Polymerase Chain Reaction History of PCR Instrumentation of PCR Principle of PCR Components of PCR Steps of PCR Optimal PCR Factors Applications of PCR

1. Polymerase Chain Reaction
&
its Applications

2. Review Literature
History of PCR
METHODOLOGY
Polymerase Chain Reaction
Principle of PCR
Instrumentation of PCR
Components of PCR
Applications of PCR
Steps of PCR
Optimal PCR Factors
Presentation
Outline

3. • Sometimes called "molecular photocopying"
• Polymerase chain reaction is a fast and inexpensive technique that
results in exponential amplification of a desired region (small
segments ) of a DNA molecule in vitro.
• This reaction allows a single or a few copies of DNA to be
replicated into millions or billions of copies.
• Why PCR ? Because the significant amounts of DNA sample are
necessary for molecular and genetic analyses, studies of isolated
pieces of DNA are nearly impossible without PCR amplification.
Polymerase Chain Reaction (PCR)
PCR stands for Polymerase Chain Reaction.
Why “Polymerase”? Because the only enzyme used in the reaction is DNA polymerase.
Why “Chain” ? Because products of first reaction become substrates of following one and so on.

4. COMMUNITY LEVEL PHYSIOLOGICAL
PROFILING
• PCR technique was invented by Kary Mullis, a
Research Scientist at a California Biotech
Company, Cetus, in 1983.
• For this work, Mullis received the 1993 Noble
Prize in Chemistry.
• This tool is commonly used in the molecular
biology and biotechnology labs.
Kary B. Mullis
The Nobel Prize in Chemistry 1993
Prize motivation: "for his invention of the
polymerase chain reaction (PCR) method."
History of PCR

5. Instrumentation of PCR

6. • The PCR technique is based on the enzymatic replication of
DNA.
• In PCR, a short segment of DNA is amplified using primer
mediated enzymes. DNA Polymerase synthesises new strands
of DNA complementary to the template DNA.
• The DNA polymerase can add a nucleotide to the pre-
existing 3’-OH group only. Therefore, a primer is required.
Thus, more nucleotides are added to the 3’ prime end of the
DNA polymerase.
COMMUNITY LEVEL PHYSIOLOGICAL
PROFILING
Principle of PCR

7. 1) Target DNA - contains the sequence to be amplified.
2) Pair of Primers - oligonucleotides that define the
sequence to be amplified.
3) Nucleotides (dNTPs) - single units of the bases A,
T, G, and C, which are essentially "building blocks" for
new DNA strands.
4) Thermo-stable DNA Polymerase - enzyme that
catalyzes the reaction
5) Mg++ ions - cofactor of the enzyme
6) Buffer solution – maintains pH and ionic strength of
the reaction solution suitable for the activity of the enzyme
Components of PCR

8. Steps of PCR

10. Optimal PCR Factors are
1. PCR Primers
2. DNA Polymerase
3. Annealing Temperature
4. Melting Temperature
5. G/C content
Optimal PCR Factors
PCR Primers
 Correctly designed pair of primers is required
 Typical primers are 18-28 bases in length ,Having
50-60% GC composition
 Have a balanced distribution of G/C and A/T rich
domains
 Are not complementary to each other at the 3' ends
to avoid primer- dimer forming artifacts.
 Not self complementary “Hairpin” formation.

11. Optimal PCR Factors are
1. PCR Primers
2. DNA Polymerase
3. Annealing Temperature
4. Melting Temperature
5. G/C content
Optimal PCR Factors
DNA Polymerase
 The most widely characterized polymerase is from
Thermus aquaticus (Taq).
 This thermophilic bacterium lives in hot springs
and consist of a single polypeptide chain has an
optimum polymerization temperature of 70 – 80 C.
 It lacks proof reading exonuclease activity.
 Other polymerases can be used, e.g.:
1) Tma DNA Polymerase from Thermotoga maritama,
2) Pfu DNA Polymerase from Pyrococcus furiosus.

12. Optimal PCR Factors are
1. PCR Primers
2. DNA Polymerase
3. Annealing Temperature
4. Melting Temperature
5. G/C content
Optimal PCR Factors
Annealing Temperature
 Very important since the success and specificity of
PCR depend on it because DNA-DNA hybridization is
a temperature dependent process.
 If annealing temperature is too high, pairing
between primer and template DNA will not take place
then PCR will fail.
 Ideal Annealing temperature must be low enough to
enable hybridization between primer and template but
high enough to prevent amplification of non- target
sites.
 Should be usually 1-2°C or 5°C lower than melting
temperature of the template-primer duplex.

13. Melting Temperature
 Temperature at which 2 strands of the duplex
dissociate.
 It can be determined experimentally or calculated
from formula
Tm = (4(G+C)) + (2(A+T))
Optimal PCR Factors are
1. PCR Primers
2. DNA Polymerase
3. Annealing Temperature
4. Melting Temperature
5. G/C content
Optimal PCR Factors

14. Optimal PCR Factors are
1. PCR Primers
2. DNA Polymerase
3. Annealing Temperature
4. Melting Temperature
5. G/C content
Optimal PCR Factors
G/C content
 Ideally a primer should have a near random mix
of nucleotides, a 50% GC content.
 There should be no PolyG or PolyC stretches that
can promote non-specific annealing

15. The following are the applications of PCR
Applications of PCR
 Used as a tool in
genetic fingerprinting.
 Identifying the
criminal from millions
of people.
 Paternity tests
 Compare the genome of
two organisms in genomic
studies.
 In the phylogenetic
analysis of DNA from any
source such as fossils.
 Analysis of gene
expression and Gene
Mapping
 Testing of genetic
disease mutations.
 Monitoring the gene
in gene therapy.
 Detecting disease-
causing genes in the
parents.
Medicine Forensic Science Research

16. REFERANCES
 Satyanarayana, U., & Chakrapani, U. (2008). Essentials of biochemistry. Book and Allied, Kolkata, India,
.
 David Hames and Nigel Hooper (2005). Biochemistry. Third ed. Taylor & Francis Group: New York.
 Bailey, W. R., Scott, E. G., Finegold, S. M., & Baron, E. J. (1986). Bailey and Scott’s Diagnostic
microbiology. St. Louis: Mosby.
 Sastry A.S. & Bhat S.K. (2016). Essentials of Medical Microbiology. New Delhi : Jaypee Brothers Medical
Publishers.
References