PUC-IVT, a modified pUC19 based in vitro transcription vector

Abstract

The popular cloning vector, pUC19, was modified into an in vitro transcription system, pUC-IVT, by incorporation of T7 promoter sequence and appropriate restriction sites for cloning of gene of interest and generation of transcript. Strategy was also devised to eliminate incorporation of undesirable sequences at both 5′ and 3′ ends of the transcript for its use in many sensitive downstream applications. All the major restriction sites, except SphI and PstI present in the MCS of pUC19, were retained in pUC-IVT for facile cloning. Any DNA fragment having 5′ blunt end, and cohesive 3′ end, generated by restriction enzymes present in MCS of pUC19, except SphI and PstI, can be directionally cloned into pUC-IVT backbone generated by restricting with StuI and the same restriction enzyme used for 3′-end generation of target DNA fragment. EcoRI digested modified gene coding for archaebacterial (Synechocystis sp.) precursor tRNA-glutamine (ptRNAGln), harbouring recognition site for a type IIS restriction enzyme, FokI, was ligated onto the StuI and EcoRI digested pUC-IVT to generate pUC-IVT::ptRNAGln. In vitro run-off transcription was performed using FokI digested linearized pUC-IVT::ptRNAGln plasmid DNA as template to get ptRNAGln transcript. Reconstituted Escherichia coli ribonuclease P (RNase P) efficiently cleaved 5′ leader sequence of ptRNAGln to generate mature tRNAGln.