Methods of analysis and manipulating of DNA utilizing mismatch repair systems

• US 5,702,894 A
• Filed: 06/02/1995
• Issued: 12/30/1997
• Est. Priority Date: 05/12/1989
• Status: Expired due to Term

• 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): Jones, W. Gary
• Assistant Examiner(s): REES, DIANE
• Application Number:
  US08/460,663
• Time in Patent Office: 942 Days
• Field of Search: 435/6, 435/91.1, 435/91.2, 435/174, 435/7.1-7.9, 536/22.1, 536/23.1, 536/24.3-24.33
• US Class Current: 435/6.11
• 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 method for removing DNA molecules containing one or more base pair mismatches in a population of DNA duplexes that have been produced by enzymatic amplification, potentially containing one or more base pair mismatches, comprising the steps of:

contacting said population of DNA duplexes with a mismatch repair system under conditions such that one or more components of said mismatch repair system form a specific complex with a base pair mismatch contained in a DNA duplex having a base pair mismatch, and

removing said DNA duplex containing said complex or the product of said complex from the population of duplex molecules without the use of an additional agent capable of enzymatic digestion of said DNA duplexes which is not a component of said mismatch repair system, prior to said removal.

2. A method for removing DNA duplex molecules containing one or more base pair mismatches in a population of heteroduplex DNA molecules produced from different sources, comprising the steps of:

contacting said population of DNA duplex molecules potentially containing base pair mismatches with some or all components of a mismatch repair system under conditions such that said component or components form a complex with DNA duplex molecules containing a base pair mismatch, and not with DNA duplex molecules lacking a base pair mismatch, and

removing DNA molecules containing said complex or the product of said complex from said population of heteroduplex DNA molecules without the use of an additional agent capable of enzymatic digestion of said DNA duplexes which is not a component of said mismatch repair system prior to said removal.

3. The method of claim 2, wherein some or all proteins of the mismatch repair system have been affixed to a solid support and removal of said complex is by adsorption.

4. The method of claim 2, wherein said complex specially binds with other cellular proteins and removal of said complex occurs through said interaction.

5. Assay kit for eliminating DNA molecules containing one or more base pair mismatches from a population of heterohybrid duplex molecules formed by base pairing of single-stranded DNA molecules obtained from a first and a second source comprising:

an aliquot of proteins of a mismatch repair system,

an aliquot of a recombinase protein whose ability to catalyze duplex formation is inhibited by said proteins of a mismatch repair system, and

a means for removing said DNA molecules containing one or more base pairing mismatches.

6. The kit of claim 5, wherein the proteins of the mismatch correction system are the MutS and MutL proteins of Escherichia coli.

7. Kit for fractionating a heteroduplex DNA population into two parts, one consisting of duplexes which contain a mismatch prior to fractionating and the other consisting of duplexes that do not contain a mismatch prior to fractionating comprising:

an aliquot of components of a mismatch repair system;

an aliquot of biotinylated nucleotides; and

an aliquot of avidin or avidin linked to a support.

8. The kit of claim 7, wherein the mismatch repair system is from Escherichia coli and consists of products of the mutH, mutL, and mutS genes, DNA helicase II, single-strand DNA binding protein, DNA polymerase III holoenzyme, exonuclease I, Exo VII exonuclease or RecJ exonuclease, DNA ligase, and ATP.