DNA polymerase to act.
DNA polymerase extends the primers, adding nucleotides onto the primer in a sequential
manner, using the target DNA as a template.
• With one cycle, a single segment of double-stranded DNA template is amplified into two
separate pieces of double-stranded DNA. These two pieces are then available for
amplification in the next cycle. As the cycles are repeated, more and more copies are
generated and the number of copies of the template is increased exponentially.
•

5. • What is the principle of PCR?
• Its principle is based on the use of DNA polymerase which is an in vitro replication
of specific DNA sequences.
• This method can generate tens of billions of copies of a particular DNA fragment (the
sequence of interest, DNA of interest, or target DNA) from a DNA extract (DNA
template).
• What are the 5 components of PCR?
In general, a complete PCR reaction requires five basic PCR reagents; DNA/RNA template,
DNA polymerase, primers (forward and reverse), deoxynucleotide triphosphates
(dNTPs) and PCR buffers.
• What are the types of PCR?
• Types of polymerase chain reaction-PCR
Real-Time PCR (quantitative PCR or qPCR) Reverse-Transcriptase (RT-PCR) Multiplex
PCR. Types of PCR
• Real-time PCR.
• Quantitative real time PCR (Q-RT PCR)
• Reverse Transcriptase PCR (RT-PCR)
• Multiplex PCR.
• Nested PCR.
• Long-range PCR.
• Single-cell PCR.
• Fast-cycling PCR.

6. Types of PCR
1. Real-time PCR
2. Quantitative real time PCR (Q-RT PCR)
3. Reverse Transcriptase PCR (RT-PCR)
4. Multiplex PCR
5. Nested PCR
6. Long-range PCR
7. Single-cell PCR
8. Fast-cycling PCR
9. Methylation-specific PCR (MSP)
10. Hot start PCR
11. High-fidelity PCR
12. In situ PCR
13. Variable Number of Tandem Repeats (VNTR) PCR
14. Asymmetric PCR
15. Repetitive sequence-based PCR
16. Overlap extension PCR
17. Assemble PCR
18. Intersequence-specific PCR(ISSR)
19. Ligation-mediated PCR
20. Methylation –specific PCR
21. Miniprimer PCR
22. Solid phase PCR
23. Touch down PCR, etc

7. Long range PCR refers
to the amplification of
DNA targets over 5kb in
length which typically
cannot be amplified
using routine PCR
methods or reagents.
Single cell RT PCR
Fast Cycling PCR kit
What is Fast Cycling PCR?
Fast PCR enzymes shorten
cycling times, thus
reducing time from sample
to results and increasing
throughput. Phusion and
Phire DNA Polymerases
incorporate more nucleotides
per binding event as
compared to other
polymerases.
Long range PCR

9. Methylation-specific PCR (MSP) is a method for
analysis of DNA methylation patterns in CpG
islands. For performing MSP, DNA is modified by
and PCR performed with two primer pairs,
which are detectable methylated and
unmethylated DNA
Methylation-specific PCR
Hot start PCR is a modified form of conventional polymerase
chain reaction (PCR) that reduces the presence of undesired
products and primer dimers due to non-specific DNA
amplification at room (or colder) temperatures.
What are the benefits of hot-start technology?
Prevents extension of primers binding to template sequences
with low homology (mispriming)
Prevents extension of primers binding to each other (primer-
dimer formation) during reaction setup.
Increases sensitivity and yield of the desired target fragments.

10. High-fidelity PCR enzymes
are used for applications
requiring high accuracy
during DNA amplification
such as cloning,
sequencing or
mutagenesis. Thermo
Scientific Phusion high-
fidelity DNA polymerases
were created by fusing a
dsDNA-binding domain to
a Pyrococcus
polymerase–like
proofreading enzyme
https://youtu.be/-
UP5A08UOQE
In Situ RT Polymerase Chain Reaction (In Situ RT-
PCR) is a powerful method that detects minute
quantities of rare or single-copy number nucleic
acid sequences in frozen or paraffin-embedded
cells or tissue sections for the localization of those
sequences within the cells.

11. Variable number tandem repeats (VNTRs) are short
nucleotide sequences (20–100 bp) that vary in copy number
in bacterial genomes. They are thought to arise through DNA
strand slippage during replication and are of unknown
function.
VNTRs are used as an important source of RFLP
genetic markers for linkage analysis of diploid genomes.
VNTR analysis is used to study genetic diversity and breeding
patterns of wild and domestic animals. They are also used to
distinguish the strains of bacterial pathogens.
VNTR-PCR
Asymmetric PCR can be used to form
single stranded DNA from double
stranded DNA, which is then used
for DNA sequencing in the
mutagenesis method. Single
stranded DNA is also important for
aptamer generation.

