Lambda Therapeutic Research

Polymerase chain reaction or PCR is a method of multiplying DNA copies and was invented in 1983. This process depends on denaturation of DNA at high temperatures, annealing of primers to the DNA, and elongation and synthesis of new DNA strands by the heat-resistant Thermophilus aquaticus polymerase. It utilizes nucleotides and magnesium chloride as a cofactor. PCR can generate more than 30 billion copies of DNA in a couple of hours. Its advanced method, real-time PCR (RT-PCR), can quantify simultaneously while multiplying the DNA and is, hence, also known as quantitative PCR (qPCR). It uses fluorescence-emitting dyes such as SYBR green and TaqMan to quantify DNA multiplication. PCR is used in forensics to multiply DNA evidence, in diagnosing genetic diseases and mutations from body fluids like saliva and blood, and in genetic engineering.

Southern blotting, discovered in 1975 by E.M. Southern, represents a technique to detect a gene of interest in the DNA sample. The steps involved are isolation of DNA, its separation by electrophoresis, transfer to a suitable medium, hybridization to probes and visualization of the gene if it is present. Western blotting is the counterpart which is used to detect proteins. The difference lies in the visualization process. In Western blotting, this is made possible by primary and secondary antibodies, whereas in Southern blotting, a radiolabeled (fluorescent) probe or dye that binds to the DNA is used. Application of Western blotting includes identifying HIV antigens or Hepatitis B surface antigen in blood. Northern blotting is used to detect mRNA of interest, where after separation by electrophoresis, cDNA is used as a probe that binds to the RNA strand; the application includes finding alternate transcript size. The term ‘blotting’ in all the three techniques represents the transfer of material after separation to nitrocellulose paper by means of diffusion.