Tuesday, July 10, 2007

Principles of Various Microbiological Techniques

Principles of Polymerase Chain Reaction (PCR):

PCR is based on the mechanism of DNA replication in vivo: dsDNA is unwound to ssDNA, duplicated and rewound. This technique consists of repetitive cycles of denaturation of DNA through melting at elevated temperature to convert double-stranded DNA to single-stranded DNA, annealing (hybridization) of two oligonucleotides used as primers to the target DNA and extension of the DNA chain by nucleotide addition from the primers using DNA polymerase as catalyst in the presence of MG2+ ions.

Principles of ELISA:

Enzyme-Linked-Immunosorbent Assay (ELISA) is also known as Enzyme Immunoassay (EIA). This method depends on the exquisite specificity of antigen-antibodies reactions, biological amplification of the antigen-antibody reaction by an enzyme and the antibody’s ability to retain its immunoreactivity after conjugation with an enzyme.

Principle of DNA Hybridization:

This technique includes the separation of DNA by gel electrophoresis, followed by immobilization on a membrane with subsequent probe hybridization and detection though either radiolabeling or non-radiolabeling methods (e.g. chemiluminescence). In addition, the generation of a specific signal is highly dependent on parameters such as transfer efficiency, sequence homology, buffer condition, temperature and incubation time.

Principles of Agarose Gel Electrophoresis:

Agarose gel electrophoresis is a standard method to separate, identify and purify DNA fragments. The principles of this technique are; in high pH environment, protein molecules tend to be negatively charged and vice versa. A protein’s isoelectric pH is the level of pH at which it has neither a positive nor a negative charge. This level varies depending on the chemical structure of the particular protein.