Polymerase chain reaction (pcr)

Base information on PCR, Phases of PCR and Application

1. Polymerase Chain Reaction (PCR) Dr. T. Ramesh Assistant Professor of Zoology Vivekananda College

2. Contents

3. Introduction PCR, polymerase chain reaction, is an in-vitro technique- Large quantities of specified DNA Cell free Amplification techniques-Synthesis of multiple copies of DNA 1966, Thomas Brock discovers Thermus aquaticus 1984, Kary Mullis postulated the concept of PCR (Nobel Prize in 1993) 1985, Saiki publishes the first application of PCR 1985 , Cetus Corp . Scientists isolate Thermostable Taq Polymerase (from T . aquaticus ), which revolutionized PCR

4. PCR- Principles Double strand DNA- denatured Each strand then allow to Hybridize with primers Template duplex is used for synthesis of DNA Repeat these again and again with optimum condition of Temp.-Multiple copies can produced PCR instrument

5. Reaction Components DNA template - The double stranded DNA of interest separated from the sample. Primers - Short pieces of single stranded DNA ( often 20-30 bp ) which are complementary to the 3’ ends. Enzyme - Usually a thermostable Taq polymerase that does not rapidly denature at high temperatures. Deoxynucleotides - Single units of the bases A, T, G, and C ( dATP , dTTP , dGTP , dCTP ). Buffers- Magnesium & Potassium

6. dsDNA The double stranded DNA of interest separated from the sample Size range 100-35000 bp length

7. PRIMERS 2 sets of primers Generally 20-30 nucleotides long Synthetically produced complimentary to the 3’ ends of target DNA not complimentary to each other

8. Enzymes Usually Taq DNA Polymerase or anyone of the natural or Recombinant thermostable polymerases Stable at T ° up to 95 ° C High processivity Taq Pol has 5’-3’ exo only Thermus aquaticus

9. Deoxynucleotides Single units of the bases A, T, G, and C ( dATP , dTTP , dGTP , dCTP ) They provide the energy for polymerization and the building blocks for DNA synthesis.

10. Buffers Magnesium & Potassium to provide the optimal conditions for DNA denaturation and renaturation . It also important for polymerase activity, stability.

11. Cycles in PCR On rising the temperature to about 95° C/1 min The DNA gets denatured During this, the double stranded DNA is denatured to single strands Due to breakage in weak hydrogen bonds . Denaturation

12. 2. Renaturation The reaction temperature is rapidly lowered to 54-60°C for 20-40 seconds . This allows the primers to bind (anneal) to their complementary sequence in the template DNA. Renaturation or Annealing. Binding depends conc. of primers.

13. 3. Synthesis Also known at extension or elongation This step usually occurs at 72-80°C ( most commonly 72°C ). The polymerase enzyme sequentially adds bases to the 3′ each primer, extending the DNA sequence in the 5′ to 3′ direction. Under optimal conditions, DNA polymerase will add about 1,000 bp /minute .

14. Standard T hermocycle

15. Target Amplification

16. Real-time PCR Quantitative real time PCR (Q-RT PCR) Reverse Transcriptase PCR (RT-PCR) Multiplex PCR Nested PCR Long-range PCR Single-cell PCR Fast-cycling PCR Methylation-specific PCR (MSP) Hot start PCR High-fidelity PCR In situ PCR Variable Number of Tandem Repeats (VNTR) PCR Asymmetric PCR Repetitive sequence-based PCR Overlap extension PCR Assemble PCR Inter sequence-specific PCR(ISSR) Ligation-mediated PCR Methylation – specifin PCR Mini primer PCR Solid phase PCR Touch down PCR, etc. Types of PCR

17. Applications Medical 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. Helps to monitor the gene in gene therapy Analyzing clinical specimens for the presence of infectious agents, including HIV, hepatitis, malaria, tuberulosis , and now COVID 19 etc. Forensic 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 , rule out suspects during police investigation, paternity testing even in case of availbility of very small amount of specimens ( stains of blood, semen, hair etc )

18. Research and Molecular Genetics 1. In genomic studies : PCR helps to compare the genomes of two organisms and identify the difference between them . 2. In phylogenetic analysis . Minute quantities of DNA from any source such a fossilized material, hair, bones, mummified tissues . 3. In study of gene expression analysis, PCR based mutagenesis 4. In Human genome project for aim to complete mapping and understanding of all genes of human beings.

19. References U. Satyanarayana (2018) Biotechnology, Book and Allied publication, Kolkata Laboratory Info: https ://laboratoryinfo.com/polymerase-chain-reaction-pcr / Accessed on 05.07.2020

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