Background technology

T-A cloning (Clark, J.M.Novel non-templated nucleotide addition reactionscatalyzed by procaryotic and eucaryotic DNA polymerases (nucleic acid of new protokaryon or the catalytic non-template dependent form of eukaryotic DNA polysaccharase adds reaction) .Nucleic Acids Res.1988,16,9677-9686; Marchuk, D., Drumm, M., Saulino, A.and Collins, F.S.Construction ofT-vectors, a rapid and general system for direct cloning of unmodified PCR product (structure of T carrier, a kind of system of the general original PCR product of direct clone fast) .Nucleic Acids Res.1991,19,1154; Holton, T.A.and Graham, M.W.A simple and efficient method fordirect cloning of PCR products using ddT-tailed vectors (a kind of simple and effective method of utilizing the direct clone PCR products of ddT overhang carrier) .Nucleic Acids Res.1991,19,1156; Mead, D.A., Pey, N.K., Herrnstadt, C., Marcil, R.A.and Smith, L.M.A universal method forthe direct cloning of PCR amplified nucleic acid (a kind of universal method that is used for direct clone PCR products) .Biotechnology (N Y) .1991,9, be to be accompanied by a kind of cloning process that round pcr grows up 657-663).PCR product cloning to 3 ' end that so-called T-A clone has 3 ' end single A tail exactly has on the T carrier of single T tail.T-A clone's theoretical foundation is: in the pcr amplification process, because the Taq polysaccharase has the terminal enzyme (DNA) activity of the template of not relying on and adds single deoxynucleotide at 3 of PCR product ' end, and preference is added the deoxyadenylic acid (A) in four kinds of deoxynucleotides, make 3 ' end of the PCR product more than 50% have single deoxyadenylic acid, i.e. 3 ' end A tail.Therefore people develop carrier---the T carrier of a kind of 3 ' end with an outstanding T, do the linear DNA segment of A-tailing operation with the Taq enzyme with the PCR product that is used to clone the taq enzymatic amplification or other.

At present, the preparation method of T carrier has three kinds.First method is to utilize the restriction endonuclease that produces flat end that the cyclic plasmid enzyme is cut into the wire plasmid, utilize terminal enzyme (DNA) to add single ddTTP to 3 of wire carrier ' end (Holton then, T.A.and Graham, M.W.A simple and efficient method for directcloning of PCR products using ddT-tailed vectors (a kind of simple and effective method of utilizing the direct clone PCR products of ddT overhang carrier) .NucleicAcids Res.1991,19,1156).Second method is to utilize the terminal enzyme (DNA) activity that does not rely on template of Taq enzyme to add single dTTP to 3 of wire carrier ' end (Marchuk, D., Drumm, M., Saulino, A.and Collins, F.S.Construction ofT-vectors, the a rapid and general system for direct cloning of unmodified PCR product (structure of T carrier, a kind of system of the general original PCR product of direct clone fast) .Nucleic Acids Res.1991,19,1154).The third method is to introduce specific restriction enzyme site in the multiple clone site of plasmid vector, produces 3 ' end with corresponding endonuclease digestion and has the outstanding linear T carrier of single T.In theory, the restriction endonuclease that can be used for preparing the T carrier comprises: XcmI, AhdI/Eam11051/EclHKl/AspEI/NruG0/BspOVI, BfiI/BmrI, HphI/AsuHPI, MboII/NcuI, BfuI/BciVI, HpyAV/Hin4II etc.Wherein XcmI and AhdI and isoschizomers thereof widespread use in preparation T carrier.Other restriction endonuclease or because too expensive, or because too many recognition site is arranged on common cloning vector, thereby the application in preparation T carrier is restricted.

Preceding two kinds of methods exist tailing efficient sequences lower and linear plasmid its two ends in the tailing process problem (Shuman such as to be partially removed sometimes in preparation T carrier process, S.Novel approach tomolecular cloning and polynucleotide synthesis using vaccinia DNA topoisomerase (a kind of vaccinia virus topoisomerase that utilizes carries out molecular cloning and polynucleotide synthetic novel method) .J.Biol.Chem.1994,269,32678-32684).Owing to the strict quality control of production firm, its efficient also improves greatly now, can satisfy conventional clone operations, but because its operation steps is more, quality is not easy control, only is applied to several frequently seen carrier at present, and must producing, so price comparison costliness by the company of specialty.The third method also is applied in preparation T carrier at present, has a potential problem but make up the T carrier with restriction endonuclease, and partially digested pre-T carrier is mingled in and can causes the background of non-recombinant conversion too high in the T carrier.In addition, the T carrier that this method produces, look like natural, but be not widely used in practice, do not adopted by a lot of biotech firms yet, except too expensive because of restriction endonuclease, perhaps having on the common cloning vector outside the too many recognition site, a prior reason may be to be used for connecting relatively difficulty behind these enzyme cutting DNAs, dna fragmentation as Ahd I and Xcm I generation has the outstanding of a base at 3 ' end, even than the more difficult connection of flat end (http://www.neb-china.com/cn).Along with the continuous progress of biotechnology, the method for preparing the T carrier is also improved gradually now, and the problem that prepare high quality, stablize, inexpensive T carrier remains vast biological study person's care also is a key of effectively using the T-A clone technology.