Polymerase Contents

Chain •
•
What is PCR?
History of PCR

Reaction
• Components of PCR
• Principles of PCR
• Basic Requirements
• Instrumentation
• PCR Programme
• Advantages of PCR
• Applications of PCR

What is PCR? Short History of PCR

• PCR is a technique that takes specific
• 1983: Dr. Kary Mullis developed PCR
sequence of DNA of small amount and • 1985: First publication of PCR by Cetus Corporation
amplifies it to be used for further testing. appears in Science.
• In vitro technique • 1986: Purified Taq polymerase is first used in PCR
• 1988: PerkinElmer introduces the automated thermal
cycler.
• 1989: Science declares Taq polymerase "molecule of
the year.
 Short History of PCR Short History of PCR
• 1990: amplification and detection of specific DNA • 1993:Dr. Kary Mullis shares Nobel Prize in Chemistry
sequences using a fluorescent DNA-binding dye, for conceiving PCR technology.
laying the foundation for future "real-time" or
• 1999: AMPLICOR® CT/NG Test for Chlamydia
"kinetic" PCR.
trachomatis, Neisseria gonorrhoeae.
• 1991: RT-PCR is developed using a single • 1999: Japanese Red Cross Society implemented
thermostable polymerase, rTth, facilitating nucleic acid technology (NAT) testing to screen 100%
diagnostic tests for RNA viruses. of donated blood for HIV, HCV and HBV using
• 1992: AMPLICOR Chlamydia trachomatis Test and AmpliNAT MPX system.
AMPLICOR HIV-1 MONITOR Test were introduced
outside of the USA.

Short History of PCR Principle of PCR

• 2000: US National Human Genome Research Institute
(NHGRI) of the National Institutes of Health (NIH)
awarded three-year, $1.2 million grant for development •Purpose:
of SNP genotyping program using kinetic thermocycler
technology.
• 2003: Roche and deCODE genetics identified specific
•Condition:
variations within a single gene that confer significant
increased risk of osteoporosis using PCR.
• 2004: PCR has been widely used in paternity testing.
•Components:
• 2006: PCR and Taq polymerase patents expire.
• 2020: qRT-PCR routinely used for COVID-19 detection
 Purpose Condition
• To amplify a lot of double-stranded DNA 1. Denaturation of dsDNA template
molecules (fragments) with same (identical)
size and sequence by enzymatic method and 2. Annealing of primers
cycling condition.
3. Extension of ds DNA molecules

Denaturation Annealing
• Temperature: 92-94C • Temperature: ~50-70C (dependant on the
• Double stranded DNA melts single melting temperature of the expected duplex)
stranded DNA • Primers bind to their complementary
5’ 3’ sequences
3’ 5’ 5’ 3’
92C
Forward primer Reverse primer
5’ 3’
3’ 5’
+
3’ 5’
 Extension Cycling
• Temperature: ~72C
• Time: 0.5-3min
• DNA polymerase binds to the annealed
primers and extends DNA at the 3’ end of the
chain

Taq

5’ Taq

3’
5’

Products of Extension Overall Principle of PCR

5’ 3’
• DNA – 1 copy
Taq
3’ 5’

• Known sequence Sequence of interest Known sequence

5’ 3’ • PCR

3’ 5’

Taq
 Basic requirements for PCR
Chemical Components
reaction
• Magnesium chloride: .5-2.5mM • 1) DNA sequence of target region must
be known.
• Buffer: pH 8.3-8.8
• dNTPs: 20-200µM 2) Primers - typically 20-30 bases in
size.
• Primers: 0.1-0.5µM These can be readily produced by
• DNA Polymerase: 1-2.5 units commercial companies. Can also be
prepared using a DNA synthesizer
• Target DNA: ≤ 1 µg

Basic requirements for PCR

Primer Design
reaction
• 3) Thermo-stable DNA polymerase - eg • Needs to get the target DNA sequence (e.g.
Taq polymerase which is not from GenBank)
inactivated by heating to 95C • Choice of the length of the primers and their
melting temperature (Tm) depends on a
4) DNA thermal cycler - machine which number of considerations:
can be programmed to carry out • melting or annealing temperature of a primer
heating and cooling of samples over a is defined as the temperature below which the
number of cycles. primer will anneal to the DNA template and
above which the primer will dissociate from
DNA template.
• Primers that are too short would anneal at Instrumentation
several positions on a long DNA template,
which would result in non-specific copies.
• Optimum length of primer is 20 to 30
nucleotides with a Tm between 55°C and 65°C.
• Simple formula: Tm = 2(A+T) + 4(G+C) (if bases
<14 nucleotides).

• Ta=Tm-5
Three Aspects of PCR
• Specificity

• Efficiency

• Fidelity
Things to try if PCR does not work Things to try if PCR does not work
• A) If no product ( of correct size ) produced: • B) If extra spurious product bands present
– 1 Check DNA quality – 1 Increase annealing temperature
– 2 Reduce annealing temperature – 2 Reduce magnesium concentration
– 3 Increase magnesium concentration – 3 Reduce number of cycles
– 4 Add dimethylsulphoxide ( DMSO ) to – 4 Try different enzyme
assay ( at around 10% )
– 5 Use different thermostable enzyme
– 6 Throw out primers - make new stocks

Example of PCR programme Advantages of PCR

• Initial denaturation 95C for 5 mins • Small amount of DNA is required per test
• Thermo-cycle file - 30 cycles of • Result obtained more quickly - usually within 1
• Denaturation : 95C for 30 secs day for PCR
• Usually not necessary to use radioactive
• Annealing : 55C for 30 secs
material (32P) for PCR.
• Extension : 72C for 45 secs • PCR is much more precise in determining the
• Final extension 72C for 5 mins sizes of alleles - essential for some disorders.
• Holding ( soak ) file usually 4C • PCR can be used to detect point mutations.
 Applications of PCR Applications of PCR
Neisseria gonorrhea - STD
Chlamydia trachomatis - STD Molecular Identification Sequencing Genetic Engineering
Molecular Archaeology Bioinformatics Site-directed mutagenesis
HIV-1 > AIDS Molecular Epidemiology Genomic Cloning Gene Expression Studies
Molecular Ecology Human Genome Project
Factor V Leiden – blood clot gene DNA fingerprinting
Classification of organisms
mutation Genotyping
Pre-natal diagnosis
SARS-CoV-2 > COVID-19 Mutation screening
Drug discovery
Forensic testing and many others Genetic matching
Detection of pathogens

Conclusion
PCR is not only vital in the clinical laboratory by
amplifying small amounts of DNA for STD
detection, but it is also important for genetic
predisposing for defects such as Factor V
Leiden.
The PCR technology can also be employed in law
enforcement, genetic testing of animal stocks
and vegetable hybrids, and drug screening
along with many more areas.