A biochemical technique used in Molecular Biology to amplify a specific fragment of target DNA. PCR is used in medical and biological research, including cloning, genetic analysis, genetic fingerprinting, diagnostics, pathogen detection and genetic fingerprinting

1. Technique of Polymerase Chain
Reaction (PCR)- Experimental
Biotechnology
Presented by
Dr. B. Victor., Ph.D.,
email : bonfiliusvictor@gmail.com
blog : bonvictor.blogspot.com

2. Presentation out line
• Polymerase chain reaction (PCR)
(Enzymatic Amplification of DNA)
• Origin and definition of PCR
• PCR –a DNA copying machine
• Requirements of PCR
• Thermostable DNA polymerases
• Critical steps in PCR
• Step cycle programme
• PCR protocol
• Variants of PCR
• Characterization of PCR product
• Problems and limitations
• Merits and applications

3. Polymerase chain reaction (PCR)
(Enzymatic Amplification of DNA)
PCR is a novel molecular
technique involving in
vitro enzymatic
replication of defined
DNA sequences.

4. Definitions of PCR
Definition-1
• A technique for amplifying DNA sequences in
vitro by separating the DNA into two strands
and incubating it with oligonucleotide
primers and DNA polymerase.
Definition -2
• A biochemical technique used in Molecular
Biology to amplify a specific fragment of
target DNA.

5. Origin of Polymerase chain reaction
(PCR)
In vitro DNA synthesis
• PCR was discovered by Kary B. Mulis in
1983 of Cetus Corporation, a Biotech
company in California, USA.
• He won the Nobel Prize for Chemistry in
1993 for ‘contributions to the developments
of methods within DNA-based chemistry’.
• ‘Taq polymerase’ an enzyme used in PCR was
described as ‘molecule of the year’ 1989.

6. A copying machine for DNA molecules
PCR multiplies a single, microscopic
strand of the DNA molecule into
billions of times within hours.
PCR had a major impact on
recombinant DNA technology.
PCR has multiple applications in
medicine, genetics, biotechnology, and
forensics.

7. PCR-A DNA multiplication technology
PCR is a powerful technique, in which from a
single copy of a DNA molecule, millions of copies
can be obtained with high accuracy, specificity and
in a very short time.
DNA amplification process in PCR is cyclical and
the concentration of DNA doubles at each cycle.
The total amount of DNA concentration
increases exponentially during the cyclical
process of PCR machine.

8. The ‘master mix’ components
for PCR machine
1. A thermostable DNA polymerase:
tag polymerase
2. A template DNA
3. A complete set of deoxynucleotide
triphosphates e.g. dATP, dCTP,
dGTP and dTTP
4. Tris buffer of pH 8.8
5. A pair of oligonucleotide primers
6. Mg 2+ and detergents
7. 2-mercaptoethanol to stabilize
proteins during thermal cycle.

9. Requirements for PCR
DNA template – DNA segment to be amplified.
Two primers- a short segment of DNA ( forward and
reverse primers) about 20–25 bases long .
Taq polymerase – an enzyme to synthesize DNA copies.
Deoxynucleotide triphosphates – the building blocks for
new DNA strand.
Buffer solution – a suitable chemical environment.
Divalent cations – Mg 2+ ions
Monovalent ions – Potassium ions
PCR machine – a thermal cycler

10. Thermostable DNA polymerase
• The thermophilic DNA polymerases catalyze
template-directed synthesis of DNA from
nucleotide triphosphates.
• Several thermostable polymerase enzymes are
used in PCR
Pfu DNA polymerase- Pyrococcus furiosus
Vent polymerase- Thermococcus litoralis
Taq polymerase- Thermus aquaticus

11. Oligonucleotide primers
• They are synthesized chemically to be
complementary to sequences which flank the
region of DNA to be amplified.
• They are usually about 20-25 nucleotides in
length.
• The primers are designed to anneal specifically
to the opposite strands of the template molecule.
• It is the specificity of the primer annealing
reaction which ensures that the PCR amplifies
the appropriate region of the template DNA.

12. Critical steps in PCR
Sample Target Primer
Preparation selection selection

13. 3 – temperature cycle in PCR
Temperature - 90-980C - separates two
strands of target DNA.
Temperature – 40-600C anneals two
complementary primers to the ends of
separated single strands of target DNA
Temperature 720 C allows taq
polymerase to use ss target DNA and
primers to synthesize new strands.

14. DNA thermal cycler use
‘Step cycle’ programme
Denature Anneal at
at 940C 550C for
For 20 sec 20 sec
Extend at 720C
for 30 sec
*For a total of 30 cycles *Overall single cycle time is 3.75 min.

