The Polymerase Chain Reaction (PCR) is a technique used to amplify a specific region of DNA to produce enough copies to be adequately tested. It involves using a special DNA polymerase (Taq) to make many copies of a short length of DNA defined by primers. Kary Mullis invented PCR and was awarded the 1993 Nobel Prize in Chemistry for his invention, which revolutionized research and forensics by allowing DNA to be amplified.

1. • The Polymerase Chain Reaction
(PCR) was not a discovery, but
rather an invention
• A special DNA polymerase
(Taq) is used to make many
copies of a short length of DNA
(100-10,000 bp) defined by
primers
• Kary Mullis, the inventor of
PCR, was awarded the 1993
Nobel Prize in Chemistry

2. Polymerase Chain Reaction (PCR)
• PCR is a technique which is used to amplify the number
of copies of a specific region of DNA, in order to
produce enough DNA to be adequately tested.
• The purpose of a PCR is to make a huge number of
copies of a gene. As a result, it now becomes possible to
analyze and characterize DNA fragments found in minute
quantities in places like a drop of blood at a crime scene

3. PCR Thermocycler

4. What all PCR Can Do ?
• Starting with one original copy an almost infinite
number of copies can be made using PCR
• “Amplified” fragments of DNA can be sized using
electrophoresis
• Defective genes can be amplified to diagnose any
number of illnesses
• Genes from pathogens can be amplified to identify
them (i.e., HIV, Vibrio sp., Salmonella sp. etc.)

5. PROCEDURE …..

6. PCR Reagents
• 1X Buffer
– 10mM Tris-HCl, 50mM KCl
• MgCl2
– 1mM - 4mM (1.5mM)
• dNTPs
– 200μM
• Primers
– 100nM-1μM, 200nm (or less) for real time analysis
• DNA polymerase
– Taq DNA polymerase is thermostable
– 1-4 Units (1 unit)
• DNA
– 10pg-1μg (20ng)

8. Polymerase Chain Reaction

10. More Cycles = More DNA
Number of cycles
0 10 15 20 25 30
Size
Marker

11. • Traditional PCR has advanced from detection at
the end-point of the reaction to detection while the
reaction is occurring (Real-Time).
• Real-time PCR uses a fluorescent reporter signal
to measure the amount of amplicon as it is
generated. This kinetic PCR allows for data
collection after each cycle of PCR instead of only
at the end of the 20 to 40 cycles.

12. Real-time PCR advantages
* amplification can be monitored real-time
* no post-PCR processing of products
(high throughput, low contamination risk)
* ultra-rapid cycling (30 minutes to 2 hours)
* wider dynamic range of up to 1010-fold
* requirement of 1000-fold less RNA than conventional
assays
(6 picogram = one diploid genome equivalent)
* detection is capable down to a two-fold change
* confirmation of specific amplification by melting curve
analysis
* most specific, sensitive and reproducible
* not much more expensive than conventional PCR
(except equipment cost)

13. Applications of PCR
• Classification
of organisms
• Genotyping
• Molecular
archaeology
• Mutagenesis
• Mutation
detection
• Sequencing
• Cancer research
• Detection of
pathogens
• DNA
fingerprinting
• Drug discovery
• Genetic
matching
• Genetic
engineering
• Pre-natal
diagnosis

14. Thank you
THANK YOU