Cloning method and kit

US5525493A · kind A · utility

Assignee

• Dynal AS · NO

Inventors

• Erik Hornes · Oslo, NO
• Mathias Uhlen · Uppsala, SE

Key dates

• Filing date: May 19, 1992
• Grant date: Jun 11, 1996
• Expiry date: May 19, 2012

Classification

• Technology area (CPC C): Chemistry; Metallurgy
• CPC primary: C12Q2600/156
• WIPO field: Biotechnology
• WIPO sector: Chemistry

Abstract

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.