Reverse transcriptase-polymerase chain gene primers

The amplification of cDNA by combining a reverse transcriptase (RT) reaction with PCR is known as reverse transcription polymerase chain reaction (RT-PCR). A first-strand of cDNA is generated from RNA with RT in the presence of dNTP, Mg + by the use of either gene-specific RT primers or random hexamer primer at 50°C for 1 h. The reaction mixture is heated (e.g. 70°C for 15 min) and cooled. The resulting gene-specific RT reaction mixture is column purified and subjected to PCR amplification of cDNA. 

A set of sense and antisense primers should be selected to synthesize specific cDNAs and also to detect the amplified messages of the genes, complementary to their specific gene sequences. It is important to consider the following points while designing the primers for reverse transcriptase and polymerase chain reaction ... 

Ribosomal genes can be cloned or PCR amplified and then sequenced as other DNA sequences. However, the products of the genes, ribosomal RNAs, can be used as template for direct sequencing with the reverse transcriptase as polymerase following a protocol derived from the Sanger s technics for DNA (dideojiynucleotide chain termination). Universal primers can be used to sequence the chosen region of the rRNA. 

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