Cloning of phage SP6 RNA polymerase gene

Abstract

To clone the phage SP6 RNA polymerase gene, phage SP6 genome was cleaved with DraI and BstXI and 2.9 kb fragment containing SP6 RNA polymerase gene was obtained. We attempted to clone this fragment into pUC19 and pBR322, but it was unsuccessful. We speculate that this is because of an unclonable sequence which seems to have lethal effect on host cells. The location of the sequence is close upstream of the $5``$ end of the structural gene. To overcome this problem, we split the DraI/BstXI fragment with KpnI which cleaves the fragment into about $\frac{2}{5}$ and $\frac{3}{5}$ of the total length. The downstream $\frac{3}{5}$ of the gene was inserted into pUC19. To clone the 5`` portion of the gene, PCR technique was used to synthesize DNA from the beginning of the structural gene to the KpnI site. Also, to find out the function of the $5``$ proximal region of the gene, a fragment was obtained by cleavage with Sau3AI which cleaves at 63 bp downstream from transcription initiation site. The fragment was inserted into pUC18. In turn, this fragment and the fragment which is downstream three fifths of the gene was joined together and inserted into expression vector pTTQ19.