XcmI site-containing vector for direct cloning and in vitro transcription of PCR product.
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.
Arashi-Heese
N
N
Department of Biochemistry and Molecular Oncology, University of Tsukuba, Ibaraki, Japan.
Miwa
M
M
Shibata
H
H
D000595:Amino Acid Sequence; D016375:Antisense Elements (Genetics) / Q000235:genetics; D003001:Cloning, Molecular / Q000379:methods*; D015252:Deoxyribonucleases, Type II Site-Specific / Q000235:genetics* / Q000378:metabolism; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D019914:Taq Polymerase / Q000378:metabolism; D014158:Transcription, Genetic
D016375:Antisense Elements (Genetics); D019914:Taq Polymerase; C075914:endodeoxyribonuclease Eam1105I; C084267:endodeoxyribonuclease XcmI; D015252:Deoxyribonucleases, Type II Site-Specific