US 5,792,640 AGranted

General method to clone hybrid restriction endonucleases using lig gene

AssigneeThe Johns Hopkins University

InventorSrinivasan Chandrasegaran

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Abstract

The present invention reveals methods for cloning hybrid restriction endonucleases and for enzymatically inactivating a target DNA. The method for cloning hybrid restriction endonucleases involves co-expression of a ligase. A first plasmid contains a gene encoding a DNA ligase. A second plasmid contains a gene encoding a hybrid restriction endonuclease and is compatible with the first plasmid. The method involves transfecting host cells with the first plasmid, so that DNA ligase is produced, followed by transfecting the cells with the second plasmid. The method for enzymatically inactivating a target DNA involves preparing a plasmid, phage, virus or any other delivery vehicle such as a liposome containing a gene encoding a nuclease, delivering the gene into cells, inducing the cells to produce the nuclease and enzymatically inactivating the target DNA.

Claims32

Dependent claims

A method for producing a nuclease in a cell wherein

said nuclease, when produced in a cell comprising a target nucleic acid which comprises a target nucleotide sequence specifically binds to said target nucleotide sequence and cleaves said target nucleic acid specifically bound to said nuclease,
said method comprising the steps of:
a) providing a preparation of a first polynucleotide encoding said nuclease, wherein said preparation does not comprise a second polynucleotide encoding an enzyme which protects said target nucleic acid from said nuclease by specifically binding to said target nucleotide sequence and enzymatically modifying said target nucleic acid such that said target nucleic acid is not cleaved by said nuclease;
and
b) delivering said preparation of said first polynucleotide encoding said nuclease into said cell under conditions such that said first polynucleotide expresses said nuclease, thereby producing said nuclease.

2 more elements

Dependencies are inferred from the claim text.

Facts

Application: 08/575,361Filed 24 Dec 1995 · continuation in part
Priority: 3 Apr 1992
Granted: 11 Aug 1998
Term ends: Not estimatedThe source records don’t support an estimate.

Classification8 codes

C12N 9/22
Acting on ester bonds: ribonucleases [RNase]; Deoxyribonucleases [DNase]
C12Q 1/683
For detection of mutation or polymorphism: involving restriction enzymes, e.g. restriction fragment length polymorphism [RFLP]
C07K 2319/09
Containing a localisation/targetting motif: containing a nuclear localisation signal
C07K 2319/60
Fusion polypeptide: containing spectroscopic/fluorescent detection, e.g. green fluorescent protein [GFP]

Family

Serial862,831Parent of this continuation-in-part · filed 3 Apr 1992
Serial17,493Parent of this continuation-in-part · filed 12 Feb 1993
Serial126,564Parent of this continuation-in-part · filed 27 Sep 1993
Serial346,293Parent of this continuation-in-part · filed 23 Nov 1994
US 5,436,150 AParent of this continuation-in-part · filed 25 Jul 1995
US 5,487,994 AParent of this continuation-in-part
US 5,356,802 AParent of this continuation-in-part
US 5,792,640 AThis patent

From the US filing history. Foreign family members aren’t listed.