[Use of thermostable DNA polymerase from Thermus thermophilus KTP in a combined reverse transcription and amplification reaction of detecting interleukin 2alpha RNA and determining expression of the multidrug resistance gene (MDR-1)].

Abstract:

According to our data native Tth DNA polymerase displays higher reverse transcription activity than Taq DNA polymerase. This allows one to use Tth DNA polymerase in the complete reaction of reverse transcription and amplification (RT/PCR). We used this enzyme to synthesize the interleukine (IL-2 alpha) RNA template synthesized by the RT/PCR method in vitro. The conditions for RNA IL-2 alpha detection were optimized. The maximum yield of the specific product was obtained at pH 8.5-9.0. The influence of bivalent cations on the efficiency of RT reaction of coupled RT/PCR can be expressed as: Mn2+ > or = Cu2+ > Mg2+ > Cd2+ >> Co2+. The optimal ratio is 1.25-1.88 for Mn2+/dNTPs and 1.88-2.5 for Cu2+/dNTPs and Cd2+/dNTPs. The maximum yield of the RT/PCR product is found at Mg2+/dNTPs = 3.75. When Mn2+ is used instead of Mg2+ in the PCR reaction the efficiency of RT/PCR decreases. The RT/PCR method embracing thermostable Tth DNA-polymerase provides detection of 10(3) copies of RNA IL-2 alpha. An efficient method of the express-diagnostics of MDR-1 gene expression by coupled RT/PCR using Tth DNA polymerase is described.

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