PCR
by
Jeremiah
Bennett J
“Polymerase
Chain
Reaction”
CERTIFICATE
PAGE 1
INDEX
SNO. Content Page
numbe
r
1 Objective 3
2 History 4
3 Types of PCR 5
4 Thermus aquaticus 7
5 Requirements 8
6 Steps involved in PCR 9
7 Results of PCR 12
8 Applications of PCR 13
9 Advantages of PCR 14
10 Disadvantages of PCR 15
11 Summary 16
12 Bibliography 17
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Objective
To obtain in-depth knowledge about a PCR (Polymerase chain
reaction), its working mechanism, and its application in the field of
molecular biotechnology.
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History
The 1993 Nobel Prize in chemistry was awarded to American
Kary Banks Mullis.
Mullis received the award for his invention of the polymerase
chain reaction (PCR), a simple technique that allows a
specific stretch of DNA to be copied billions of times in a few
hours.
Mullis began his Nobel Prize-winning research while at the
Cetus Corporation. He described his technique for the first
time in the December 20, 1985, issue of Science and received
a patent in 1987.
The technique can be used to amplify DNA sequences from
any type of organism. It has been adapted over the years to
allow amplification of RNA samples, as well as quantification
of the amount of DNA or RNA in a sample.
The isolation of a thermal stable DNA polymerase (Taq) from
an archaebacterium named Thermus aquaticus, isolated from
a geothermal vent in Yellowstone National Park allowed the
reaction to be carried out in a single closed tube driven by
varying temperatures.
Why “Polymerase”? It is called “polymerase” because the
only enzyme used in this reaction is DNA polymerase.
Why “Chain”? It is called “chain” because the products of the
first reaction become substrates of the following one, and so
on.
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TYPES OF PCR
1. Amplified fragment length polymorphism (AFLP) PCR
2. Allele-specific PCR
3. Alu PCR
4. Assembly PCR
5. Asymmetric PCR
6. COLD PCR
7. Colony PCR
8. Conventional PCR
9. Digital PCR (dPCR)
10. Fast-cycling PCR
11. High-fidelity PCR
12. High-Resolution Melt (HRM) PCR
13. Hot-start PCR
14. In situ PCR
15. Intersequence-specific (ISSR) PCR
16. Inverse PCR
17. LATE (linear after the exponential) PCR
18. Ligation-mediated PCR
19. Long-range PCR
20. Methylation-specific PCR (MSP)
21. Miniprimer PCR
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22. Multiplex-PCR
23. Nanoparticle-Assisted PCR (nanoPCR)
24. Nested PCR
25. Overlap extension PCR
26. Real-Time PCR (quantitative PCR or qPCR)
27. Repetitive sequence-based PCR
28. Reverse-Transcriptase (RT-PCR)
29. Reverse-Transcriptase Real-Time PCR (RT-qPCR)
30. RNase H-dependent PCR (rhPCR)
31. Single-cell PCR
32. Single Specific Primer-PCR (SSP-PCR)
33. Solid-phase PCR
34. Suicide PCR.
35. Thermal asymmetric interlaced PCR (TAIL-PCR)
36. Touch down (TD) PCR
37. Variable number of Tandem repeats (VNTR) PCR
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Thermus aquatics
Thermophilus aquatics is a species of bacterium that can
tolerate high temperatures; it is the source of the heat-
resistant enzyme Taq DNA Polymerase, one of the most
important enzymes in molecular biology because of its use in
the polymerase chain reaction.
Thermophilus aquatics is one of several thermophilic
bacteria that belong to the Deinococcus-Thermus group.
It was first isolated in 1969 from Yellowstone National Park
T. aquaticus shows best growth at 65–70 °C (149–158 °F) but
can survive at temperatures of 50–80 °C (122–176 °F).
It primarily scavenges for protein from its environment as is
evidenced by the large number of extracellular and
intracellular proteases and peptidases as well as transport
proteins for amino acids and oligopeptides across its cell
membrane.
This bacterium is a chemotroph—it performs chemosynthesis
to obtain food.
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PAGE 9
REQUIREMENTS
Target DNA (the template DNA)
Two oligonucleotide primers
2+
Mg ions
dNTP (deoxy nucleotide triphosphate
Thermally stable Taq-DNA Polymerase
Buffer solution
Each PCR assay requires the presence of template DNA,
primers, nucleotides, and DNA polymerase. The DNA
polymerase is the key enzyme that links individual
nucleotides together to form the PCR product. The
nucleotides include the four bases – adenine, thymine,
cytosine, and guanine (A, T, C, G) – that are found in DNA.
These act as the building blocks that are used by the DNA
polymerase to create the resultant PCR product. The primers
in the reaction specify the exact DNA product to be
amplified. The primers are short DNA fragments with a
defined sequence complementary to the target DNA that is to
be detected and amplified. These serve as an extension point
for the DNA polymerase to build on.
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Steps Involved In PCR
1) Denaturation
During this stage the cocktail containing the template DNA
and all the other core ingredients is heated to 94-95⁰C.
The high temperature causes the hydrogen bonds between
the bases in two strands of template DNA to break and the
two strands to separate.
This results in two single strands of DNA, which will act as
templates for the production of the new strands of DNA.
It is important that the temperature is maintained at this
stage for long enough to ensure that the DNA strands have
separated.
This usually takes between 15-30 seconds.
2)Annealing
During this stage the reaction is cooled to 50-65⁰C. This
enables the primers to attach to a specific location on the
single-stranded template DNA by way of hydrogen bonding
(the exact temperature depends on the melting temperature
of the primers you are using).
