this presentation is about the polymerase chain reaction and all of its types

1. Polymerase Chain Reaction and
its Variants
Presented bye: Irfanullah
Reg No: CUI Atd /SP21/RBT/003
Presented to: Dr Amjid Hassan
1

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
4

5. Principle of PCR
• Purpose
• Component
• Steps
5

6. Component of PCR
• DNA template
• Primers
• Nucleotides (dNTPs or deoxynucleotide
triphosphates.
• DNA polymerase
• Mgcl2
• Buffer
6

7. There are three main steps:
• Denaturing
• Annealing
• Extending
https://www.yourgenome.org/sites/default/fil
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8. https://www.google.com/url?sa=i&url=https%3A%2F%2Fwww.youtube.com%
2Fwatch%3Fv%3Dvi7MeqD2_FY&psig=AOvVaw2dEP5H6hAlQWqf8knqFuRD&u
st=1638898630467000&source=images&cd=vfe&ved=0CAsQjRxqFwoTCPCWys
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9. Advantages
• Valuable for detecting specific pathogens.
• Significantly more rapid in providing results.
• Valuable screening tool.
(Yang,et al ,2004)
9

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
https://www.google.com/url?sa=i&url=https%3A%2F%2Fg
eneticeducation.co.in%2Fwhat-is-nested-
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https://www.google.com/url?sa=i&url=https%3A%2F%2Fwww.yo
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12. Multiplex PCR Inverse PCR
https://www.google.com/url?sa=i&url=https%3A%2F%2Fwww.yo
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ved=0CAsQjRxqFwoTCKDdk53kz_QCFQAAAAAdAAAAABAD
https://www.google.com/url?sa=i&url=https%3A%2F%2Fsli
detodoc.com%2Fpolymerase-chain-reaction-genetics-
course-dr-shagufta-naz%2F&psig=AOvVaw1sX3fZlkBB-
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13. Reverse transcriptase PCR Hot start PCR
https://www.google.com/url?sa=i&url=https%3A%2F%2Fwww.iae
a.org%2Fbulletin%2Finfectious-diseases%2Fhow-is-the-covid-
19-virus-detected-using-real-time-rt-
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https://www.google.com/url?sa=i&url=https%3A%2F%2Fge
neticeducation.co.in%2Fwhat-is-a-hot-start-
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14. In situ PCR Long PCR
https://www.google.com/url?sa=i&url=https%3A%2F%2Fge
neticeducation.co.in%2Fwhat-is-a-long-range-
pcr%2F&psig=AOvVaw1VQZcco4FfR2q1VaEUg1kH&ust=1
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https://www.google.com/url?sa=i&url=https%3A%2F%2Fwww.nature.
com%2Farticles%2Fnprot.2007.395&psig=AOvVaw2DlET1ExNgZgK9u9
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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)
15

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)
16

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)
17

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)
18

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)
19

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.
24

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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