PCR

 PCR = Polymerase Chain Reaction

PCR is a very effective technique of obtaining multiple identical copies of a certain DNA strand (amplifying DNA).  The method relies on thermal cycling, consisting of cycles of repeated heating and cooling of the reaction for DNA melting and enzymatic replication of the DNA.

Primers (short DNA fragments) containing sequences complementary to the target region along with a DNA polymerase (after which the method is named) are key components to enable selective and repeated amplification. As PCR progresses, the DNA generated is itself used as a template for replication, setting in motion a chain reaction in which the DNA template is exponentially amplified.

Almost all PCR applications employ a heat-stable DNA polymerase, such as Taq polymerase (an enzyme originally isolated from the bacterium Thermus aquaticus). This DNA polymerase enzymatically assembles a new DNA strand from DNA building-blocks, the nucleotides, by using single-stranded DNA as a template and DNA oligonucleotides (also called DNA primers), which are required for initiation of DNA synthesis.

A basic PCR set up requires several components and reagents. These components include:

• DNA template that contains the DNA region (target) 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.
• Taq polymerase or another DNA polymerase with a temperature optimum at around 70 °C.
• Deoxynucleoside triphosphates (dNTPs) the building-blocks from which the DNA polymerase synthesizes a new DNA strand.
• 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.

 
PCR procedure:

1. Initialization step: Heating the reaction to a temperature of 94–96 °C, because DNA polymerases require heat activation.

2. Denaturation step: Heat-denaturation of a DNA sample into single strands(94–98 °C for 20–30 seconds).

3. 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. These specific oligonucleotides, which are at a very high concentration, will hybridize with their complementary sequences in the DNA sample, whereas the long strands of the sample DNA remain apart because of their low concentration. 

4. Elongation step:  At 75–80 °C  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. The extension time depends both on the DNA polymerase used and on the length of the DNA fragment to be amplified.

5. 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. 

6. Final hold: This step at 4–15 °C for an indefinite time may be employed for short-term storage of the reaction.

The PCR product is analyzed on an agarose gel and is abundant enough to be detected with an ethidium bromide stain.
Beacasue this method of analysis is at best semi-quantitative and, in many cases, the amount of product is not related to the amount of input DNA making this PCR, thus real time PCR was developed