Polymerase Chain Reaction (PCR) is a technique used to make millions of copies of a specific DNA sequence. It was invented in 1983 by Kary Mullis. The basic steps of PCR include denaturation of DNA, annealing of primers, and extension of primers by DNA polymerase. There are many variants of PCR that have been developed, including real-time PCR, nested PCR, multiplex PCR, and long PCR. While PCR is a powerful technique, it also has limitations such as sensitivity to contamination and inability to amplify unknown DNA sequences.

1. Polymerase Chain Reaction and
its Variants
Presented bye: Irfanullah
Reg No: CUI Atd /SP21/RBT/003
Presented to: Dr Amjid Hassan
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2. Outline
• Definition
• Background
• Principle
• Processes
• Applications
• limitations
2

3. Definition
• PCR is a technique used in the lab to make
millions of copies of a particular section of
DNA.
(Sylvia,et al,2012)
3

4. History of PCR
• PCR is coping machine for DNA molecule
• Invented by Kary Mullis and his colleague in
1983.
• Nobel price in 1993
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5. Principle of PCR
• Purpose
• Component
• Steps
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6. Component of PCR
• DNA template
• Primers
• Nucleotides (dNTPs or deoxynucleotide
triphosphates.
• DNA polymerase
• Mgcl2
• Buffer
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7. There are three main steps:
• Denaturing
• Annealing
• Extending
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9. Advantages
• Valuable for detecting specific pathogens.
• Significantly more rapid in providing results.
• Valuable screening tool.
(Yang,et al ,2004)
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10. Limitations of PCR
• PCR cannot be used to amplify unknown
targets.
• DNA polymerases are prone to error.
• PCR is very sensitive to contamination.
( Yang , et al ,2004)
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11. Variants of the PCR
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12. Multiplex PCR Inverse PCR
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13. Reverse transcriptase PCR Hot start PCR
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14. In situ PCR Long PCR
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15. Advantages
Colony PCR
• Rapid and cost- effective
• Accuracy & specificity is higher
• Set up is simple
• cDNA library screening
(Zhang,et al,2019)
Nested PCR
• Very low probability of
nonspecific amplification
(Yamamoto,et el, 2019)
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16. Advantages
Multiplex PCR
• Technique is rapid,
• Time-saving
• Forensic Studies
• Set of primers used as
internal control.
• Needs less consumables,
chemicals and other
utilities.
(Poritz,et al,2011)
Inverse PCR
• Used to identified flanking
sequence around gnomic
insert.
• Identified and amplify
transposable element.
(Yang,et al,2012)
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17. Advantages
Reverse Transcriptase PCR
• it is used for gene
expression studies.
• easy to use, rapid and cost-
effective.
(Kucirka,et al,2020)
Hot start PCR
• prevent primer-dimer
formation
• requires less handling
• reduces the risk of
contamination.
(Tang,et al,2021)
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18. Advantages
In situ PCR
• Single copy of DNA can be
measured or amplified.
• Localizing and visualizing
the amplicon within the cell
(Zagklavara,et al,2021)
Long PCR
• cost effective tool for
detecting genetic variations
• To clone large genes not
possible with conventional
pcr.
(Min Hu,et al,2007)
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19. Limitations
Multiplex PCR
• High risk of contamination
• lack of detection of a single
target sequence
• the chances of non-specific
bindings
• It can’t amplify longer
templates effectively
(Poritz,et al,2011)
Inverse PCR
• many enzymatic steps.
• The cost of the overall
experiment is higher
• it cannot be used into the
routine genetic diagnostic
labs.
(Yang,et al,2012)
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20. limitations
Colony PCR
• High risk of contamination
• It can't give us sequence
information
• very high chance false-
positive results .
(Zhang,et al,2019)
Nested PCR
• time-consuming
• Required more reagents
• an increased risk of
contamination
(Yamamoto,et el, 2019)
20

