Polymerase Chain Reaction (PCR)

1. Polymerase Chain Reaction (PCR) JonyMallik

2. PCR • PCR is stands for ‘Polymerase Chain Reaction”. PCR is a very essential molecular biological, qualitative & quantitative analytical technique, that helps to amplify the small piece of DNA or segment of a DNA to a large piece under the agency of In vitro replication process. • PCR is a cell free amplification technique to synthesizing multiple identical copies of the DNA . • It utilizes the principle of DNA replication.

3. History of PCR • 1983: Dr. Kary Mullis developed PCR • 1985: First publication of PCR by Cetus Corporation appears in Science. • 1986: Purified Taq polymerase is first used in PCR • 1988: PerkinElmer introduces the automated thermal cycler. • 1989: Science declares Taq polymerase "molecule of the year.

4. History of PCR… • 1990: amplification and detection of specific DNA sequences using a fluorescent DNA-binding dye, laying the foundation for future "real-time" or "kinetic" PCR. • 1991: RT-PCR is developed using a single thermostable polymerase, rTth, facilitating diagnostic tests for RNA viruses. • 1993:Dr. Kary Mullis shares Nobel Prize in Chemistry for conceiving PCR technology.

5. Components of PCR • 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 withstand at a temperature upto 95 ° C (derived from Thermusaquaticus)

6. Components of PCR…. • Buffer solution, providing a suitable chemical environment for optimum activity and stability of the DNA polymerase. • Divalentcations, magnesium or manganese ions; generally Mg2+ is used, but Mn2+ can be utilized for PCR-mediated DNA mutagenesis, as higher Mn2+ concentration increases the error rate during DNA synthesis. • Monovalentcationpotassium ions.

7. Process of 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.

8. Steps in PCR • Initialization step: This step consists of heating the reaction to a temperature of 94–96 °C (or 98 °C if extremely thermostable polymerases are used), which is held for 1–9 minutes. It is only required for DNA polymerases that require heat activation by hot-start PCR. • Denaturation step: This step is the first regular cycling event and consists of heating the reaction to 94–98 °C for 20–30 seconds. It causes DNA melting of the DNA template by disrupting the hydrogen bonds between complementary bases, yielding single-stranded DNA molecules. • Annealing step: The reaction temperature is lowered to 50–65 °C for 20–40 seconds allowing annealing of the primers to the single-stranded DNA template. Typically the annealing temperature is about 3-5 degrees Celsius below the Tm of the primers used. Stable DNA-DNA hydrogen bonds are only formed when the primer sequence very closely matches the template sequence. The polymerase binds to the primer-template hybrid and begins DNA synthesis.

9. Extension/elongation step: The temperature at this step depends on the DNA polymerase used; Taq polymerase has its optimum activity temperature at 75–80 °C,and commonly a temperature of 72 °C is used with this enzyme. At this step the DNA polymerase synthesizes a new DNA strand complementary to the DNA template strand by adding dNTPs that are complementary to the template in 5' to 3' direction, condensing the 5'-phosphate group of the dNTPs with the 3'-hydroxyl group at the end of the nascent (extending) DNA strand. The extension time depends both on the DNA polymerase used and on the length of the DNA fragment to be amplified. As a rule-of-thumb, at its optimum temperature, the DNA polymerase will polymerize a thousand bases per minute. Under optimum conditions, i.e., if there are no limitations due to limiting substrates or reagents, at each extension step, the amount of DNA target is doubled, leading to exponential (geometric) amplification of the specific DNA fragment. • Final elongation: This single step is occasionally performed at a temperature of 70–74 °C for 5–15 minutes after the last PCR cycle to ensure that any remaining single-stranded DNA is fully extended. • Final hold: This step at 4–15 °C for an indefinite time may be employed for short-term storage of the reaction.

10. Diagrammatic Presentation

11. 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. • The thermal cycler heats and cools the reaction tubes to achieve the temperatures required at each step of the reaction An older model three-temperature thermal cycler for PCR Modern thermo cycler

12. PCR relation to temperature vs time

13. Types of PCR • Nested PCR • Nested primers increases the specificity and selectively amplifies the target DNA. • Inverse PCR • Can be study the unknown sequences using known sequence. • Anchored PCRthis is particularly useful when the sequence surrounding the target is not known. It can be done by using adaptors.

14. Reverse transcription PCR • The mRNA converted to cDNA by reverse transcriptase , this cDNA serve as the template for PCR • Real time PCR • Commonly used technique for measuring the quantity of • DNA by employing fluorescence compound ethidium bromide. • Asymmetric PCR • This technique can be used for the synthesis of single stranded DNA , particularly used for DNA sequencing.

15. Applications PCR in clinical diagnosis: Prenatal diagnosis of inherited diseases: Using chorionic villus samples or cell from amniocentesis. Thus diseases like sickle-cell anemia, β- thalassemia can be detected by PCR. Diagnosis of retroviral and bacterial infections: PCR from cDNA is valuable tool for diagnosis and monitoring of retroviral infection.eg: HIV infection. Also the tuberculosis (TB) by mycobacterium tuberculosis.

16. 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.

17. 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.