VERFAHREN ZUM KLONIEREN UND REAGENZIENSATZ.

摘要

PCT No. PCT/EP90/02013 Sec. 371 Date May 19, 1992 Sec. 102(e) Date May 19, 1992 PCT Filed Nov. 20, 1990 PCT Pub. No. WO91/07505 PCT Pub. Date May 30, 1991.A method of amplifying target DNA is disclosed wherein said DNA is first amplified by PCR, the amplified DNA then being contacted with a single stranded linearised plasmid vector having terminal regions which are complementary to terminal regions of the PCR amplified DNA, whereby a cyclic product is formed comprising single stranded sequences from said target DNA and said vector and two double stranded regions from the overlapping terminal regions of the vector and the PCR amplified DNA; the cyclic product then being introduced into a host organism. Two-stage PCR may be performed and site-specific mutagenesis may be effected between PCR amplification and formation of the cyclic product. The single-stranded linearised plasmid vector and/or the target DNA may be immobilised. Kits are disclosed for performing various aspects of the method which can be used in a method of diagnosis wherein the target DNA is characteristic of a physiological condition.