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| Many RT-PCR kits contain inferior enzymes, such as reverse transcriptases with an associated RNase H activity that limits cDNA yield, or low-accuracy PCR enzymes that introduce errors which inhibit gene expression and sequence determination. | ||||||||
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New System Improves RT-PCR Reverse transcription followed by PCR (RT-PCR) is a common and efficient laboratory technique for generating cDNAs for cloning, library construction, and in vitro transcription. These applications rely on producing full-length, highly representative cDNA. We previously measured the frequency of mutations in RT-PCR reactions performed with different PCR enzymes1 and various competitors’ RT-PCR systems2. These results clearly indicate that although RTs exhibit relatively high error rates, the accuracy of the PCR enzyme influences overall mutation frequency as a result of exponential amplification of the cDNA during the PCR step. In these studies, mutation frequencies were shown to be significantly lower in RT-PCR reactions carried out with PfuTurbo™ DNA polymerase compared to Taq DNA polymerase or Taq-based blends1,2. To provide you with improved RT-PCR methods, we now offer a one-tube method designed to facilitate simple RT-PCR cloning into TA/UA vectors with high accuracy. Convenient One-Tube Format Delivers High-Fidelity The new StrataScript™ one-tube RT-PCR system is ideal for rapid, high-fidelity RT-PCR cloning applications with reduced sample contamination risk. It is particularly useful when amplifying the same target from multiple RNA samples. The kit features our robust RNase H minus StrataScript reverse transcriptase for first-strand cDNA synthesis, followed by amplification with the Easy-A™ highfidelity PCR cloning enzyme.* This proofreading enzyme has the same high fidelity as PfuTurbo DNA polymerase; that is, it’s six-fold more accurate than Taq DNA polymerase. It also can amplify a wide variety of target lengths from 0 to 5 kb. Same TA/UA Cloning Efficiencies As Taq Unlike other proofreading polymerases, the Easy-A PCR cloning enzyme preferentially adds a single 3’-A overhang to the ends of PCR products. The combination of StrataScript RT plus the Easy-A enzyme means that RT-PCR products can be cloned directly into T- or U-modified vectors without additional steps required by other proofreading DNA polymerases. As shown in Table 1, RTPCR products generated with the StrataScript one tube RT-PCR system are cloned into T- or U-modified vectors with the same high efficiencies as Taq-based cloning.
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