Methods and kits for fractionating a population of DNA molecules based on the presence or absence of a base-pair mismatch utilizing mismatch repair systems

• US 5,556,750 A
• Filed: 11/01/1993
• Issued: 09/17/1996
• Est. Priority Date: 05/12/1989
• Status: Expired due to Term

• Current Assignee: Duke University
• Original Assignee: Duke University
• Inventors: Cooper, Deani L., Modrich, Paul L., Su, Shin-San, Lahue, Robert S., Au, Karin G., Worth, Leroy Jr.
• Primary Examiner(s): Jones, W. Gary
• Assistant Examiner(s): Marschel, Ardin H.
• Application Number:
  US08/145,837
• Time in Patent Office: 1,051 Days
• Field of Search: 435/6, 435/91.1, 435/91.2, 435/810, 436/501, 536/22.1, 536/23.1, 536/24.1, 536/24.3-.33, 935/77, 935/78, 935/88
• 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 eliminating DNA molecules containing one or more base pairing mismatches from a population of heterohybrid duplex DNA molecules formed by base pairing of single-stranded DNA molecules obtained from a first source and a second source, comprising the steps of:

digesting genomic DNA from said first and said second source with a restriction endonuclease,

methylating the DNA from one of said sources,

denaturing said DNA from said first and said second source,

mixing DNA from said first and said second source in the presence of a recombinase protein, proteins of a mismatch repair system that modulate said recombinase protein, single-strand binding protein, and ATP, such that DNA duplexes form in homologous regions of DNA molecules from said first and said second source and the presence of a base pair mismatch results in regions that remain single-stranded, and

removing molecules that contain a said single-stranded region from said population.

2. The method of claim 1, wherein said recombinase protein is the Escherichia coli RecA protein.

3. The method of claim 1, wherein said mismatch repair system comprises the Escherichia coli methyl-directed mismatch repair system and consists of the MutS and MutL proteins.

5. The method of claim 1 or 4, wherein the removal of said molecules containing a single-stranded region is by treatment with a single-strand specific nuclease.

6. The method of claim 1 or 4, wherein the removal of said molecules containing a single-stranded region is by chromatography on benzoylated naphthoylated DEAE.

9. The method of claim 1 or 4, wherein said sources of DNA are selected from the group consisting of:

individuals of the same species, individuals of different species, individuals of different kingdoms, different tissue types, the same tissue type in different states of growth, different cell types, cells of the same type in different states of growth, cells of the same origin in different stages of development and cells of the same origin have undergone differential somatic mutagenesis.

10. The method of claim 9, wherein one of said sources consists of a probe sequence that has been at least partially characterized.

4. A method for eliminating DNA molecules containing one or more mismatches from a population of heterohybrid duplex DNA molecules formed by a strand transfer reaction between duplex DNA molecules obtained from a first source and denatured DNA molecules obtained from a second source, comprising the steps of:

restriction digesting genomic DNA from said first and said second sources,

methylating the DNA of said first or said second source,

denaturing DNA from said second source,

mixing DNA molecules from said first and said second source in the presence of a protein which catalyzes a strand transfer reaction, proteins of a mismatch repair system that modulate said protein which catalyzes a strand transfer reaction, single-strand binding protein, and ATP, such that DNA heteroduplexes form in homologous regions of DNA molecules from the first and the second source and the presence of a base pair mismatch results in regions that remain single-stranded, and

removing molecules that contain a said single-stranded region from said population.

7. The method of claim 4, wherein said strand transferase protein is the Escherichia coli RecA protein.

8. The method of claim 4, wherein mismatch repair system comprises the Escherichia coli methyl-directed mismatch repair system and consists of the MutS and MutL proteins.

11. A method of genomic mismatch scanning, wherein heterohybrid DNA molecules containing base pair mismatches are removed, without the use of exonuclease III, comprising the steps of:

contacting a population of heterohybrid DNA molecules potentially containing a base pair mismatch with a DNA mismatch repair system in the presence of one or more dideoxynucleoside triphosphates such that a single-stranded region is generated in a DNA molecule that contained a base pair mismatch, without the use of exonuclease III, and,

removing said molecule containing a single-stranded region from the population.

12. The method of claim 11, wherein said DNA mismatch repair system is the Escherichia coli methyl-directed mismatch repair system.

13. The method of claim 11, wherein the removal of said molecule containing a single-stranded region is by chromatography on benzoylated naphthoylated DEAE.

14. The method of claim 11, wherein the removal of said molecule containing a single-stranded region is by treatment with a single-strand specific nuclease.

15. A method for fractionating a population of DNA molecules based upon a mismatch in a subset of said molecules, wherein a heterohybrid DNA molecule containing a base pair mismatch is separated from non-mismatch-containing molecules, without the use of exonuclease III, comprising the steps of:

contacting a population of heterohybrid DNA molecules potentially containing a base pair mismatch with a DNA mismatch repair system and biotinylated nucleotide triphosphates, in the absence of exonuclease III, such that biotinylated nucleotides are incorporated into DNA molecules that contained a base pair mismatch and,

separating said molecule containing biotinylated nucleotides from those not containing said nucleotides by binding to avidin.

16. The method of claim 15, wherein said DNA mismatch repair system is the Escherichia coli methyl-directed mismatch repair system.

17. A method for removing DNA duplex molecules containing 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 and biotinylated nucleotides such that said nucleotides are incorporated into duplex molecules that contain a base pair mismatch and not into DNA duplex molecules lacking a base pair mismatch, and

removing said duplexes with incorporated biotinylated nucleotides by binding to avidin.