POLYMERASE CHAIN REACTION TECHNIQUES (1) Standard PCR: @ Kary Mullis invented the PCR in 1983 (USA) and synthesized short chains of DNA. Given Nobel Prize in Chemistry in 1993. @ He invented a method of amplifying DNA through repeated cycles of duplication driven by DNA polymerase enzyme. @ PCR technique used an automated process for DNA amplification & separation of the DNA strands.

PCR Components and Reagents: @ DNA template that contains the DNA fragment to be amplified. @ Two Primers that are complementary to the 3' (three prime) ends of each of the sense and anti-sense strand of the DNA target. @ Deoxynucleoside triphosphates (dNTPs), the building-blocks from which DNA polymerase synthesizes a new DNA strand.

@ Taq DNA polymerase with a temperature optimum at around 70 °C. @ Buffer solution, providing a suitable chemical environment for optimum activity and stability of the DNA polymerase. @ Divalent cations (magnesium or manganese) for DNA synthesis @ Monovalent cation (potassium) for DNA synthesis

PCR Principle: @ PCR is a biochemical technology to amplify a single or a few copies of a piece of DNA to generate millions of copies of a certain DNA sequence. @ The technique depends on thermal cycling, with repeated heating and cooling to melt and amplify (replicate) the DNA. @ Repeated amplification of DNA requires short DNA fragments (Primers) and a heat- stable DNA polymerase (Taq polymerase) obtained from bacterium Thermus aquaticus.

A thermal cycler for PCR

@ This DNA Taq polymerase enzyme assembles a new DNA strand from DNA nucleotides primers by using the single stranded DNA present in the reaction as a template. @ Thus DNA synthesis is initiated and a chain of reactions develops, resulting in massive DNA amplification.

PCR Procedure: @ PCR consists of a series of 20-40 repeated temp. changes, called cycles, each cycle consists of 2-3 temp. steps. @ These cycles is preceded by a single temp. step (called hold) equaling >90°C, and followed by another hold temp. at the end. @ The temp. used and the length of time they are applied in each cycle depend on: # The enzyme used for DNA synthesis # The concentration of divalent ions and Deoxynucleoside triphosphates (dNTPs). # The melting temp. (Tm) of the primers.

Initialization step: This step consists of heating the reaction to a temp. of 94–96°C and held for 1–9 minutes. Denaturation step: @ This step consists of heating the reaction to 94–98°C for 20–30 seconds. @ It causes DNA melting of the DNA template yielding single-stranded DNA molecules. Annealing step: The reaction temp. is lowered to 50–65°C for 20–40 sec. to allow annealing (attaching) DNA primers to the single stranded DNA template

Extension/elongation step: @ The temp. at this step is 75–80°C which is required by the Taq DNA polymerase. @ This DNA polymerase synthesizes a new DNA strand similar to the DNA template strand. @ This is done by adding dNTPs to the template in 5' to 3' direction, then replaces the 5'-phosphate group of the dNTPs with the 3'-hydroxyl group at the end of the chain to extend DNA strand. @ At each extension step, the DNA amount is doubled, leading to exponential (geometric) amplification of the DNA fragment.

PCR cycle. (1) Denaturing (2) Annealing (3) Elongation PCR cycle. (1) Denaturing (2) Annealing (3) Elongation. Blue lines represent DNA template to which primers (red arrows) anneal; extended by DNA polymerase (light green circles); to give shorter DNA products (green lines); which themselves are used as templates as PCR progresses.

Final elongation step: This step is performed at a temp. of 70–74°C for 5–15 minutes to ensure that any remaining single-stranded DNA is fully extended. Extension/elongation step: @ This step is performed at 4–15°C for an indefinite time. @ It is used for short-term storage of the reaction.

@ To check whether PCR generated good amplification agarose gel electrophoresis is employed for size separation of the PCR products. @ The size of PCR products is determined by comparison with a DNA ladder (a molecular weight marker), which runs on the gel alongside the PCR products.

PCR products after gel electrophoresis. No amplification in sample 1; Sample 2 & 3 indicate amplification; Positive control & DNA ladder are shown

False PCR: Contamination causes false amplification of DNA products. This is prevented by: @ Separation of PCR area from areas for analysis or purification of PCR products. @ Use of disposable plastic ware @ Thorough cleaning of the work surface between reaction steps. @ Proper primer design techniques by using electronic PCR @ Usage of other buffers and polymerase enzymes.

PCR Applications: @ DNA extraction: From cell genome DNA by selective amplification of a specific region of DNA. This is done by hybridization probes and DNA cloning, to create copies of DNA fragments. @ DNA sequencing: To determine unknown PCR-amplified sequences by the insertion of a DNA sequence into a genetic material of another organism. @ DNA-based phylogeny: To determine the evolutionary relationships among organisms.

@ Amplification and quantification of DNA: To estimate the amount of a given sequence present in a sample (gene expression), using Real-time PCR. @ Identification of uncultivable or slow- growing organisms: Such as mycobacteria, anaerobic bacteria, or viruses. @ Early diagnosis of infectious diseases: Such as leukemia and lymphomas; detecting malignant cells at a high sensitivity.

Genes are expressed by being transcribed into RNA, and this RNA transcript is then translated into protein

@ Quantification viral load in a patient (HIV) @ Diagnosis of hereditary diseases @ Identification of genetic fingerprints. This is used in forensic sciences and paternity testing

Paternity fingerprints (1) Father. (2) Child. (3) Mother.

(2) Real-time PCR: @ Also called quantitative real time PCR (qPCR) @ It is a laboratory technique based on the PCR technique that is used to amplify and quantify a DNA molecule. @ In a DNA sample, qPCR enables at the same time both detection and quantification. @ Its procedure is same as PCR, But in qPCR the amplified DNA is detected as the reaction progresses in real time (immediate). @ qPCR technique can either use non-specific fluorochromes or hybridization probes

Applications of qPCR: @ Clinical quantification and genotyping @ Quantification of microbial load in foods or on vegetables. @ Rapid detection of cancer, newly emerging diseases, and genetic abnormalities. @ Microbial assessment of water quality. @ Quantitative measure of gene transcription @ Production of plant seedlings that are free of pathogens.