Why different amounts of forward and reverese primers in RT-QPCR - (May/24/2005 )

Hi
I am optimizing the primer concentration for a RT-QPCR expt. using strategen protocol. I am confused why we should use different amounts of forward and reverese primers? Theoritically it should be same???

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Asymmetric PCR: A PCR in which the predominant product is a single-stranded DNA, as a result of unequal primer concentrations. As asymmetric PCR proceeds, the lower concentration primer is quantitatively incorporated into double-stranded DNA. The higher concentration primer continues to primer synthesis, but only of its strand.

Asymmetric amplification may help reduce the decline in the fluorescent plateau (hook effect) that is sometimes seen with HybProbe Probes. Depending on base composition and length of an amplicon its strands may tend to anneal faster than the HybProbe Probes have the opportunity to bind to their target sites. This is also avoided by favouring the production of one strand. The drawback of asymmetric amplification is a reduced PCR efficiency because the reaction shifts from exponential to linear amplification as one of the primers becomes limiting.

If no fluorescent signal is obtained in the PCR reaction containing HybProbe Probes, and one is confident that the primers are successfully amplifying DNA, the problem might be caused by competition between probe-target hybridization and target-target hybridization that results in out-competition of the probes by the complementary strand of the target DNA. This can be overcome by skewing the primer concentration in the reaction to favor the formation of the DNA strand that the probes bind to.

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Thank you

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