US 5,334,522 AGranted

System for isolating and producing new genes, gene products and DNA sequences

AssigneeUnited States/National Institutes of Health

InventorsMichael A. Resnick, Miroslav Radman

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Abstract

A method of producing plasmids with heteroduplex DNA sequences, in which probe plasmids containing DNA inserts that correspond to probe sequences and test plasmids containing DNA inserts that correspond to a population of test sequences are first constructed. The vector portions of the plasmids used in these constructions have similar sequences. When test and probe plasmids are cleaved with appropriate restriction enzymes and then denatured to separate strands, complementary regions of the linear strands which correspond to vector sequences can anneal. The molecules that harbor test and probe inserts that are related by sequence complementarity form non-covalently closed circular molecules. These molecules can replicate after transformation into an appropriate host organism. There is a replication bias against plasmids which anneal through vector sequences, but which do not contain homologous probe and test inserts.

A method for producing plasmids with heteroduplex DNA sequences, comprising the steps of:

(a) providing a first cloning vector with a first backbone molecule of DNA and having cloned therein a probe molecule of DNA wherein a first restriction site is present at an end of the probe DNA, said first restriction site being different from any other restriction site present anywhere in said first backbone or said probe DNA;
(b) providing a second cloning vector having a second backbone molecule of DNA which is capable of annealing to said first backbone of said first cloning vector at between 75.degree. C. and 95.degree. C. in the presence of 0.2M NaCl, said second backbone having a test molecule of DNA cloned therein, the test DNA having a second restriction site, said second restriction site being present at an end of said test DNA, wherein the end of said test DNA having said second restriction site is distal to the end of said probe DNA having said first restriction site when said probe DNA and test DNA are annealed to each other, wherein said second restriction site is different from any other restriction site present anywhere in said first or second backbones, probe DNA or test DNA;
(c) cutting said first cloning vector at said first restriction site with a restriction enzyme, thereby producing a first linear molecule;
(d) cutting said second cloning vector at said second restriction site with a restriction enzyme, thereby producing a second linear molecule;
(e) heat-denaturing said first and second linear molecules to obtain single-stranded DNA;
and
(f) hybridizing said linear molecules at between 75.degree. C. and 95.degree. C. in the presence of 0.2M NaCl to allow annealing of backbone DNA, followed by hybridization at between 22.degree. C. and 75.degree. C. in the presence of 0.2M NaCl to allow annealing of probe DNA and test DNA strands so that circular plasmids containing heteroduplex DNA sequences are obtained, wherein each strand of said circular plasmids contains a length of single-stranded DNA not annealed to the other strand of said plasmid, and wherein one end of each of said single-stranded lengths is not joined to the strand of said plasmid of which it is a part.

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Facts

Application

07/860,233Filed 27 Mar 1992 · continuation

Priority

27 Dec 1989

Granted

2 Aug 1994

Term ends

Not estimatedThe source records don’t support an estimate.

Classification4 codes

C12N 15/10
Recombinant DNA-technology: processes for the isolation, preparation or purification of DNA or RNA
C12N 15/00
Mutation or genetic engineering
C12N 15/66
Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression: general methods for inserting a…
C12N
Microorganisms or enzymes

Abstract

Claims9