PCR is a technique used to amplify a specific segment of DNA. It involves repeated cycles of heating and cooling of the DNA sample in the presence of DNA polymerase, primers, and nucleotides. During each cycle, the DNA denatures, the primers anneal to the DNA, and the polymerase extends the primers to make copies of the DNA segment. This allows millions of copies of the target DNA sequence to be generated from a very small initial sample. PCR is widely used in applications like disease diagnosis, DNA fingerprinting, and molecular cloning.

1. POLYMERASE CHAIN REACTION (PCR)
Merin alice george

2. WHAT IS PCR
• Cloning technique ;Used for making copies or amplifying a specific sequence of Dna in
a short period of time
OR
• It is a technique used to amplify a segment of DNA of interest or produce lots and lots
of copies.
• PCR enables produces millions of copies of a specific DNA sequence from an initially
small sample – sometimes even a single copy.
• Karry Mullis (1985)

3. PRINCIPLE
• Its principle is based on the use of DNA polymerase
involved in the replication of specific DNA sequences.
• In PCR, a short segment of DNA is amplified using
primer.
• DNA Polymerase synthesizes 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.

4. STEPS IN PCR
Denaturation
Annealing
Extension

5. Requirements
• DNA sample containing the desired segment to be amplified.
• Two nucleotide primers (20 bp long) specific for the two 3’ ends of the desired
segment.
• Four deoxyribonucleoside triphosphates ATP, d GTP, d CTP, d TTP
• A heat stable DNA polymerase
• Taq polymerase ( isolated from the bacterium Thermus aquaticus)
• Pfu ( from Pyrococcus furiosus)
• Vent (from Thermococcus litoralis )

6. STEPS
1. DENATURATION
The reaction mixture is heated to a temperature (usually 90 – 98⁰C→ denaturation of
DNA).
2. ANNEALING – The mixture is cooled to 40 ⁰C- 60⁰C - →annealing of the primer to the
complementary sequences in the DNA
3. EXTENSION OR POLYMERIZATION – The temperature is adjusted (For Taq
polymerase – optimum 70⁰C - 75⁰C) – DNA polymerase synthesizes the complementary
strands – first cycle is completed.
Repeat 20 – 30 times.
Automated PCR machines – Thermal Cyclers – specify the Number, duration of cycles etc.
after placing the complete reaction mixture for incubation

12. APPLICATIONS OF PCR
• In vitro amplification of genes and other DNA sequences
• To obtain definitive structural data on genes and DNA sequences when very
small amounts of DNA are available.
• Diagnosis of diseases by the identification of pathogens, such as viruses or
bacteria.
• Pre natal diagnosis of genetic diseases- Sickle cell anemia
Tay – Sachs disease ( a lethal autosomal recessive disorder- normal at birth but
undergo rapid neurological degeneration).
Cystic fibrosis – due to autosomal recessive mutation – salty sweat, infections
of lungs,pancreas, liver etc.

13. • Dna fingerprinting in forensic medicine.
• Molecular mapping
• Preparation of molecular markers.
• Monitoring of genetic engineering and gene therapy experiments
• Study of Dna Polymorphism.
• Gene tagging
• Determination of sex of embryos
• Study of molecular evolution