Primers are single strands of DNA or RNA sequence that are
around 20 to 30 bases in length.
The primers are designed to be complementary in sequence
to short sections of DNA on each end of the sequence to be
copied.
Primers serve as the starting point for DNA synthesis. The
polymerase enzyme can only add DNA bases to a double
strand of DNA. Only once the primer has bound can the
polymerase enzyme can only add DNA bases to a double
strand of DNA. Only once the primer has bound can the
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polymerase enzyme attach and start making the new
complementary strand of DNA from the loose DNA bases.
The two separated strands of DNA are complementary and
run in opposite directions (from one end – the 5’ end – to the
other – the 3’
Steps Involved In PCR
end); as a result, there are two primers – a forward primer
and a reverse primer
This step usually takes about 10-30 seconds.
3)Extension / Elongation
During this final step, the heat is increased to 72⁰C to enable
the new DNA to be made by a special Taq DNA polymerase
enzyme which adds DNA bases.
Taq DNA polymerase is an enzyme taken from the heat-
loving bacteria Thermus aquaticus.
72⁰C is the optimum temperature for the Taq polymerase to
build the complementary strand. It attaches to the primer
and then adds DNA bases to the single strand one by one in
the 5’ to 3’ direction.
The result is a brand-new strand of DNA and a double-
stranded molecule of DNA.
The duration of this step depends on the length of the DNA
sequence being amplified but usually takes around one
minute to copy 1,000 DNA bases (1Kb).
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Steps Involved In PCR
Results of PCR
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Each cycle gives 2 copies of the same DNA and therefore the total
number of copies due to n cycles is 2n
Applications of PCR
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Medicine - Detecting infectious organisms
-Discovering variations and mutations in genes, especially
human genes.
Human Genome Project – For the sequencing of the
isolated DNA
The Law – DNA fingerprinting
Evolutionary biology – DNA analysis for taxonomical
classification
Zoology – Research on animal behavior
Ecology
– Study of seed dispersal
1)Reducing illegal trade in endangered species
2)Monitoring the release of genetically engineered organisms
Archaeological and Paleontology – “Ancient DNA”
3)Analysing genetic variation in plants and animals
In Phylogenetic analysis of fossils and living
organisms.
It’s used in gene mapping as well as paternity tests.
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ADVANTAGES OF PCR
1. Highly specific: PCR can distinguish DNA sequences
by just one nucleotide, making it a very accurate
technique.
2. Sensitive: PCR is a very useful technique when the
amount of DNA sample is limited because it allows the
detection of even a single copy of a specific DNA
template.
3. Versatile: The PCR technique can be used for various
applications like genetic testing, criminal
investigations, and paternity tests.
4. Rapid and efficient: PCR can efficiently and rapidly
amplify a small amount of DNA sample to million copies
in just a few hours.
Multiple reports have shown PCR to be comparable, if not
better than, traditional staining and culturing. Gram
staining can correctly identify bacteria and fungi 60-75%
and 35-90% of the time, respectively. Cultures have a
similar result, identifying 59% of bacteria and 45% of
fungi. The sensitivities produced by these tests are
relatively low and show considerable variation, leaving
room for diagnostic improvement. Polymerase chain
reaction demonstrated higher positivity rates and
sensitivity than culture and stains for both bacteria and
fungi.
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Moreover, PCR has been seen to be several times faster
than cultures.
DISADVANTAGES OF PCR
1. Contamination: The PCR technique is very
susceptible to contamination from other sources of
DNA or RNA or the environment. This can mislead data
interpretation.
2. Cost and complexity: PCR can be expensive and
requires expert knowledge for high-throughput
projects.
3. Lack of novel information: Since PCR can only
amplify and target specific DNA sequences targeted by
the primers, PCR provides limited information and
cannot detect novel DNA sequences.
4. Inhibition from sample content: The whole PCR cycle
can be disrupted by inhibitors that co-purify with DNA,
such as heme from blood samples, reducing the
sensitivity of the process.
5. Errors in amplification: Base substitutions, indels,
and other alterations in DNA sequences can lead to
inaccurate amplification and hence, false results. Cross-
contamination is also an issue in culture and staining,
but perhaps the sensitive nature of PCR increases that
risk
Overall, PCR significantly impacts many research areas
but careful quality measures should be performed while
designing and interpreting PCR experiments
Its specificity is potentially lower than culturing and
staining, implying an increased risk of false positives
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Summary
In several cases, there is often very less amount of DNA present
such as on a crime scene and to identify the suspect behind it
several tests have to be done but it cannot be done on such a
small amount of DNA. PCR provides several copies of the DNA
which allows for conducting all of the tests.
PCR has been seen to be vital in the field of genetics with
enormous future scope. PCR allows for exponential amplification
of specific segments of the DNA. It allows us to make billions of
copies of the genetic material available in a short period as well.
It has proved its usefulness in several fields such as mapping
genes, identifying genotypes, and so on.
Through the making of this project, I have gained an in-depth
understanding of the various aspects of a PCR.
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Bibliography
National human genome research institute
https://www.britannica.com/science/polymerase-chain-
reaction
https://ib.bioninja.com.au
https://microbenotes.com/types-of-pcr/
https://www.bionity.com/
Wikipedia
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6778471/
https://facellitate.com/advantages-and-disadvantages-of-pcr-
technology/
www.yourgenome.org
J Invest Dermatol. 2013 March ; 133(3): e6.
doi:10.1038/jid.2013.1
American Society for Microbiology
2016,mgcub.ac.in
Mayo Clin Proc. 2002;77:606
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