Brief discussion about PCR. will help you if you quickly want to learn the basics for an exam or a presentation.

1. Polymerase Chain Reaction (PCR)
Presented by :
Ashes Karki Chhetri
201503015

2. What is PCR?
- In vitro technique for amplification of a region of DNA
- Allows amplification of specific DNA fragments
- Possible to generate thousands to million copies

3. BRIEF HISTORY OF PCR
1966 - Thomas Brock, Thermus aquaticus
1983 - Kary Mullis, PCR
1985 - Saiki publish first application of PCR
1989 - Taq pol, “molecula of the year”

4. COMPONENTS OF PCR
1. DNA template
- DNA target region
2. Primers
- Complimentary to the 3’ end of sense and anti sense strand of target
DNA
3. DNA polymerase
- Enzyme that polymerizes new DNA strand
4. dNTPs
- Compounds with nitrogenous base, sugar, tri phosphate
5. Buffer
- Provide suitable chemical environment

5. 6. Bivalent cations
- Typically Mg and Mn
7. Thermal cycler
- Heating and cooling of reactants

6. PRINCIPLE
PCR relies on the principle of thermal cycling.

7. STEPS IN PCR
Relies on three thermal cycling steps to achieve amplification of DNA.
1. DENATURATION
- Initial step
- During this step reaction chamber is heated to 94-98°C, causes melting or
denaturation of ds DNA, breaking H bonds
- Yields two ss DNA molecules

8. 2. ANNEALING
- In this step reaction temp. is lowered
- Facilitates annealing of primer to each template, H bonding
- Primers are complimentary to 3’ end of each strand, serve as starting point

9. 3. EXTENSION
- In this step, primers are extended
- Action of DNA pol, adds free dNTPs to the primer

11. 4. Final hold
- Final step cools the reaction chamber, 4-15°C
- May be employed for short term storage of PCR products
The final quantification of fragments are done by separating
them in usually agarose gel electrophoresis.

12. TYPES OF PCR
1. Inverse PCR
- Amplification of DNA of unknown sequence
- Identification of genomic inserts
2. RT PCR ( reverse transcription)
- For amplifying DNA from RNA
- Reverse transcriptase reverse transcribes RNA into cDNA
3. Quantitative Real Time PCR
- Quantitatively measures starting amount, DNA, cDNA or
RNA
- Commonly used to determine whether DNA seq present
in sample

13. APPLICATIONS OF PCR
1. Disease diagnosis
- Analysing clinical specimens for the presence of infectious agents
- Detection of new virulent subtype of organisms
2. Forensic application
- Can be used as a tool in genetic fingerprinting
- Can be used to evaluate evidence at the scene of crime
3. Research and modular genetics
- In genomic studies : helps to compare the genomes of two organism and
identify difference between them
- In phylogenetic analysis : minute quantity of DNA from any source
- In study of gene expression and PCR based mutagenesis.