DETECTION OF INTERLEUKIN-2-ALPHA RNA AND DETERMINATION OF MDR-1 GENE-EXPRESSION BY COMBINED REVERSE TRANSCRIPTION AND AMPLIFICATION USING THERMUS-THERMOPHILUS DNA-POLYMERASE

It was shown that native Thermus thermophilus DNA polymerase possesses a higher reverse transcriptase activity than the Tag enzyme and can t herefore be used in combined reverse transcription and polymerase chai n reaction (RT/PCR). In this work, it was employed for RT/PCR on inter leukin-2 alpha RNA synthesized by in vitro transcription. The conditio ns of IL-2 alpha RNA detection were optimized. The yield of the specif ic product was maximal at pH 8.5-9.0. The effect of Mg2+, Mn2+, Cu2+, Co2+, and Cd2+ on RT/PCR was studied. It was found that the stimulatin g effect of divalent cations on reverse transcription decreases in the following order. Mn2+ greater than or equal to Cu2+ > Mg2+ > Cd2+ >> Co2+. The optimal [Me(2+)]/[dNTP] ratio for Mg2+ and Mn2+ was found to be 3.75 and 1.25-1.88, respectively. For Cu2+ and Cd2+, this paramete r ranged from 1.88 to 2.5. Replacement of Mg2+ with Mn2+ reduced the e fficiency of PCR. It was shown that the RT/PCR method using T. thermop hilus DNA polymerase allows detection of 10(3) copies of IL-2 alpha RN A. A quick and efficient method was developed for determining the mult idrug resistance gene 1 (MDG-1) expression in human ovary sarcoma cell s (SKLVLB line).