PCR-What is that ??

 Polymerase chain reaction (PCR) is a relatively simple and widely used molecular biology technique to amplify and detect DNA and RNA sequences. Compared to traditional methods of DNA cloning and amplification, which can often take days, PCR requires only a few hours. PCR is highly sensitive and requires minimal template for detection and amplification of specific sequences. Basic PCR methods have further advanced from simple DNA and RNA detection. For standard PCR, all  one need  is a DNA polymerase, magnesium, nucleotides, primers, the DNA template to be amplified and a thermocycler. The PCR mechanism is as simple as its purpose: 1) double-stranded DNA (dsDNA) is heat denatured, 2) primers align to the single DNA strands and 3) the primers are extended by DNA polymerase, resulting in two copies of the original DNA strand. The denaturation, annealing, and elongation process over a series of temperatures and times is known as one cycle of amplification. Each step of the cycle should be optimized for the template and primer set used. This cycle is repeated approximately 20-40 times and the amplified product can then be analyzed. PCR is widely used to amplify DNA for subsequent experimental use. PCR also has applications in genetic testing or for the detection of pathogenic DNA.

As PCR is a highly sensitive method and very small volumes are required for single reactions, preparation of a master mix for several reactions is recommended. The master mix must be well mixed and then split by the number of reactions, ensuring that each reaction will contain the same amount of enzyme, dNTPs and primers. Many suppliers, such as Enzo Life Sciences, also offer PCR mixes that already contain everything except primers and the DNA template.

Guanine/Cytosine-rich (GC-rich) regions represent a challenge in standard PCR techniques. GC-rich sequences are more stable than sequences with lower GC content. Furthermore, GC-rich sequences tend to form secondary structures, such as hairpin loops. As a result, GC-rich double strands are difficult to completely separate during the denaturation phase. Consequently, DNA polymerase cannot synthesize the new strand without hindrance. A higher denaturation temperature can improve this and adjustments towards a higher annealing temperature and shorter annealing time can prevent unspecific binding of GC-rich primers. Additional reagents can improve the amplification of GC-rich sequences. DMSO, glycerol and betaine help to disrupt the secondary structures that are caused by GC interactions and thereby facilitate separation of the double strands