Arashi-Heese N, Miwa M, Shibata H
Department of Biochemistry and Molecular Oncology, University of Tsukuba, Ibaraki, Japan.
Mol Biotechnol. 1999 Oct;12(3):281-3. doi: 10.1385/MB:12:3:281.
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.
为了进行TA克隆以及逆转录聚合酶链反应（RT-PCR）产物的直接体外转录，我们构建了一种源自pGEM-3Zf(+)的新型T载体pGEMTA.miwa。该载体经限制性内切酶Xcml消化后可产生单胸腺嘧啶（T）突出端。在直接TA克隆系统的一项有用改进中，这种新型T载体可用于从克隆序列直接体外转录核糖探针。该载体的优点是在载体的多克隆位点排除了富含GC的额外序列，以减少原位杂交中的非特异性杂交信号。使用该载体还提供了一个用于克隆未修饰PCR产物的快速系统、重组克隆的颜色筛选以及随后对克隆片段的测序分析。这些功能使我们能够直接从RT-PCR产物或cDNA序列合成RNA探针，并且对杂交或原位杂交分析很有用。
