Use of immobilized mismatch binding protein for detection of mutations and polymorphisms, and allele identification

• US 6,114,115 A
• Filed: 04/28/1995
• Issued: 09/05/2000
• Est. Priority Date: 11/04/1993
• Status: Expired due to Fees

• Current Assignee: Valigen (US), Inc.
• Original Assignee: ValiGene Corporation
• Inventors: Wagner, Robert E. Jr.
• Primary Examiner(s): Myers, Carla J.
• Assistant Examiner(s): JOHANNSEN, DIANA B
• Application Number:
  US08/431,081
• Time in Patent Office: 1,957 Days
• Field of Search: 435/6, 435/91.2, 935/77, 935/78
• US Class Current: 435/6.12
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 1/6858 Allele-specific amplification

Abstract

A method for detecting mutations, such as a single base change or an addition or deletion of about one to four base pairs, is based on the use of an immobilized DNA mismatch-binding protein, such as MutS, which binds to a nucleic acid hybrid having a single base mismatch or unpaired base or bases, thereby allowing the detection of mutations involving as little as one base change in a nucleotide sequence. Such a method is useful for diagnosing a variety of important disease states or susceptibilities, including the presence of a mutated oncogene and the presence of DNA containing triplet repeat sequences which characterize several genetic diseases including fragile X syndrome. The present method is used to isolate or remove by affinity chromatography duplex DNA molecules containing mismatches such as error-containing molecules in PCR-amplified DNA samples. Also provided are compositions and kits useful for practicing the methods of the present invention.

Claims

1. A method for detecting heterozygosity or the presence of a mutation or polymorphism in heterozygous form in a test DNA sample, which heterozygosity, mutation or polymorphism is the result of a deletion or addition of up to four nucleotides, which method comprises:

(a) detectably labeling said test DNA any time prior to step (c) below;

(b) denaturing double stranded DNA in said sample into single strands and allowing said single strands to reanneal into duplexes;

(c) contacting said duplexes with immobilized E. coli MutS protein, a homologue thereof or a functional derivative of said MutS or of said homologue, under conditions in which heteroduplexes containing one to four unpaired bases bind to said immobilized protein, homologue or derivative; and

(d) detecting any bound heteroduplexes of step (c),

wherein the presence of bound heteroduplex DNA is indicative of the presence of said heterozygosity, mutation or polymorphism in said test DNA.

2. A method according to claim 1, wherein said test DNA in said sample is amplified prior to step (a).

3. A method according to claim 1 wherein said immobilized protein, homologue or derivative is immobilized to a solid support selected from the group consisting of natural cellulose, nitrocellulose or other modified cellulose, polystyrene, polypropylene, polyethylene, dextran, nylon, polyacrylamide, polyvinylidene difluoride, agarose and magnetic beads.

4. A method according to claim 1 wherein said detectably labeled DNA has a detectable label which is a chromogenic compound, a chemiluminescent compound, a bioluminescent compound, a fluorescent compound, a radiolabel or biotin.

5. A method for detecting any specific DNA sequence in a test DNA sample, which method comprises:

(a) detectably labeling anytime prior to step (c) below,

(i) test DNA in said sample, or

(ii) an added DNA polynucleotide or oligonucleotide as recited in step (b), below, or

(iii) both (i) and (ii);

(b) denaturing double stranded DNA in said sample into single strands and allowing said single strands to anneal into duplexes in the presence of an added DNA polynucleotide or oligonucleotide having a sequence such that said added DNA forms a perfectly matched duplex with said specific sequence;

(c) contacting said duplexes with immobilized E. coli MutS protein, a homologue thereof or a functional derivative of said MutS or of said homologue, under conditions in which heteroduplexes containing one to four unpaired bases bind to said immobilized protein, homologue or derivative; and

(d) detecting bound heteroduplexes of step (c),

wherein the absence of bound heteroduplexes is indicative of the presence of said specific sequence in said test DNA.

6. A method according to claim 5, wherein said sample DNA is amplified prior to step (b).

7. A method for detecting heterozygosity or the presence of a mutation or polymorphism in heterozygous form in a test DNA sample, which heterozygosity, mutation or polymorphism is the result of a deletion or addition of up to four nucleotides, which method comprises:

(a) denaturing double stranded DNA in said sample into single strands and allowing said single strands to reanneal into duplexes;

(b) contacting the denatured and reannealed duplexes of step (a) with E. coli MutS protein, a homologue thereof or a functional derivative of said MutS or of said homologue, immobilized to a sold support, either

(i) in the presence of a detectably labeled double stranded DNA oligonucleotide containing one to four unpaired bases, wherein said oligonucleotide is capable of binding to said protein, homologue or derivative, or

(ii) wherein said immobilized protein, homologue or derivative was previously allowed to bind to a detectably labeled double stranded DNA oligonucleotide containing one to four unpaired bases,

under conditions in which heteroduplexes containing one to four unpaired bases bind to said immobilized protein, homologue or derivative; and

(c) detecting the amount of bound detectably labeled oligonucleotide,

wherein a decrease in the amount of bound detectably labeled oligonucleotide is indicative of the presence of said heterozygosity, mutation, or polymorphism.