15. Sources of sample material for PCR
Drop of
Hospital dried from Cells of Sperm or Mouth wash
tissue hair blood from mummified sperm
specimens the scene of material lysates Etc.
crime

16. PCR procedure-cycle of amplification
Denaturation
reaction
Extension Annealing
reaction reaction

17. PCR protocol
1. Denaturation of ds DNA template –melting target DNA-it is
the thermal denaturation of the dsDNA at 950C for 1 min.
2. Annealing of two oligonucleotide primers – 680C for 60 sec.
The annealing temperature is dependent on the length and
G+C content of the primer sequences.
3. Polymerase extension of dsDNA molecules – temp. raised
at 750C for about 30 sec.
The step cycle programme makes the instrument to heat
and cool to the set temperatures due to solid state Peltier-
effect device , which actively modulates the desired
temperature. There may be as many as 30-35 cycles.

18. PCR protocol

19. PCR-DNA synthesis cycle

20. Laboratory PCR technique
Prepare the Master Mix of reagents
and aliquot into tubes
Add DNA template(s)
Program thermal cycler, load with tubes
and start
Remove tubes and analyze results

21. Variants of PCR
1. Standard PCR – sequences of both ends of target
DNA have to be known. Two primers define the
ends of target DNA and only that part is amplified.
2. Single sided PCR – Here DNA is rearranged before
amplification so that only one primer is needed.
This is also called Anchored PCR.
3. Inverse PCR – DNA at primer sites rather than
between two primers is amplified because primer
sites which are bracketing may have important
sequence like promoter for triggering target gene
into action.

22. Characterization of PCR product
• Contamination of the reagents by foreign DNA
or annealing of primers to alternative sites in the
template DNA may produce unwanted DNA
molecules.
• Gel electrophoresis – to assess purity of product.
• Multiple bands in the electropherogram suggest
primers annealing to multiple sites.
• Smear of DNA suggests presence of excess
template DNA.

23. Uses of PCR
Cloning
Detection Forensic
of ancient DNA
DNA detection
Uses
of PCR
Detection Identifying
of viral transgenic
infection plants

24. Problems and limitations
Contamination of reaction PCR can not substitute for
mixture by bacteria, viruses, cell- based gene cloning ,
and our own DNA presents a when large amounts of a gene
real problem . are desired.
Taq polymerase used in
PCR often lack 3' to 5' PCRs of longer products are
exonuclease activity. This less efficient due to enzyme
enzyme lacks the ability to activity loss. Applies only to
correct mis-incorporated short DNA fragments
nucleotides.

25. Merits of PCR
Simplicity
Specificity
Much faster
Generate and modify DNA fragments of
defined length and sequence

26. Applications of PCR -1
 Detection of pathogens in food, water and
tissue specimens.
 Detection of tuberculosis, AIDS and other
microbial diseases.
 Diagnosis of genetic diseases-e.g. sickle cell
anemia, β-thalasemia, hemophilia
 Identification of criminals, disputed
parentage.
 Monitor gene expression in genetic
engineering or gene therapy experiments.

27. Applications of PCR -2
To study genetic profile of animals and to trace
evolutionary and cultural lineage of human
beings.
To study DNA polymorphism.
To determine orientation and location of
restriction fragments relative to one another.
To conduct microbial surveillance of the
environment.

28. Summary - applications of PCR
PCR is used in medical and biological
research, including cloning, genetic analysis,
genetic fingerprinting, diagnostics, pathogen
detection and genetic fingerprinting
PCR is valuable to scientists in gene
mapping, the study of gene functions and cell
identification.

29. References
• Saiki, R., Scharf, S., Faloona, F., Mullis, K., Horn, G., and
Erlich, H. (1985). Enzymatic amplification of beta-globin
genomic sequences and restriction site analysis for
diagnosis of sickle cell anemia. Science 230: 1350-54
• Mullis, K. and Faloona, F. (1987). Specific synthesis of
DNA in vitro via a polymerase-catalyzed chain
reaction. Methods Enzymol 155: 335-350.
• Mullis, K. (1990). The unusual origin of the polymerase
chain reaction. Scientific American April 56-65
• Rabinow, P. (1996). Making PCR: A story of
biotechnology. University of Chicago Press

30.  Dr.B.Victor is a highly experienced professor,
recently retired from the reputed educational
institution- St. Xavier’ s College, Palayamkottai,
India-627001.
 He was the dean of sciences, IQAC coordinator
and assistant controller of examinations.
 He has more than 32 years of teaching and
research experience
 He has taught a diversity of college courses and
guided 12 Ph.D scholars.
 Send your comments to :
bonfiliusvictor@gmail.com