21. Limitations
Reverse transcriptase PCR
• Extremely sensitive
• Huge experience and
expertise are required
(Kucirka,et al,2020)
Hot start PCR
• The overall cost of the
reaction is increased due to
the use antibody.
• can not amplify the larger
DNA templates more than
2kb.
• Due toDNA can damage or
break down badly.
(Tang,et al,2021)
21

22. Limitations
In situ PCR
• Higher the chance of non-
specific binding .
• The precision of the
reaction is also low.
(Zagklavara,et al,2021)
Long PCR
• Time-consuming.
• The success rate is also very
low.
• The chance of
misincorporation of dNTPs
is high.
• The technique is not
suitable for routine use.
(Min Hu,et al,2007)
22

23. References
• Yeh, S. H., & Mink, C. M. (2012). Bordetella pertussis and Pertussis (Whooping
Cough). In Netter’s Infectious Diseases (pp. 11-14). WB Saunders.
• Yang, S. and R.E. Rothman. 2004. PCR-based diagnostics for infectious diseases:
uses, limitations, and future applications in acute-care settings.
https://www.academia.edu/3577385/PCR-based_Diagnostic_for_Infectious_Diseases
• Guidance for Clinicians on the Use of RT-PCR and Other Molecular Assays for
Diagnosis of Influenza Virus Infection.
http://www.cdc.gov/flu/professionals/diagnosis/molecularassays.htm
• Yang, S., & Rothman, R. E. (2004). PCR-based diagnostics for infectious diseases:
uses, limitations, and future applications in acute-care settings. The Lancet infectious
diseases, 4(6), 337-348.
• Zhang, D., Lu, X., Liao, Y., Xia, Z., Peng, Z., Yang, X., & Yang, R. (2019). Rapid and
Simple Detection of Trichosporon asahii by Optimized Colony PCR. BioMed research
international, 2019.
• Yamamoto, M., Kashiwamura, S., Ohuchi, A., & Furukawa, M. (2008). Large-scale DNA
memory based on the nested PCR. Natural Computing, 7(3), 335-346.
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24. • Poritz, M. A., Blaschke, A. J., Byington, C. L., Meyers, L., Nilsson, K., Jones, D. E., ...
& Ririe, K. M. (2011). FilmArray, an automated nested multiplex PCR system for
multi-pathogen detection: development and application to respiratory tract
infection. PloS one, 6(10), e26047.
• Yang, J. J., Marschalek, R., Meyer, C., & Park, T. S. (2012). Diagnostic usefulness of
genomic breakpoint analysis of various gene rearrangements in acute leukemias: a
perspective of long distance-or long distance inverse-PCR-based approaches. Annals
of laboratory medicine, 32(4), 316-318.
• Kucirka, L. M., Lauer, S. A., Laeyendecker, O., Boon, D., & Lessler, J. (2020).
Variation in false-negative rate of reverse transcriptase polymerase chain reaction–
based SARS-CoV-2 tests by time since exposure. Annals of internal
medicine, 173(4), 262-267.
• Tang, Y., Chen, X., Zhang, J., Wang, J., Hu, W., Liu, S., ... & Xu, H. (2021).
Generation and Characterization of Monoclonal Antibodies Against Tth DNA
Polymerase and its Application to Hot-Start PCR. Protein and Peptide Letters.
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25. • Zagklavara, F., Jimack, P. K., Kapur, N., Querin, O. M., & Thompson, H. M. (2021).
Numerical Modelling and Analysis of a Microfluidic PCR Device. In Proceedings of the
6th World Congress on Momentum, Heat and Mass Transfer (MHMT'21), Lisbon,
Portugal Virtual Conference–June (pp. 17-19).
• Hu, M., Jex, A. R., Campbell, B. E., & Gasser, R. B. (2007). Long PCR amplification
of the entire mitochondrial genome from individual helminths for direct
sequencing. Nature protocols, 2(10), 2339-234
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26. Thank You
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