Efficient directional genetic cloning system

US5595895A · kind A · utility

Assignee

• The United States of America, as represented by the Department of Health and Human Services · US

Inventors

• Toru Miki · North Bethesda, US
• Stuart A. Aaronson · Great Falls, US

Key dates

• Filing date: Jun 24, 1992
• Grant date: Jan 21, 1997
• Expiry date: Jun 24, 2012

Classification

• Technology area (CPC C): Chemistry; Metallurgy
• CPC primary: C12N15/66
• WIPO field: Biotechnology
• WIPO sector: Chemistry

Abstract

A highly efficient genetic cloning system is disclosed which is particularly useful for cloning cDNA copies of eukaryotic mRNAs and can direct the orientation of inserts in .lambda.-plasmid composite vectors with large cloning capacities. Cleavage of such vector DNA, by the restriction enzyme SfiI, for example, creates two different non-symmetrical 3' extensions at the ends of vector DNA. Using a linker-primer and an adaptor, cDNA is prepared to have two different sticky ends which can be ligated to those of the vector. When the cDNA fragments and the vector DNAs are mixed, both the molecules can assemble without self-circularization due to base-pairing specificity. This system provides (1) high cloning efficiency (10.sup.7 -10.sup.8 clones/.mu.g poly(A).sup.+ RNA), (2) low background (more than 90% of the clones contain inserts), (3) directional insertion of cDNA fragments into the vectors, (4) presence of a single insert in each clone, (5) accommodation of long inserts (up to 10 kb), (6) a mechanism for rescue of the plasmid part from a .lambda. genome, and (7) a straightforward protocol for library preparation.