A COUPLED ONE-STEP METHOD OF REVERSE TRANSCRIPTION(RT)FOR GENERATION OF OPEN READING OF G PROTEIN αSUBUNIT GENE

It is reported that a one-step reverse transcription and polymerase chain reaction (RT-PCR)procedure for amplification of open reading frame of G-protein αsubunit gene from Arabidopsis thaliana which is larger than 1kb. In the procedure developed, manual work is minimized, larger numbers of samples can be handled. After denaturation of template RNAand the primers and addition of other regents, no further manual steps are needed, It was not found that AMVreverse transcriptase inhibited the activity of Taq-DNA polymerase, so the coupled one-step procedure of RT-PCRrapidly amplifies ORFjust only from 0.5μg amount of total RNA.