Biotechnology and Bioinformatics (1+1)

4.1.4. Different versions of PCR

The basic protocol of PCR has been improved to develop several versions.

4.1.4.1. Two step PCR - the assay is done in two steps. In the second step 1-5% of the product developed in the first step is used for amplification using same set of primer s for further increasing the sensitivity.

4.1.4.2. Nested PCR - the assay is carried out in two steps. In the second step internal primers are used to amplify to increase the specificity and sensitivity.

4.1.4.3. RT PCR where RNA is converted to cDNA for amplification.

4.1.4.4. In-situ PCR- DNA in tissue is detected in situ.

4.1.4.5. Single tube PCR with several set of primers directed to different regions of pathogen or primers for two pathogens are used.

4.1.4.6. Real time PCR

Real-time polymerase chain reaction or quantitative real time PCR (Q-PCR/qPCR) is used to amplify and simultaneously quantify a targeted DNA molecule. For one or more specific sequences in a DNA sample, Real Time-PCR enables both detection and quantification. The quantity can be either an absolute number of copies or a relative amount when normalized to DNA input or additional normalizing genes. The procedure follows the general principle of PCR and its key feature is that the amplified DNA is detected as the reaction progresses in real time. Frequently, real-time PCR is combined with reverse transcription to quantify messenger RNA and Non-coding RNA in cells or tissues.

4.1.4.7. Hot-start PCR

In this technique, initial denaturation is performed in the absence of polymerase or primers. The temperature of the reaction mix is then maintained at 70–90 ° C until all the components are combined.