Sorbent Assay Coating Antigen is adsorbed onto the wells in ELISA plate in coating buffer Blocking A buffer containing unrelated protein is used to block free sites in the wells Remove buffer and wash plate Readout Substrate is catalyzed by the enzyme to generate colored readout Key Remove buffer and wash plate Analyte/ Antigen Detection Enzyme conjugated detection antibody binds antigen Remove buffer and wash plate Enzyme Directly conjugated primary antibody Conjugated secondary antibody Capture antibody There are four kinds of ELISA namely, Direct ELISA, Indirect ELISA, sandwich ELISA and competitive ELISA. It is a highly sensitive and specific method used for diagnosis. ELISA possesses the added advantages of not requiring radioisotopes or a radiation counting apparatus. PCR (Polymerase Chain Reaction) The polymerase chain reaction (PCR) is an invitro amplification technique used for synthesising multiple identical copies (billions) of DNA of interest.
The technique was developed by Kary Mullis (Nobel laureate, ) in the year . Denaturation, renaturation or primer annealing and synthesis or primer extension, are the three steps involved in PCR (Fig. . ) .
The double stranded DNA of interest is denatured to separate into two individual strands by high temperature . This is called denaturation . Each strand is allowed to hybridize with a primer (renaturation or primer annealing). The primer template is used to synthesize DNA by using Taq – DNA polymerase (isolated from the bacterium Thermus aquaticus).
During denaturation the reaction mixture is heated to C for a short time to denature the target DNA into single strands that will act as a template for DNA synthesis. Annealing is done by rapid cooling of the mixture, allowing the primers to bind to the sequences on each of the two strands flanking the target DNA. During primer extension or synthesis the temperature of the mixture is increased to C for a sufficient period of time to allow Taq DNA polymerase to extend each primer by copying the single stranded template. At the end of incubation both single template strands will be made partially double stranded.
The new strand of each double stranded