Types of PCR

What is PCR?
History of PCR
Components of PCR
Principles of PCR
Basic Requirements
Instrumentation
PCR Programme
Advantages of PCR
Applications of PCR
Conclusion
References

1. TYPES OF PCR By KAUSHAL KUMAR SAHU Assistant Professor (Ad Hoc) Department of Biotechnology Govt. Digvijay Autonomous P. G. College Raj-Nandgaon ( C. G. )

2. SYNOPSIS What is PCR? History of PCR Components of PCR Principles of PCR Basic Requirements Instrumentation PCR Programme Advantages of PCR Applications of PCR Conclusion References

3. What is PCR? PCR is a technique that takes specific sequence of DNA of small amount and amplifies it to be used for further testing. In vitro technique

4. History In 1983 Karry Mullis conceived the idea of PCR. Developed in 1984. In 1993 Karry Mullis was awarded the Nobel Prize for the discovery of PCR.

5. 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 (renature). The polymerase enzyme that starts synthesizing new strands. These three steps are repeated to get more copies of DNA.

6. Requirements Target DNA (100-35,000 bp in length) Primers (synthetic oligonucleotides of 17-30 nucleotides in length ) Four deoxyribonucleotides (dATP, dCTP, dGTP, dTTP) Thermo stable DNA polymerase that can with stand at a temperature up to 95 ° C (derived from Thermus Aquaticus ).

7. Buffer solution - providing a suitable chemical environment for optimum activity and stability of the DNA polymerase. Divalent cations , magnesium or manganese ions- generally Mg 2+ is used, but Mn 2+ can be utilized for PCR-mediated DNA mutagenesis , as higher Mn 2+ concentration increases the error rate during DNA synthesis . Monovalent cation potassium ions.

8. Thermo cycler This whole process was done by using an automated machine called as thermo cycler. It can raises and lowers the temperature automatically. The PCR is commonly carried out in a reaction volume of 10–200  μl in small reaction tubes (0.2–0.5 ml volumes) in a thermal cycler . Modern thermo cycler

9. Major processes in PCR Denaturation: The temperature is raised at 94–98 °C for 1minute to separate the double stranded DNA . Renaturation: Decrease the temperature at 55 ° C . This helps the primer to bind with target DNA . This step is also known as annealing . 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 at 75 ° C. Note : This whole steps are considered as one cycle

10. PCR relation to temperature vs time

11. Types of PCR 1.Inverse PCR Can be study the unknown sequences using known sequence.

12. Nested primers increases the specificity and selectively amplifies the target DNA. 2.Nested PCR

13. 3.Reverse transcription PCR The mRNA converted to cDNA by reverse transcriptas , this cDNA serve as the template for PCR

14. 4.Real-Time or Quantitative PCR ( qPCR ) Real time PCR Commonly used technique for measuring the quantity of DNA by employing fluorescence compound ethidium bromide. Detection of signal in real time allows quantification of starting material. Performed in specialized thermal cyclers with fluorescent detection systems.

15. DNA Detection: SYBR Green I Dye DENATURATION STEP: DNA + PRIMERS + DYE WEAK BACKGROUND FLUORESCENCE ANEALING STEP:DYE BINDS dsDNA, EMITS LIGHT EXTENSION STEP: MEASURE LIGHT EMMISSION

16. Types of PCR Anchored PCR this is particularly useful when the sequence surrounding the target is not known. It can be done by using adaptors. Asymmetric PCR This technique can be used for the synthesis of single stranded DNA , particularly used for DNA sequencing.

17. Advantages of PCR Small amount of DNA is required per test Result obtained more quickly - usually within 1 day for PCR Usually not necessary to use radioactive material (32P) for PCR. PCR is much more precise in determining the sizes of alleles - essential for some disorders. PCR can be used to detect point mutations.

18. PCR IN FORENSICS: A single molecule of DNA from any sources like blood ,hair, small tissue etc can be amplified by PCR. The PCR is useful in the DNA finger printing technology. Application

19. PCR in comparative study of genomes: The differences in the genomes can be measured after electrophoresis. The closely related organisms can give similar bands. PCR is very useful in the study of evolutionary biology, more specifically referred as phylogenetics.

20. Applications of PCR Molecular Identification Sequencing Genetic Engineering Molecular Archaeology Bioinformatics Site-directed mutagenesis Molecular Epidemiology Genomic Cloning Gene Expression Studies Molecular Ecology Human Genome Project DNA fingerprinting Classification of organisms Genotyping Pre-natal diagnosis Mutation screening Drug discovery Genetic matching Detection of pathogens

21. Limitations Sequence Information Amplicon size Error rate during amplification Sensitivity to inhibitors Contamination 25

22. References Books 1- Gene cloning and DNA analysis = T.A. Brown( sixth edition ) 2- An Introduction of Genetic Engineering=Desmond S. T. Nicholl (Third edition) PDF & Internet 1.M Wiedmann , W J Wilson, J Czajika , et al. 1994 , Ligase chain reaction overview and applications Genome Res, 3:S51-S64 2.http:// groups.Molbiosci.northwestern.edu / holmgren /Glossary/ DefL /Ligase_chain_raction.html