Procedures for PCR

The PCR usually consists of a series of 20 to 35 cycles. Most commonly, PCR is carried out in three steps (Fig. 2), often preceded by one temperature hold at the start and followed by one hold at the end.

1) Prior to the first cycle, during an initialization step, the PCR reaction is often heated to a temperature of 94-96°C (or 98°C if extremely thermostable polymerases are used), and this temperature is then held for 1-9 minutes.

-This first hold is employed to ensure that most of the DNA template and primers are denatured, i.e., that the DNA is melted by disrupting the hydrogen bonds between complementary bases of the DNA strands, yielding two single strands of DNA.
-Also, some PCR polymerases require this step for activation (see hot-start PCR).[5] Following this hold, cycling begins, with one step at 94-98°C for 20-30 seconds (denaturation step).

2) The denaturation is followed by the annealing step.

-In this step the reaction temperature is lowered so that the primers can anneal to the single-stranded DNA template. Brownian motion causes the primers to move around, and DNA-DNA hydrogen bonds are constantly formed and broken between primer and template.
-Stable bonds are only formed when the primer sequence very closely matches the template sequence, and to this short section of double-stranded DNA the polymerase attaches and begins DNA synthesis. The temperature at this step depends on the melting temperature of the primers, and is usually between 50-64°C for 20-40 seconds.

3) The annealing step is followed by an extension/elongation step during which the DNA polymerase synthesizes new DNA strands complementary to the DNA template strands.

-The temperature at this step depends on the DNA polymerase used. Taq polymerase has a temperature optimum of 70-74°C; thus, in most cases a temperature of 72°C is used. The hydrogen bonds between the extended primer and the DNA template are now strong enough to withstand forces breaking these attractions at the higher temperature.
- Primers that have annealed to DNA regions with mismatching bases dissociate from the template and are not extended. 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 in one minute.
-A final elongation step of 5-15 minutes (depending on the length of the DNA template) after the last cycle may be used to ensure that any remaining single-stranded DNA is fully extended.
-A final hold of 4-15°C for an indefinite time may be employed for short-term storage of the reaction, e.g., if reactions are run overnight.


~To check whether the PCR generated the anticipated DNA fragment (also sometimes referred to as amplimer), agarose gel electrophoresis is commonly employed for size separation of the PCR products. The size(s) of PCR products is thereby determined by comparison with a DNA ladder, which contains DNA fragments of known size, ran on the gel alongside the PCR products


*Extras: `Uses of PCR`
=>Isolation of genomic material

Part of PCR's power lies in its ability to easily isolate particular regions of DNA sequence from whole genomic material. Many techniques need a pool of DNA molecules isolated from a particular DNA fragment, and the use of PCR has enabled these techniques more widespread in usage. Because PCR also amplifies the isolated region, the techniques are more powerful, applicable to samples otherwise too small for analysis.

1) Sequencing and the detection of genetic diseases

2) Recombinant DNA techniques

3) Genetic fingerprinting and paternity testing


Credits: http://en.wikipedia.org/wiki/Polymerase_chain_reaction