Polymerase chain reaction (PCR)

Polymerase Chain Reaction
History of PCR
Instrumentation of PCR
Principle of PCR
Components of PCR
Steps of PCR
Optimal PCR Factors
Applications of PCR

1. Polymerase Chain Reaction & its Applications  

2. Review Literature History of PCR METHODOLOGY Polymerase Chain Reaction Principle of PCR Instrumentation of PCR Components of PCR Applications of PCR Steps of PCR Optimal PCR Factors Presentation Outline

3. Sometimes called "molecular photocopying" Polymerase chain reaction is a fast and inexpensive technique that results in exponential amplification of a desired region (small segments ) of a DNA molecule in vitro. This reaction allows a single or a few copies of DNA to be replicated into millions or billions of copies. Why PCR ? Because the significant amounts of DNA sample are necessary for molecular and genetic analyses, studies of isolated pieces of DNA are nearly impossible without PCR amplification. Polymerase Chain Reaction ( PCR) PCR stands for P olymerase C hain R eaction. Why “Polymerase”? Because the only enzyme used in the reaction is DNA polymerase. Why “Chain” ? Because products of first reaction become substrates of following one and so on.

4. COMMUNITY LEVEL PHYSIOLOGICAL PROFILING PCR technique was invented by Kary Mullis, a Research Scientist at a California Biotech Company, Cetus , in 1983. For this work, Mullis received the 1993 Noble Prize in Chemistry. This tool is commonly used in the molecular biology and biotechnology labs. Kary B. Mullis The Nobel Prize in Chemistry 1993 Prize motivation : "for his invention of the polymerase chain reaction (PCR) method." History of PCR

5. Instrumentation of PCR

6. The PCR technique is based on the enzymatic replication of DNA. In PCR, a short segment of DNA is amplified using primer mediated enzymes. DNA Polymerase synthesises 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. Thus, more nucleotides are added to the 3’ prime end of the DNA polymerase. COMMUNITY LEVEL PHYSIOLOGICAL PROFILING Principle of PCR

7. 1) Target DNA - contains the sequence to be amplified. 2) Pair of Primers - oligonucleotides that define the sequence to be amplified. 3) Nucleotides ( dNTPs ) - single units of the bases A, T, G, and C, which are essentially "building blocks" for new DNA strands. 4) Thermo-stable DNA Polymerase - enzyme that catalyzes the reaction 5) Mg++ ions - cofactor of the enzyme 6) Buffer solution – maintains pH and ionic strength of the reaction solution suitable for the activity of the enzyme Components of PCR

8. Steps of PCR

9. Optimal PCR Factors are PCR Primers DNA Polymerase Annealing Temperature Melting Temperature G/C content Optimal PCR Factors PCR Primers Correctly designed pair of primers is required Typical primers are 18-28 bases in length ,Having 50-60% GC composition Have a balanced distribution of G/C and A/T rich domains Are not complementary to each other at the 3' ends to avoid primer- dimer forming artifacts. Not self complementary “Hairpin” formation.

10. Optimal PCR Factors are PCR Primers DNA Polymerase Annealing Temperature Melting Temperature G/C content Optimal PCR Factors DNA Polymerase The most widely characterized polymerase is from Thermus aquaticus ( Taq ). This thermophilic bacterium lives in hot springs and consist of a single polypeptide chain has an optimum polymerization temperature of 70 – 80 C. It lacks proof reading exonuclease activity. Other polymerases can be used, e.g.: 1) Tma DNA Polymerase from Thermotoga maritama , 2) Pfu DNA Polymerase from Pyrococcus furiosus .

11. Optimal PCR Factors are PCR Primers DNA Polymerase Annealing Temperature Melting Temperature G/C content Optimal PCR Factors Annealing Temperature Very important since the success and specificity of PCR depend on it because DNA-DNA hybridization is a temperature dependent process. If annealing temperature is too high, pairing between primer and template DNA will not take place then PCR will fail. Ideal Annealing temperature must be low enough to enable hybridization between primer and template but high enough to prevent amplification of non- target sites. Should be usually 1-2°C or 5°C lower than melting temperature of the template-primer duplex.

12. Melting Temperature Temperature at which 2 strands of the duplex dissociate. It can be determined experimentally or calculated from formula Tm = (4(G+C)) + (2(A+T)) Optimal PCR Factors are PCR Primers DNA Polymerase Annealing Temperature Melting Temperature G/C content Optimal PCR Factors

13. Optimal PCR Factors are PCR Primers DNA Polymerase Annealing Temperature Melting Temperature G/C content Optimal PCR Factors G/C content Ideally a primer should have a near random mix of nucleotides, a 50% GC content. There should be no PolyG or PolyC stretches that can promote non-specific annealing

14. The following are the applications of PCR Applications of PCR Used as a tool in genetic fingerprinting. Identifying the criminal from millions of people. Paternity tests Compare the genome of two organisms in genomic studies. In the phylogenetic analysis of DNA from any source such as fossils. Analysis of gene expression and Gene Mapping Testing of genetic disease mutations. Monitoring the gene in gene therapy. Detecting disease-causing genes in the parents. Medicine Forensic Science Research

15. REFERANCES Satyanarayana , U., & Chakrapani , U. (2008). Essentials of biochemistry.  Book and Allied, Kolkata, India, . David Hames and Nigel Hooper (2005). Biochemistry. Third ed. Taylor & Francis Group: New York. Bailey, W. R., Scott, E. G., Finegold , S. M., & Baron, E. J. (1986). Bailey and Scott’s Diagnostic microbiology. St. Louis: Mosby. Sastry A.S. & Bhat S.K. (2016). Essentials of Medical Microbiology. New Delhi : Jaypee Brothers Medical Publishers. References