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prostar RT-PCR Systems for Robust High-Fidelity RNA Amplification

nds to RT-PCR can lead to improved sensitivity, higher product yields, and amplification of longer complex targets.5 To date, little attention has been directed toward the accuracy of current RT-PCR procedures and the use of high-fidelity proofreading PCR enzymes. Although the frequency of mutations in the final product is thought to be influenced by the fidelity of both the RT and the DNA polymerase, the relative contributions of each enzyme are presently unknown.

Besides differences in enzymes employed, various procedures have been used for coupling cDNA synthesis and PCR amplification. RT-PCR is essentially carried out using either a two-step or a one-step procedure. In the two-step technique, cDNA synthesis is performed by the RT under optimal conditions, followed by PCR amplification with an appropriate thermostable DNA polymerase. The reaction tube must be opened after cDNA synthesis. PCR reagents are then added to the reaction tube (known as two-step/one-tube procedures) or the synthesized cDNA is transferred to a second tube for the PCR portion of the procedure. Stratagenes ProSTAR Ultra HF RT-PCR system (described below) uses the latter approach, called a two-step/two-tube procedure. Two-tube techniques are particularly useful when amplifying multiple targets from a single cDNA synthesis reaction.

In one-step RT-PCR procedures, cDNA synthesis and PCR amplification are performed in the same tube in a common buffer; there is no need to add reagents between the cDNA synthesis and PCR steps. One-step techniques are convenient, fast, and reduce the risk of sample contamination; they are particularly useful when amplifying the same target from multiple RNA samples. Only a few commercial one-tube RT-PCR kits are truly one-step kits, such as Stratagenes ProSTAR HF Single-Tube RT-PCR system. Other kits marketed as single tube/single buffer RT-PCR kits are actually two-step kits since
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