Methods of analysis and manipulation of DNA utilizing mismatch repair systems

• US 5,858,754 A
• Filed: 02/13/1996
• Issued: 01/12/1999
• Est. Priority Date: 05/12/1989
• Status: Expired due to Fees

• Current Assignee: Duke University
• Original Assignee: Duke University
• Inventors: Modrich, Paul L., Su, Shin-San, Lahue, Robert S., Cooper, Deani Lee, Au, Karin G., Worth, Leroy Jr.
• Primary Examiner(s): Wax, Robert A.
• Assistant Examiner(s): Nashed, Nashaat T.
• Application Number:
  US08/600,450
• Time in Patent Office: 1,064 Days
• Field of Search: 435/191, 435/6, 435/91.1, 435/91.2, 435/195, 435/252.3, 435/252.33, 536/24.3, 536/24.31, 536/24.32, 536/23.1, 536/23.2, 536/23.4, 536/23.7
• US Class Current: 435/195
• CPC Class Codes: C07K 14/245 Escherichia (G)
• CPC Class Codes: C12N 15/00 Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor mutants or genetically engineered microorganisms, per se C12N1/00, C12N5/00, C12N7/00; new plants per se A01H; plant reproduction by tissue culture techniques A01H4/00; new animals per se A01K67/00; use of medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases, gene therapy A61K48/00
• CPC Class Codes: C12N 15/102 Mutagenizing nucleic acids
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 2521/514 Mismatch repair protein
• CPC Class Codes: C12Q 2535/131 Allele specific probes
• CPC Class Codes: C12Q 2537/113 Heteroduplex formation
• CPC Class Codes: C12Y 302/0202 DNA-3-methyladenine glycosylase I (3.2.2.20), i.e. adenine DNA glycosylase
• CPC Class Codes: Y10S 435/81 Packaged device or kit

Abstract

A diagnostic method for detecting a base pair mismatch in a DNA duplex, comprising the steps of contacting at least one strand of a first DNA molecule with the complementary strand of a second DNA molecule under conditions such that base pairing occurs contacting a DNA duplex potentially containing a base pair mismatch with a mispair recognition protein under conditions suitable for the protein to form a specific complex only with the DNA duplex having a base pair mismatch, and not with a DNA duplex lacking a base pair mismatch, and detecting any complex as a measure of the presence of a base pair mismatch in the DNA duplex.

Claims

1. A conjugated DNA mispair recognition protein consisting of a mispair recognition function and a DNA cleaving function, wherein said cleaving function is selected from the group consisting of a hydroxyl radical cleaving function and an endonuclease cleaving function, such that said conjugated DNA mispair recognition protein is able to modify at least one strand of the DNA duplex in the vicinity of the bound mispair recognition protein.

2. The conjugated DNA mispair recognition protein of claim 1 wherein said protein is the product of the mutS gene of E. coli which is attached to a hydroxyl radical cleaving function.

3. The conjugated DNA mispair recognition protein of claim 2, wherein the hydroxyl radical cleaving function is selected from the group consisting of the 1,10-phenanthroline-copper complex, the EDTA iron complex, and the copper binding domain of serum albumin.

4. The conjugated DNA mispair recognition protein of claim 1, wherein said protein is the product of the mutS gene of E. coli which is attached to a DNA endonuclease activity capable of cleaving double-stranded DNA.

5. The conjugated DNA mispair repair protein of claim 4, wherein said endonuclease activity is provided by the DNA cleavage domain of Flavobacterium okeanokoites I endonuclease.