12. • Repetitive element sequence-based
PCR (rep-PCR) is a new typing method
that differentiates microbes by using
primers complementary to interspersed
repetitive consensus sequences that
enable amplification of diverse-sized
DNA fragments consisting of sequences
between the repetitive elements (16,
25, 26, 28)

13. • How does overlap
extension PCR work?
An overlap is formed
during PCR
reaction. When induced
polymerase chain
reaction conditions, the
common sequence
allows strands from two
different fragments to
hybridize to one
another. This forms an
overlap. When this
overlap is extended by
DNA polymerase yields a
recombinant molecule.

14. Assembly PCR
• Assembly PCR, using synthetically derived DNA,
is a flexible technique for producing novel gene
sequences. Single-stranded oligos or a mix of
single- and double-stranded DNA are used to
produce longer genes of up to several thousand
base pairs. Assembly PCR can be used to assemble
two gene-sized pieces of DNA into one piece for
easier cloning of fusion genes/parts. Briefly, it
essentially involves PCR'ing the two pieces
separately with primers that have a 20bp
overlap and then doing an extra PCR step using
the two products as the template.

15. Inter simple sequence repeat (ISSR)-PCR
• Inter simple sequence repeat (ISSR)-PCR
is a technique that involves the use of
microsatellite sequences as primers in a
polymerase chain reaction to generate
multi-locus markers.

16. Ligation-mediated polymerase chain
reaction (LM-PCR)
• Ligation-mediated
polymerase chain reaction
(LM-PCR) is a genomic
analysis technique for
determination of (1) primary
DNA nucleotide sequences
(2) cytosine methylation
patterns (3) DNA lesion
formation and repair, and (4)
in vivo protein–DNA
footprints

17. Methylation-specific PCR (MSP)
• Methylation-specific PCR (MSP) is a
method for analysis of DNA
methylation patterns in CpG islands.
For performing MSP, DNA is modified
by and PCR performed with two primer
pairs, which are detectable methylated
and unmethylated DNA.
• The basic principle of MSP is
the specific PCR amplification of
bisulfite-converted DNA. Treatment of
genomic DNA with sodium bisulfite
results in the conversion of
unmethylated cytosines into uracil in
the DNA sample, while methylated
cytosines are resistant to this
modification and remain unchanged.

18. Miniprimer PCR
• Miniprimer PCR amplified a
greater proportion than did
standard primers of
sequences that were novel
or that poorly matched a
database of previously
isolated 16S rRNA gene
sequences. This confirmed a
prediction based on a
computational search of the
NCBI environmental
sequence database

19. Solid-phase PCR (SP-PCR)
• Solid-phase PCR (SP-PCR)
has attracted
considerable interest in
different research fields
since it allows parallel
DNA amplification on
the surface of a solid
substrate. However, the
applications of SP-PCR
have been hampered by
the low efficiency of the
solid-phase amplification.

20. "Touchdown polymerase chain reaction (PCR)"
• "Touchdown polymerase chain
reaction (PCR)" is a method to
decrease off-target priming and
hence to increase the specificity of
PCRs.
• In touchdown PCR the temperature
selected for the annealing step is
initially set 5°C-10°C higher than the
calculated Tm of the primers.
• It is a method for increasing specificity
of PCR reactions.
• Touchdown PCR uses a cycling
program where the annealing
temperature is gradually
reduced (e.g. 1-2°C /every second
cycle).
• The initial annealing temperature
should be several degrees above the
estimated Tm of the primers.

21. Applications of PCR
A. PCR is used in analyzing clinical specimens for the presence of infectious
agents, including HIV, hepatitis, malaria, anthrax, etc.
B. PCR can provide information on a patient’s prognosis, and predict response or
resistance to therapy. Many cancers are characterized by small mutations in
certain genes, and this is what PCR is employed to identify.
C. PCR is used in the analysis of mutations that occur in many genetic diseases
(e.g. cystic fibrosis, sickle cell anaemia, phenylketonuria, muscular
dystrophy).
D. PCR is also used in forensics laboratories and is especially useful because
only a tiny amount of original DNA is required, for example, sufficient DNA
can be obtained from a droplet of blood or a single hair.
E. PCR is an essential technique in cloning procedure which allows generation of
large amounts of pure DNA from tiny amount of template strand and further
study of a particular gene.
F. The Human Genome Project (HGP) for determining the sequence of the 3
billion base pairs in the human genome, relied heavily on PCR.
G. PCR has been used to identify and to explore relationships among species in
the field of evolutionary biology. In anthropology, it is also used to understand
the ancient human migration patterns. In archaeology, it has been used to spot
the ancient human race. PCR commonly used by Paleontologists to amplify
DNA from extinct species or cryopreserved fossils of millions years and thus
can be further studied to elucidate on.

22. References
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mediated+PCR&gs_lcp=CgNpbWcQAzIFCAAQgAQ6BAgjECdQhQ9YhQ9ghxhoAHAAeACAAVyIAbYBkgEBMpgBAKABAaoBC2d3cy13aXotaW1nuAEDwAEB&sclient=img&ei=gRIKY7ruLu6n4t4P-
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23. THANK YOU