9. A method according to claim 7, wherein said sample DNA is amplified prior to step (a).

8. A method for detecting a homozygous or heterozygous mutation or a polymorphism in test DNA in a sample, which mutation or polymorphism is the result of a deletion or addition of up to four nucleotides, which method comprises:

(a) denaturing double stranded DNA in said sample into single strands, and allowing said single strands to reanneal into duplexes in the presence of an added DNA polynucleotide or oligonucleotide having the wild type sequence for said mutation or lacking said polymorphism;

(b) contacting the denatured and reannealed duplexes formed in step (a) with E. coli MutS protein, a homologue thereof or a functional derivative of said MutS or of said homologue, immobilized to a solid support, either

(i) in the presence of a detectably labeled double stranded DNA oligonucleotide containing one to four unpaired bases, wherein said oligonucleotide is capable of binding to said protein, homologue or derivative, or

(ii) wherein said immobilized protein, homologue or derivative was previously allowed to bind to a detectably labeled double stranded DNA oligonucleotide containing one to four unpaired bases prior to said contacting,

under conditions in which heteroduplexes containing one to four unpaired bases bind to said immobilized protein, homologue or derivative; and

(c) detecting the amount of bound detectably labeled oligonucleotide,

wherein a decrease in the amount of bound detectably labeled oligonucleotide is indicative of the presence of said homozygous or heterozygous mutation or polymorphism in said test DNA of said sample.

10. A method according to claim 8, wherein said sample DNA is amplified prior to step (a).

11. A method according to claim 10, wherein said added DNA polynucleotide or oligonucleotide of step (a) is added prior to said amplifying step.

12. A method for detecting in a DNA sample specific sequences characterized in being rare or minority sequences, wherein said rare and minority sequences differ from the majority sequences of said DNA sample by one or more deletions or additions of one to four nucleotides, which method comprises:

(a) subjecting the DNA sample to conditions of denaturation followed by annealing, such that said rare or minority sequences pair with the majority sequences to form heteroduplexes containing one to four unpaired bases, thereby generating in said sample a mixture of perfectly matched duplexes and heteroduplexes;

(b) contacting the mixture of step (a) with immobilized E. coli MutS protein, a homologue thereof or a functional derivative of said MutS or of said homologue, under conditions in which said heteroduplexes formed in step (a) bind to said immobilized protein;

(c) separating any unbound DNA from said bound heteroduplexes; and

(d) detecting bound heteroduplexes,

wherein the presence of bound heteroduplexes is indicative of the presence of said specific sequences in said sample DNA.

13. A method for screening a test DNA sample for the presence of a mutation or polymorphism in a specific known region, wherein said mutation or polymorphism:

(i) results from a deletion or addition of up to four nucleotides, and (ii) results in formation of a heteroduplex when a mutant or polymorphic DNA strand is paired with a DNA strand of known sequence, which heteroduplex is bound by E. coli MutS protein, a homologue thereof or a functional derivative of said MutS or of said homologue, said method comprising;

(a) detectably labeling said test DNA any time prior to step (c) below;

(b) denaturing double stranded DNA in said sample into single strands and allowing said single strands to reanneal into duplexes;

(c) contacting said DNA duplexes with immobilized E. coli MutS protein, a homologue thereof or a functional derivative of said MutS or of said homologue, under conditions in which heteroduplexes containing one to four unpaired bases bind to said protein, homologue or derivative; and

(d) detecting any bound heteroduplexes of step (c),

wherein the presence of bound heteroduplex DNA is indicative of the presence of said mutation or polymorphism in said specific known region of said test DNA.

