1. POLYMERASE CHAIN REACTION BY AVIJIT MALLICK
THE UNIVERSITY OF THE WEST OF SCOTLAND

2. The process was first described by Kjell Kleppe and 1968 Nobel laureate H. Gobind Khorana.
In recognition of his improvement of the polymerase chain reaction (PCR) technique, Kary Banks Mullis shared the 1993 Nobel Prize in Chemistry with Michael Smith.
Dr Kary Banks Mullis

3. POLYMERASE CHAIN REACTION
The polymerase chain reaction (PCR) is a technology in molecular biology used to amplify a single copy or a few copies of a piece of DNA across several orders of magnitude, generating thousands to millions of copies of a particular DNA sequence.
Developed in 1983 by Kary Mullis,PCR is now a common and often indispensable technique used in medical and biological research labs for a variety of applications. (

4. STEPS OF THE REACTION
Initialization step: Only for polymerase enzyme that require heat activation. The reaction is heated at °C for 1-9 minutes.
Denaturation step: The reaction is heated to °C for seconds. DNA strands separate from each other thus yielding single stranded DNA.
Annealing step: The reaction temperature is lowered to 50–65 °C for 20–40 seconds allowing annealing of the primers to the single-stranded DNA template. 
Extension/elongation step: The temperature at this step depends on the DNA polymerase used; Taq polymerase has its optimum activity temperature at 75–80 °C. Polymerase reaction takes place.
Denaturation step again and the cycle continues for ~35 min.

6. Detection of PCR products
By gel electrophoresis.
is messy and dangerous
Also results very inaccurate.
By genetic analyzer. (Beckman CEQ8000)
Capillary electrophoresis. (

7. BASIC REQUIREMENTS FOR PCR SET UP
DNA template that contains the DNA region (target) to be amplified.
Two primers that are complementary to the 3' (three prime) ends of each of the sense and anti-sense strand of the DNA target.
Taq polymerase or another DNA polymerase with a temperature optimum at around 70 °C.
Deoxynucleoside triphosphate the building-blocks from which the DNA polymerase synthesizes a new DNA strand.
Buffer solution, providing a suitable chemical environment for optimum activity and stability of the DNA polymerase.
Bivalent cations, magnesium or manganese ions; generally Mg2+ is used, but Mn2+ can be utilized for PCR-mediated DNA mutagenesis, as higher Mn2+ concentration increases the error rate during DNA synthesis.
Monovalent cation potassium ions.

8. PRIMER DESIGN IS VERY CRUCIAL
 primers should be bases in length. 
It must be specific.  
Base composition should be 50-60% (G+C).
3' End Stability: It is the maximum ΔG value of the five bases from the 3' end. An unstable 3' end (less negative ΔG) will result in less false priming.
Primers with long runs of a single base should generally be avoided as they can misprime. Maximum accepted is 4bp. Repeats should also be avoided.
Tm (melting temperature) of the primer pair should be 3-5 °C of each other.
Tm in range °C.
Primers must not dimerise with each other.
Avoid secondary structure formation (hair loop).

9. APPLICATIONS OF PCR Medical Applications.
Genetic testing.
Tissue typing (organ transplantation).
Oncogenes testing.
Infectious Disease application.
Detection of diseases such as Tuberculosis through detection of very small number of disease organism.
HIV.
Forensic applications.
Genetic fingerprinting.
Parental testing.
Research Applications.
DNA cloning.
Gene expression. (qPCR)
Hybridization probes.
Etc.

10. Quantitative PCR Uses fluoroscenated probe.
Amplified DNA is detected in real time.
A relationship exists between the initial amount of target DNA and the amount produced after a given number of cycle of PCR. (

11. Reverse transcriptase PCR
While RT-PCR is used to qualitatively detect gene expression through creation of complementary DNA (cDNA) transcripts from RNA, qPCR is used to quantitatively measure the amplification of DNA using fluorescent probes (

12. IN Diagnosis
Detecting translocation in AML by RT-PCR followed by fragment analysis.
Bone marrow/Blood
(extraction of RNA)
cDNA
PCR using specific primers
Nested PCR(PCR modified to prevent nonspecific binding in products due to the amplification of unexpected primer binding sites)
Detection by Fragment analysis
[National cancer Institute

13. Detecting Chimaerism post BMT
To detect an identifiable difference between a patient and their donor
Ability to measure the proportion of donor present in the blood or bone marrow post transplant
Microsatellite analysis used.
Two sets of primers that are informative is used.
Primers labelled with fluorophore.
Result from (Dr Fiona M. Reid)

14. Polymerase error
Taq polymerase lacks a 3' to 5' exonuclease activity so has no error-proof-reading activity.
The lack in 3' to 5' proofreading of the Taq enzyme results in a high error rate (mutations per nucleotide per cycle) of approximately 1 in 10,000 bases.
Several "high-fidelity" DNA polymerases is now engineered with the 3' to 5' exonuclease activity.
For example-KOD DNA polymerase, a recombinant form of Thermococcus kodakaraensis KOD1.

15. Summary
technology in molecular biology used to amplify a single copy or a few copies of a piece of DNA across several orders of magnitude.
a common and often indispensable technique used in medical and biological research labs for a variety of applications.
consisting of cycles of repeated heating and cooling of the reaction for DNA melting and enzymatic replication of the DNA.

16. References
Joseph Sambrook and David W. Russel (2001). Molecular Cloning: A Laboratory Manual (3rd ed.). Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory Press. ISBN   Chapter 8: In vitro Amplification of DNA by the Polymerase Chain Reaction.
Sharkey, D. J.; Scalice, E. R.; Christy, K. G.; Atwood, S. M.; Daiss, J. L. (1994). "Antibodies as Thermolabile Switches: High Temperature Triggering for the Polymerase Chain Reaction". Bio/Technology 12 (5): 506–509.
Chien A, Edgar DB, Trela JM (1976). "Deoxyribonucleic acid polymerase from the extreme thermophile Thermus aquaticus". J. Bacteriol 127 (3): 1550–1557.
Lawyer, F.; Stoffel, S.; Saiki, R.; Chang, S.; Landre, P.; Abramson, R.; Gelfand, D. (1993). "High-level expression, purification, and enzymatic characterization of full-length Thermus aquaticus DNA polymerase and a truncated form deficient in 5' to 3' exonuclease activity". PCR methods and applications 2 (4): 275–287.
Applications of PCR. [Online]. Available: 16 February, 2015].

17. THANK YOU!!!!