4. Brief introduction to Polymerase Chain Reaction.pptx

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1. Introduction To PCR ( Polymerase Chain Reaction) Prepared by: Ms. Harshada R. Bafna. M. Pharm (Quality Assurances)

2. The polymerase chain reaction (PCR) is a laboratory (in vitro) technique for generating large quantities of a specified DNA. Polymerase chain reaction (PCR) is technique for generating large quantities of a specified DNA. Obviously, PCR is a cell-free amplification technique for synthesizing multiple identical copies (billions) of any DNA of interest . Developed in 1984 by Karry Mullis PCR is now considered as a basic tool for the molecular biologist.  It is an in-vitro technique to generate large quantities of a specified DNA. Polymerase Chain Reaction 2

3. Principle of PCR: The double-stranded DNA of interest is denatured to separate into two individual strands. Each strand is then allowed to hybridize with a primer (renaturation). The primer-template duplex is used for DNA synthesis (the enzyme- DNA polymerase). These three steps: denaturation, renaturation and synthesis are repeated again and again to generate multiple forms of target DNA. 3

4. The essential requirements for PCR are listed below: A target DNA (100-35,000 bp in length). Two primers (synthetic oligonucleotides of 17-30 nucleotides length) that are complementary to regions flanking the target DNA. Four deoxyribonucleotides (dATP, dGTP, dCTP, dTTP). A DNA polymerase that can withstand at a temperature upto 95° C (i.e., thermo-stable). The reaction mixture contains the target DNA , two primers (in excess), a thermo-stable DNA polymerase (isolated from the bacterium Thermus aquaticus) (i.e., Taq DNA polymerase) and four deoxyribonucleoties . The actual technique of PCR involves repeated cycles for amplification of target DNA. Technique of PCR 4

5. 1. Denaturation: On raising the temperature to about 95° C for about one minute, the DNA gets denatured and the two strands separate. 2. Renaturation or annealing: As the temperature of the mixture is slowly cooled to about 55° C, the primers base pair with the complementary regions flanking target DNA strands. This process is called renaturation or annealing. High concentration of primer ensures annealing between each DNA strand and the primer rather than the two strands of DNA. Each cycle has three stages: Denaturation Renaturation Synthesis 5

6. 3. Synthesis: The initiation of DNA synthesis occurs at 3′-hydroxyl end of each primer. The primers are extended by joining the bases complementary to DNA strands. The synthetic process in PCR is quite comparable to the DNA replication of the leading strand. However, the temperature has to be kept optimal as required by the enzyme DNA polymerase. For Taq DNA polymerase , the optimum temperature is around 75° C (for E. coli DNA polymerase , it is around 37° C). The reaction can be stopped by raising the temperature (to about 95° C). Each cycle of PCR takes about 3-5 minutes. In the normal practice, the PCR is carried out in an automated machine. 6

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8. The new DNA strand joined to each primer is beyond the sequence that is complementary to the second primer. These new strands are referred to as long templates and they will be used in the second cycle. For the second cycle of PCR, the DNA strands (original + newly synthesized long template) are denatured, annealed with primers and subjected to DNA synthesis. At the end of second round, long templates, and short templates are formed. In the third cycle of PCR, the original DNA strands along with long and short templates are the starting materials. The technique of denaturation, renaturation and synthesis are repeated. This procedure is repeated again and again for each cycle. 8

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11. Types of PCR Inverse PCR: used to amplification of those DNA sequence which are away from primer. Anchored PCR: only one primer is used. RT-PCR: Reverse transcription-mediated PCR includes a single application combining the process of cDNA synthesis and PCR amplification. Asymmetric PCR: used to generate single strand copies of DNA sequence which can be directly used for DNA sequencing. AP-PCR: Arbitrary primed PCR is type of random amplification polymorphic DNA. 11

12. Applications of PCR Medical Applications: Genetic testing for presence of genetic disease mutations. Eg: hemoglobinopathies, cystic fibrosis, other inborn errors of metabolism. Detection of disease causing genes in suspected parents who act as carriers. Study of alteration to oncogenes may help in customization of therapy Can also be used as part of a sensitive test for tissue typing, vital to organ transplantation genotyping of embryo. Helps to monitor the gene in gene therapy Infectious disease Applications: Analyzing clinical specimens for the presence of infectious agents, including HIV, hepatitis, malaria, tuberculosis etc. Detection of new virulent subtypes of organism that is responsible for epidemics. 12

13. Forensic Applications: Can be used as a tool in genetic fingerprinting. This technology can identify any one person from millions of others in case of : crime science. Research and Molecular Genetics: In genomic studies: PCR helps to compare the genomes of two organisms and identify the difference between them. In Human genome project for aim to complete mapping and understanding of all genes of human beings. 13

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