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Polymerase Chain Reaction (PCR) JONY MALLIK

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Page 1: PCR is stands for ‘Polymerase Chain Reaction”. PCR is a very essential molecular biological, qualitative & quantitative analytical technique, that helps

Polymerase Chain Reaction (PCR)

JONY MALLIK

Page 2: PCR is stands for ‘Polymerase Chain Reaction”. PCR is a very essential molecular biological, qualitative & quantitative analytical technique, that helps

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.

Page 3: PCR is stands for ‘Polymerase Chain Reaction”. PCR is a very essential molecular biological, qualitative & quantitative analytical technique, that helps

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.

Page 4: PCR is stands for ‘Polymerase Chain Reaction”. PCR is a very essential molecular biological, qualitative & quantitative analytical technique, that helps

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.

History of PCR…

Page 5: PCR is stands for ‘Polymerase Chain Reaction”. PCR is a very essential molecular biological, qualitative & quantitative analytical technique, that helps

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

Thermus aquaticus)

Page 6: PCR is stands for ‘Polymerase Chain Reaction”. PCR is a very essential molecular biological, qualitative & quantitative analytical technique, that helps

Buffer solution, providing a suitable chemical environment for optimum

activity and stability of the DNA polymerase.

Divalent cations, 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.

Monovalent cation potassium ions.

Components of PCR….

Page 7: PCR is stands for ‘Polymerase Chain Reaction”. PCR is a very essential molecular biological, qualitative & quantitative analytical technique, that helps

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.

Page 8: PCR is stands for ‘Polymerase Chain Reaction”. PCR is a very essential molecular biological, qualitative & quantitative analytical technique, that helps

Steps in PCRInitialization 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.

Page 9: PCR is stands for ‘Polymerase Chain Reaction”. PCR is a very essential molecular biological, qualitative & quantitative analytical technique, that helps

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.

Page 10: PCR is stands for ‘Polymerase Chain Reaction”. PCR is a very essential molecular biological, qualitative & quantitative analytical technique, that helps

Diagrammatic Presentation

Page 11: PCR is stands for ‘Polymerase Chain Reaction”. PCR is a very essential molecular biological, qualitative & quantitative analytical technique, that helps

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  l in μsmall 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

Modern thermo cyclerAn older model three-temperature thermal cycler for PCR

Page 12: PCR is stands for ‘Polymerase Chain Reaction”. PCR is a very essential molecular biological, qualitative & quantitative analytical technique, that helps

1 2 3 4 5 6 70

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temperature vs time

time

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PCR relation to temperature vs time

Page 13: PCR is stands for ‘Polymerase Chain Reaction”. PCR is a very essential molecular biological, qualitative & quantitative analytical technique, that helps

Types of PCRNested PCRNested primers increases the specificity and selectively amplifies the target DNA.Inverse PCRCan 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.

Page 14: PCR is stands for ‘Polymerase Chain Reaction”. PCR is a very essential molecular biological, qualitative & quantitative analytical technique, that helps

Reverse transcription PCRThe 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 PCRThis technique can be used for the synthesis of single stranded DNA , particularly used for DNA sequencing.

Page 15: PCR is stands for ‘Polymerase Chain Reaction”. PCR is a very essential molecular biological, qualitative & quantitative analytical technique, that helps

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.

Page 16: PCR is stands for ‘Polymerase Chain Reaction”. PCR is a very essential molecular biological, qualitative & quantitative analytical technique, that helps

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.

Page 17: PCR is stands for ‘Polymerase Chain Reaction”. PCR is a very essential molecular biological, qualitative & quantitative analytical technique, that helps

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.

Page 18: PCR is stands for ‘Polymerase Chain Reaction”. PCR is a very essential molecular biological, qualitative & quantitative analytical technique, that helps