14. A method for screening a test DNA sample for the presence of a homozygous or heterozygous mutation or polymorphism in a specific region, wherein said mutation or polymorphism:

(i) results from a deletion or addition of up to four nucleotides, and (ii) results in formation of a heteroduplex when a mutant or polymorphic DNA strand is paired with a DNA strand of known sequence, which heteroduplex is bound by E. coli MutS protein, a homologue thereof or a functional derivative of said MutS or of said homologue, said method comprising;

(a) denaturing double stranded DNA in said sample into single strands and allowing said single strands to reanneal into duplexes in the presence of a detectably labeled DNA polynucleotide or oligonucleotide having the known sequence, resulting in the formation of detectably labeled DNA duplexes;

(b) contacting said detectably labeled DNA duplexes formed in step (a) with immobilized E. coli MutS protein, a homologue thereof or a functional derivative of said MutS or of said homologue, under conditions in which heteroduplexes containing one to four unpaired bases bind to said protein, homologue or derivative; and

(c) detecting any bound heteroduplexes of step (c), wherein the presence of bound heteroduplex DNA is indicative of the presence of said mutation or polymorphism in said specific region of said test DNA.

15. A method for detecting in a test DNA sample a heterozygous sequence variant in a specific region having a known sequence, wherein said sequence variant differs from the known sequence by one or more deletions or additions of one to four nucleotides, which method comprises:

(a) digesting said test DNA with one or more restriction enzymes which result in the creation of a restriction fragment containing the site of said sequence variant;

(b) denaturing said restriction fragment into single strands and allowing said strands to anneal into duplexes;

(c) contacting said duplexes with immobilized E. coli MutS protein, a homologue thereof or a functional derivative of said MutS or of said homologue, under conditions in which heteroduplexes among said duplexes containing one to four unpaired bases bind to said protein, homologue or derivative; and

(d) detecting said bound heteroduplexes,

wherein the presence of said bound heteroduplexes is indicative of the presence of said heterozygous sequence variant in said test DNA.

18. A method according to claim 15, wherein said bound heteroduplexes are amplified following binding.

16. A method for detecting a homozygous or heterozygous sequence variant in a specific region of a test DNA, wherein said sequence variant differs from a known DNA sequence by one or more deletions or additions of one to four nucleotides, which method comprises:

(a) digesting said test DNA with one or more restriction enzymes which result in the creation of a restriction fragment containing the site of said sequence variant;

(b) denaturing said restriction fragment into single strands and allowing said single strands to anneal into duplexes in the presence of an added DNA polynucleotide or oligonucleotide having the known sequence;

(c) contacting said duplexes with immobilized E. coli MutS protein, a homologue thereof or a functional derivative of said MutS or of said homologue, under condition in which heteroduplexes among said duplexes containing one to four unpaired bases bind to said protein, homologue or derivative; and

(d) detecting said bound heteroduplexes,

wherein the presence of said bound heteroduplexes is indicative of the presence of said homozygous or heterozygous sequence variant in said test DNA.

19. A method according to claim 16, wherein said bound heteroduplexes are amplified following binding.

17. A method for detecting in a test DNA sample the presence of a heteroduplex having a deletion or addition of up to four nucleotides, which method comprises detecting the binding of DNA in said sample to E. coli MutS protein, a homologue thereof or a functional derivative of said MutS or of said homologue, which has been immobilized to a solid support, wherein the presence of DNA bound to the immobilized protein, homologue or derivative is indicative of the presence of said heteroduplex.

20. A method for detecting in a sample containing test DNA the presence of a specific known sequence in a selected region of a first allele of a multi-allelic system, or the presence of an unknown sequence, wherein said specific known sequence is characterized as differing from other sequences of known identity in said selected region of other known alleles of said multi-allelic system by one or more deletions or additions of up to four nucleotides, which method comprises:

(a) mixing a detectably labeled DNA probe with an excess of the test DNA under conditions of denaturation followed by annealing such that copies of the probe can form homoduplexes or heteroduplexes, which probe

(i) is perfectly complementary to said specific known sequence of said first allele and forms detectably labeled homoduplexes when annealed with said specific known sequence, and

(ii) forms detectably labeled heteroduplexes containing one to four unpaired bases when annealed with said other sequences of known identity in said selected region of other known alleles of said multi-allelic system;

(b) contacting the mixture formed in step (a) with an excess of immobilized E. coli MutS protein, or a homologue or functional derivative thereof, under conditions in which any detectably labeled heteroduplexes containing one to four unpaired bases formed in step (a) bind to said immobilized MutS protein, homologue or functional derivative;

(c) removing said immobilized protein, homologue or derivative along with any heteroduplexes containing one to four unpaired bases bound thereto from said test DNA, thereby leaving a remaining mixture; and

(d) detecting the presence of said detectably labeled DNA probe in said remaining mixture of step (c),

wherein the presence of said detectably labeled DNA probe indicates the presence in said sample containing test DNA of said specific known sequence or said unknown sequence.