DNA and RNA sequences by the polymerase chain reaction PCR

Detection of specific DNA and RNA sequences by the polymerase chain reaction (PCR) 

In the following step, the primed strands serve as templates for DNA polymerisation. The second cycle of reaction begins with renewed denaturation, followed by annealing and polymerisation as before. If the yield for each cycle is 100%, then after n cycles there will be an increase of a factor of 2 in the amount of template present. 

The oligodeoxynudeotide primers are usually at least 20 bases long, with sufficient GC content to ensure that the annealing is specific and that the primer-template hybrid does not dissotiate at the optimum polymerization temperature of 65-75 °C. 

In applications where PCR is used for the routine detection of very small quantities of nudeic adds, there is a significant problem with potential contamination. To reduce this to manageable levels, it is essential that appropriate precautions are taken. This indudes a rigorous separation of laboratory areas for sample preparation, PCR amplification and product analysis use of dedicated pipettes and pre-pre- 

The analysis of PCR products is usually carried out by electrophoresis, and the desired product is identified by length. This is not necessarily the best approach for large scale routine analysis. Alternative sequence-specific procedures are available in which the product is hybridised to labelled oligonucleotides that can be detected by fluorimetric antibody-coupled colorimetric procedures. 

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