Powerful high-fidelity RT-PCR systems in two convenient formats
Michael Borns Janice Cline Kris Connors Ronda
Allen Holly Hogrefe
Stratagenes prostar RT-PCR systems are powerful systems that permit efficient, high-fidelity amplification of RNA in two convenient formats. The ProSTAR Ultra HF RT-PCR system features pfuturbo DNA polymerase * for amplifying cDNAs with the highest replication fidelity possible using a versatile two-tube format. The ProSTAR HF Single-Tube RT-PCR system combines the sensitivity and accuracy of the TaqPlus Precision DNA polymerase ** blend with the convenience of a one-step, one-tube procedure. These two systems are ideally suited for subcloning, expression, and sequencing of DNA coding regions and for reproducibly detecting and analyzing RNA molecules.
Traditional techniques used to study gene expression include Northern blotting, RNase protection, in situ hybridization, dot blots, and S1 nuclease assays. However, because coupled reverse transcription and PCR amplification (RT-PCR) is an easy, sensitive, and versatile technique, it is the method of choice for analyzing RNA. RT-PCR can be used to determine the presence or absence of a transcript, to quantify expression levels, and to clone rare messages without needing to construct and screen cDNA libraries.
RT-PCR kits differ not only with respect to the RT and PCR enzymes employed,
but also with the procedures whereby cDNA synthesis and PCR amplification are
coupled. MMLV reverse transcriptase (MMLV-RT), AMV RT, and Tth DNA
polymerase are most commonly used to perform cDNA synthesis.1,2,3,4
Second-strand synthesis and DNA amplification can then be carried out using Taq
or Tth DNA polymerase,1,2,3,4 or more recently, Taq-based
DNA polymerase blends. Applying these ble