PCR

Polymerase chain reaction.
1. Denaturation.
2. Annealing.
3. Extension.

1. DAVANGERE UNIVERSITY MICROBIOLOGY SEMINAR ON THE TOPIC PCR SUBMITTED BY : Ponnanna . M.B M.Sc . Microbiology.

2. CONTENTS INTRODUCTION HISTORY WHAT IS PCR? WORKING PRINCIPLE OF PCR COMPONENTS OF PCR STEPS INVOLVED IN PCR TYPES OF PCR APPLICATIONS OF PCR ADVANTAGES & LIMITATIONS OF PCR SUMMARY CONCLUSION REFERENCES

3. INTRODUCTION PCR is an abbreviation for ‘polymerase chain reaction’. It is a technique used to amplify a single copy or few copies of segments of DNA across several orders of magnitude, generating thousands of copies of DNA sequences. It is an easy, cheap and reliable way to repeatedly replicate a focused segment of DNA. A concept which is applicable to numerous fields in modern biology and related sciences. PCR is probably the most widely used technique in molecular biology, criminal forensics, molecular archaeology and in clinical and biomedical research.

4. HISTORY 1983: Kary Mullis developed the polymerase chain reaction [PCR] technique. The process similar to PCR was first described by Kjell Kleppe and Nobel laureate Har Gobind Khorana in 1971, which allows the amplification of specific DNA sequences. 1993: Kary Mullis was awarded the Nobel Prize in Chemistry for his invention of PCR. Michael Smith also shared the Nobel Prize with Kary Mullis for his work in developing site-directed mutagenesis. 1986: Purified Taq polymerase was first used in PCR. 1988: DNA fingerprinting was first used for paternity testing.

5. What is PCR? PCR is an exponentially progressing synthesis of the defined target DNA sequences in vitro. PCR [ Polymerase chain reaction ] Polymerase- The only enzyme used in this reaction is DNA polymerase. Chain- The products of the first reaction becomes the substrates of the following one, and so on. Reaction components- Target DNA, pair of primers, thermostable DNA polymerase, Mg++ ions, buffer solution.

6. W orking principle of PCR   As the name implies, it is a chain reaction, a small fragment of the DNA strand of our interest needs to be identified, which serves as the template for producing the primers that initiate the reaction. One DNA molecule is used to produce two copies, then four, then eight and so forth. This continuous doubling is accomplished by specific proteins known as polymerase enzymes, that are able to string together individual DNA building blocks to form long molecular strands. To do their job polymerases require a supply of DNA building blocks, i.e.,  the nucleotides consisting of the four bases adenine (A), guanine (G), thymine (T ) and cytosine (C ). They also need a small fragment of DNA known as the primer, to which they attach the building blocks as well as a longer DNA molecule to serve as a template for constructing the new strand. If these three ingredients are supplied, the enzymes will construct exact copies of the templates.  

7. Basic Requirements for PCR DNA Template : It is made up of double helix of two complimentary strands. Each strand of the duplex acts as a template for the synthesis of new double helix. Primer : It is a short strand of RNA or DNA that serves as a starting point for DNA synthesis. It is required for DNA replication. DNA Polymerase : Enzymes that synthesize DNA molecules from deoxyribonucleotides , the building blocks of DNA. [ Taq polymerase is used in this process ] dNTPs : deoxynucleotide triphosphates are the monomeric substrates for the polymerization reaction. It is a mixture of the four monomeric units, dATP , dTTP , dCTP and dGTP that will ultimately make up the DNA that is polymerized during PCR. Divalent cations : All thermostable DNA polymerases require free divalent cations - usually Mg2+ for their activity.

8. Buffer solution : It is necessary to create optimal conditions for activity of Taq DNA polymerase.[ Tris - Hcl ] # Pfu - Pyrococcus furiosus # Vent- Thermococcus litoralis litoralis .

9. DNA Thermal cycler The thermal cycler (also known as a thermocycler ) is a laboratory apparatus which is used to amplify segments of DNA via the polymerase chain reaction(PCR). This machine can be programmed to carry out heating and cooling of samples over number of cycles.

10. Steps involved in PCR. DENATURATION The reaction mixture is heated to a temperature between 94-96ºC so that ds DNA is denatured into single stranded by disrupting the hydrogen bonds between complementary bases. This results in two single strands of DNA, which acts as a template for the production of new strands of DNA. Duration of this step is 30-60seconds.

11. ANNEALING During this stage, the reaction temperature is lowered to 55–65  °C for 20–30 seconds, allowing annealing of the primers to each of the single-stranded DNA templates. Primers serve as the starting point of DNA synthesis. Two separated strands of DNA are complementary and run in opposite directions , as a result there are 2 primers- a forward and reverse primer.

