Polymerase chain reaction
Difiniton The polymerase chain reaction (
PCR) is powerful technique used in molecular biology. It derives its name from DNA polymerase which used to amplify a piece of DNA by enzymatic replication. The DNA used as template for replication. This sets in motion a chain reaction in which the DNA template is exponentially amplified.
principle All PCR applications depend on heat-stable DNA polymerase, such as Taq polymerase, which isolated from the bacterium
Thermu saquaticus. PCR is used to amplify specific regions of a DNA strand (the DNA target). This can be a single gene, a part of a gene, or a non-coding sequence. Most PCR methods typically amplify DNA fragments of up to 10 kilo base pairs (kb),
Components These components include:
1-DNA template that contains the DNA region (target) to be amplified.
2-Primers: which are complementary to the DNA regions at the 5' prime (forward ) and 3' prime (reverse) ends of the DNA region.
3-A DNA polymerase such as
Taq polymerase
4-Deoxynucleoside triphosphates (dNTPs; also very commonly and erroneously called deoxynucleotide triphosphates), the building blocks
5-Buffer solution, providing a suitable chemical environment for optimum activity and stability of the DNA polymerase.
Divalent cations, magnesium chloride is used.
The PCR reaction volume is 25-100
μl in thermal cycler by add deionized H2O.
Procedure
Denaturation step: This step consists of heating the reaction to 94-98°C for 20-30 seconds. It causes melting of DNA template and primers by disrupting the hydrogen bonds between complementary bases of the DNA strands, yielding single strands of DNA.
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. The polymerase binds to the primer-template hybrid and begins DNA synthesis.
Extension/elongation step: commonly a temperature at72°C is used with tag polymerase enzyme. At this step the DNA polymerase synthesizes a new DNA strand complementary to the DNA template strand by adding dNTP's
Final elongation: This single step is 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.
Contamination
it may be during amplification foreign source of DNA
it can leads to false positive results
so negative control must be obtained to overcome this contamination
Application of PCR
1-Isolation of genomic DNA(Cloning) PCR allows isolation of DNA fragments from genomic DNA by selective amplification of a specific region of DNA then introduce in plasmid in presence of ligase (example insulin production).
2- Determine unknown PCR the insertion of a DNA sequence into a plasmid or the genetic material of another organism. Bacterial colonies
3-genetic fingerprinting; a forensic technique used to identify a person or organism by comparing experimental DNAs through different PCR-based methods.
4- PCR in diagnosis of diseases
Oncogen
PCR used in diagnosis of malignant diseases through band strength which mean high gene expression
Types of PCR
1- Rapid PCR:
2-RT PCR: Reverse transcriptase PCR used to change RNA of virus to cDNA
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