XcmI site-containing vector for direct cloning and in vitro transcription of PCR product

Abstract

For TA cloning and the direct in vitro transcription of reverse transcriptase-polymerase chain reaction (RT-PCR) product, we constructed a novel T-vector, pGEMTA.miwa, derived from pGEM-3Zf(+). The vector were designed to produce single thymidine (T) overhangs when digested with a restriction enzyme Xcml. In a useful modification of the direct TA cloning system, the novel T vector can be applied to the direct in vitro transcription of riboprobe from the cloned sequences. The vector has the advantage of excluding GC-rich extra sequences in the multiple cloning site of the vector for the reduction of nonspecific hybridization signals in in situ hybridization. The use of the vector also provides a rapid system for the cloning of unmodified PCR products, a color selection of the recombinant clones, and subsequent sequencing analysis of the cloned fragments. These functions allow us to synthesize an RNA probe directly from RT-PCR products or cDNA sequences, and are useful for hybridization or in situ hybridization analysis.