12. Extension/elongation : The temperature at this step depends on the DNA polymerase used; the optimum activity temperature for the thermostable DNA polymerase of Taq ( Thermus aquaticus ) polymerase is approximately 75–80°C. T hough a temperature of 72 °C is commonly used with this enzyme i n this step, the DNA polymerase synthesizes a new DNA strand complementary to the DNA template strand by adding free dNTPs from the reaction mixture that are complementary to the template in the 5'-to-3' direction, condensing the 5'- phosphate group of the dNTPs with the 3'-hydroxy group at the end of the elongating DNA strand.

13. T hese three processes of thermal cycling are repeated several times to produce many copies of DNA sequences.

14. Quantitative PCR ( qPCR / real-time PCR ): U sed to measure the quantity of a target sequence (commonly in real-time). It quantitatively measures starting amounts of DNA, cDNA , or RNA. Q uantitative PCR is commonly used to determine whether a DNA sequence is present in a sample and the number of its copies in the sample. Quantitative PCR has a very high degree of precision. Quantitative PCR methods use fluorescent dyes, such as Sybr Green, EvaGreen or fluorophore -containing DNA probes, such as TaqMan , to measure the amount of amplified product in real time.

15. Reverse Transcription PCR (RT-PCR ): F or amplifying DNA from RNA, r everse transcriptase reverse transcribes RNA into cDNA, which is then amplified by PCR. RT-PCR is widely used in expression profiling, to determine the expression of a gene or to identify the sequence of an RNA transcript, including transcription start and termination sites. If the genomic DNA sequence of a gene is known, RT-PCR can be used to map the location of exons and introns in the gene.

16. Nested PCR : It increases the specificity of DNA amplification by reducing background due to non-specific amplification of DNA. Two sets of primers are used in two successive PCRs. In the first reaction, one pair of primers is used to generate DNA products, which besides the intended target may still consist of non-specifically amplified DNA fragments. The product(s) are then used in a second PCR with a set of primers whose binding sites are completely or partially different and located 3' of each of the primers used in the first reaction. Nested PCR is often more successful in specifically amplifying long DNA fragments than conventional PCR, but it requires more detailed knowledge of the target sequences.

17. Multiplex-PCR: It consists of multiple primer sets within a single PCR mixture, it produces amplicons of varying sizes that are specific to different DNA sequences. By targeting multiple genes at once, additional information may be gained from a single test-run that otherwise would require several times the reagents and more time to perform. Annealing temperatures for each of the primer sets must be optimized to work correctly within a single reaction, and amplicon sizes. That is, their base pair length should be different enough to form distinct bands when visualized by gel electrophoresis.

18. Hot start PCR : It is a technique that reduces non-specific amplification during the initial set up stages of the PCR. It may be performed manually by heating the reaction components to the denaturation temperature (94°C ) before adding the polymerase . Specialized enzyme systems have been developed that inhibit the polymerase activity at ambient temperature, either by the binding of an antibody or by the presence of covalently bound inhibitors that dissociate only after a high-temperature activation step. When temperature raises for amplification at 72ºC, the specific antibodies detaches from the DNA polymerase and amplification begins with great specificity.

19. Inverse PCR : It is commonly used to identify the flanking sequences around genomic inserts. It involves a series of DNA digestions and self ligation, resulting in known sequences at either end of the unknown sequence .

20. Advantages of PCR: Small amount of DNA sample is required. Amplified copies of DNA are obtained quickly . Use of radioactive substances is not necessary. PCR is more precise in determining the size of alleles essential for some disorders. Limitations of PCR: It is a sensitive technique, prone to contamination from extraneous DNA , leading to false positive result. Cross contamination between samples is the potential problem.. Instrument and reagents are expensive, hence cannot be afforded by small laboratories.

21. SUMMARY PCR is the technique that amplifies a small amount of known sequence of DNA into many copies within short period of time. The process involves three steps denaturation, annealing and extension along with addition of several components required for the reaction. There are various types of PCR working under different principles and has a wide range of application in the field of molecular biology.

22. CONCLUSION PCR is one vital process to amplify specific DNA fragments from small amounts of DNA sample. The speed and ease of use, sensitivity, specificity and robustness of PCR has revolutionized molecular biology and made PCR the most widely used technique with great spectrum of research and diagnostic applications.

23. REFERENCES Clark, D. P. and Pazderinik , N. J. 2009. Biotechnology. Elsevier Academic Press, US,750 pp. Hughes, S. and Moody, A. 2007. PCR. Schion publishing limited, UK, 348 pp. Modi , H. A. 2009. Microbial Biotechnology. Pointer publishers, India, 355 pp. Schleif , R. 1993. Genetics and molecular biology, 2 nd edn . John Hopkins University press, London, 685 pp. Walker, J. M. and Raply , R. 2009. Molecular Biology and Biotechnology, 5 th edn . RSC publishers, UK, 585 pp. Watson, J. D., Caudy , A . A., Myers, R. M. and Witkowski , J. A. 2007. Recombinant DNA, 3 rd edn . Cold spring harbour press, New York, 474 pp.

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