| 摘要 |
The present invention provides vectors and methods which improve the efficiency of nucleic acid insertion into circular vectors, which generally facilitate nucleic acid cloning and specifically facilitate the preparation of DNA libraries. In general, the present invention involves separation of the cloning process into two distinct steps: (a) insertion which is done at a hi gh nucleic acid concentration favoring intermolecular joining, and (b) circularization which is performed at a low nucleic acid concentration favoring intramolecular circularization. The present vectors generally have distinct insertion ends and circularization ends which are blocked from covalent joining during the insertion step. Circularization ends contemplate d by the present invention include complementary cohesive ends and topoisomera se- linked ends. The present vectors and methods allow minute amounts of nucleic acid inserts to be efficiently cloned. Moreover, little or no insert size selection occurs with the present methods so that large as well assmall nucleic acid inserts are readily inserted into the present vectors. Thus, DN A libraries which are representative of the entire range of size of DNA insert s can be made, and, for example, full length cDNA libraires are readily obtain ed.
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