POLYMERASE CHAIN REACTION, PCR, RT-PCR, Diagnostic Test,

1. POLYMERASE
CHAIN REACTION
DR MD SHAMIM
PG 3RD YEAR
DEPARTMENT OF PHARMACOLOGY
PATNA MEDICAL COLLEGE, PATNA

2. CONTENTS
INTRODUCTION
HISTORY
PURPOSE
 PCR COMPONENTS
 PCR STEPS
 TYPES OF PCR
 APPLICTIONS
 ADVANTAGES & DISADVANTAGES
 TAKE HOME MESSAGE

3. INTRODUCTION
 Polymerase Chain Reaction (PCR) is a laboratory technique used to produce
millions of copies of a specific segment of DNA within a short period of time.

4. HISTORY OF PCR
1983: Dr Kary B. Mullis developed PCR.
1986: Taq Polymerase is first used in PCR.
1988: Perkin Elmer introduces the automated thermal cycler.
1993: Dr Karry B. Mullis awarded Nobel Prize in Chemistry
for PCR technology.
Dr Karry B. Mullis

5. PURPOSE
 To amplify the specific DNA sequences in vitro (test tube).
 Target DNA sequences to be amplified several million folds in just few hours.

6. PCR COMPONENTS
DNA Template
 DNA segment to be amplified.
 Containing target sequence of nucleotides.
DNA Polymerase
 Taq DNA Polymerase ,obtained from Thermus aquaticus (a thermophilic
bacterium). (70-720C).
 Synthesises new strands of DNA from the target sequence.
Primers
 Synthetic single DNA strand.
18-25 nucleotides complementary to 3’ end of the DNA template.

7. PCR COMPONENTS
Nucleotides
 The building blocks of DNA, from which the DNA Polymerase synthesises
a new DNA strand.
Buffers
 MgCl2
 Tris-HCl
 KCl
 Gelatin or Bovine Serum Albumin

8. PCR MASTERMIX
Premixed, ready to use solution containing,
 Taq DNA Polymerase
 dNTPs
 MgCl2
 Other Buffers
 At optimal concentrations for efficient
amplification of DNA templates by PCR

9. PCR COMPONENTS
C
G
A
T
DNA Template Primers
Nucleotides
Taq Polymerase
Buffers
Thermal Cycler
PCR Cycle

10. PCR STEPS
Taq
Taq
Primer
Primer
Denaturation
940C
1 minute
Annealing
540C
1 minute
Extension
720C
2 minutes
Extended DNA
Next Cycle
DNA Template
3’
5’
3’ 5’
5’
3’ 5’
3’
3’
3’
3’
3’
3’
3’
5’
5’
5’
5’
5’
5’
5’ 3’
3’
5’
nth Cycle
Total number of DNA after nth cycle = 2n

11. 50
100
70
90
1 2 3 4 5 6 7
60
80
TEMP
(0C)
TIME (Mins)
DENATURATION
ANNEALING
EXTENSION
DENATURATION
NEXT CYCLE
PCR STEPS

12. TYPES OF PCR
Multiplex PCR
 More than one target segment can be amplified at a time.
 Simultaneous analysis of multiple targets in a single sample.
 Used to detect multiple pathogens by using multiple primers.
 Time savings.
Reverse Transcriptase PCR
Used to reverse-transcribe and amplify RNA to cDNA.
 PCR is preceded by a reaction using reverse transcriptase, an enzyme
that converts RNA into cDNA.
 Used to detection of RNA virus like HCV, SARS CoV-2, HIV

13. TYPES OF PCR
Quantitative PCR
 Also known as real time PCR.
 Used to measure the specific amount of target DNA in a sample.
 Quantification of target species ( total bacteria) in a given sample.
 Fluorescent dye Sybr green is used.
 Fluorescence signal is proportional to amount of the DNA synthesised.
Nested PCR
 Used to increase the specificity of DNA amplification.
 Two sets of primers are used in two successive reactions.
 In the first PCR one pairs of primer is used to generate DNA products
which will be the target for the second reaction.
 Used to detection of pathogens that occur with very few amount.

14. APPLICATIONS
Diagnosis
 Genetic Disease
 Cystic Fibrosis
 Duchenne muscular dystrophy
 Infectious Disease
 COVID-19, TB, HIV
 Faster & Efficient than conventional serological tests.
Genetic Engineering
 Gene Expression Studies
 Human Genome Project

15. APPLICATIONS
Forensic Studies
 DNA Fingerprinting
 Paternity Testing
 Organ Transplantation
Molecular Studies
 Genotyping
 Pre-natal diagnosis
 Genetic matching
 Mutation screening
 Detection of Pathogens

16. ADVANTAGES
ADVANTAGES
SIMPLE
SENSITIVE
NON-
INVASIVE
FAST

17. DISADVANTAGES
DIADVANTAGES
FALSE
POSITIVE
FALSE
NEGATIVE

18. TAKE HOME MESSAGE
 PCR is a laboratory technique used to amplify a target DNA from few samples
to billions of copies within a few hours.
 PCR minimises the time to generate large quantities of specific DNA.
 It has an important role in Diagnostic, Forensic, Molecular and Genetic studies.
 It is a Simple, Fast and Non-invasive procedure.

19. THANK YOU