Methods for rapid screening of polymorphisms, mutations and methylation

• US 20030022215A1
• Filed: 06/25/2002
• Published: 01/30/2003
• Est. Priority Date: 04/23/2001
• Status: Active Grant

• Current Assignee: Dana-Farber Cancer Institute
• Original Assignee: Dana-Farber Cancer Institute
• Inventors: Makrigiorgos, Gerrasimos M.
• Granted Patent: US 7,247,428 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 1/6883 for diseases caused by alterations of genetic material
• CPC Class Codes: C12Q 1/6886 for cancer immunoassay for cancer G01N33/574
• CPC Class Codes: C12Q 2521/301 Endonuclease
• CPC Class Codes: C12Q 2521/331 Methylation site specific nuclease
• CPC Class Codes: C12Q 2521/525 Phosphatase
• CPC Class Codes: C12Q 2525/307 Circular oligonucleotides
• CPC Class Codes: C12Q 2533/107 Probe or oligonucleotide ligation
• CPC Class Codes: C12Q 2537/143 Multiplexing, i.e. use of multiple primers or probes in a single reaction, usually for simultaneously analyse of multiple analysis

Abstract

The present method is directed to methods of detecting mismatches, polymorphisms, and methylation in multiple genes or the same gene in multiple individuals.

Claims

1. A method of identifying alteration(s), including polymorphisms, mutations, and methylation, in a target nucleic acid, which comprises:

(a) preparing the target nucleic acid and a control nucleic acid;

(b) hybridizing the target nucleic acid with a control nucleic acid to create a heteroduplex, wherein the control nucleic acid is the wild type nucleic acid corresponding to the target nucleic acid;

(c) removing any 5′

phosphate groups or 3′

hydroxyl groups from the DNA ends;

(d) cleaving the nucleic acid at the site of any mismatch, alteration or methylation in said heteroduplex using any agent that generates a single or a double strand break at the sites of mismatch or methylation;

(e) ligating a linker at the 5′

phosphate group at the newly generated 5′

phosphate group(s) at the site of cleavage if 5′

phosphate groups have been removed, or ligating a linker at the newly generated 3′

hydroxyl group(s) at the site of cleavage if 3′

hydroxyl groups have been removed; and

(f) selectively amplifying the nucleic acid fragments which have a linker ligated thereto using polymerase chain reaction.

3. The method of claim 1, wherein the nucleic acid is selected from the group consisting of cDNA, genomic DNA, PCR-amplification product, and mRNA.

4. The method of claim 1, wherein the PCR-amplification product is amplified using a high fidelity polymerase and the primers used to generate the product are not phosphorylated at the 5′

end.

5. The method of claim 1, wherein the target and control nucleic acid are digested with any restriction enzyme that does not generate blunt ends.

6. The method of claim 1, wherein the preparation of the nucleic acid further comprises ligating a double stranded linker onto each nucleic acid, wherein said double stranded linkers are synthetic complementary linkers which do not contain 5′

phosphate groups.

7. The method of claim 6, wherein the preparation of the nucleic acid further comprises PCR amplification of the nucleic acid.

8. The method of claim 1, wherein the target and control nucleic acids are hybridized in the presence of hydroxylamine.

9. The method of claim 1, wherein calf intestinal phosphatase is used to remove any 5′

phosphate groups.

10. The method of claim 1, wherein after the target and control nucleic acids are hybridized to form a heteroduplex, DNA ends are removed by circularizing DNA, and any non-circularized DNA is selectively degraded.

11. The method of claim 1, wherein the agent that generates strand breaks at sites of mismatch is selected from the group consisting of chemical agents, enzymes, and a combination of a chemical agent and an enzyme.

12. The method of claim 11, wherein the chemical agent is selected from the group consisting of hydroxylamine, osmium tetroxide, potassium permanganate, tetraethyl ammonium acetate, hydrazine, and carbodiimide, and wherein the site of cleavage is further treated with piperidine to generate a 5′

P at the strand break.

13. The method of claim 11, wherein the enzyme is selected from the group consisting of glycosylase, resolvases, cleavases, ribonucleases, and nucleases.

14. The method of claim 11, wherein the enzyme selected is a restriction endonuclease that identifies a restriction site not present in the wild-type allele.

15. The method of claim 11, wherein the restriction site is an additional restriction site of a restriction site present in the wild-type allele.

24. A kit for determining an alteration in a nucleic acid, comprising the reagents used in the method of claim 1.

25. A method of using a mutation scanning array, wherein said mutation scanning array comprises a plurality of elements, wherein the elements contain immobilized oligonucleotides 8-50 bases long that collectively span at least 10 different whole genes, to identify mutations in a target nucleic acid sequence which comprises:

(a) identifying alterations, including polymorphisms, mutations, and methylation, in a target nucleic acid using the method of claims 1, 2, 14 or 19;

(b) selectively amplifying the nucleic acid fragments which have a linker ligated thereto using polymerase chain reaction;

(c) hybridizing the selectively amplified PCR product(s) to the mutation scanning array; and

(d) identifying in which gene and gene segment the selected mismatch belongs to.

26. The method of claim 25, wherein the whole gene is represented by array elements;

each element containing immobilized oligonucleotides that sample in regular or non-regular intervals the whole 3′

to 5′

mRNA sequence of each represented gene.

27. The method of claim 25, wherein each of the whole genes is represented by the coding genomic portion of the gene.

28. The method of claim 25, wherein each of the whole genes is represented by both the coding and non-coding genomic portions of a gene.

29. The method of claim 25, wherein at least 10 different genes are selected from the genome, collectively known to predispose an individual to a particular disease.

30. The method of claim 29, where the disease is a particular kind of cancer.

31. The method of claim 29, where the disease is a cardiovascular abnormality, or a neurodegenerative disorder, or diabetes.

32. The method of claim 25, where the genes selected are all known tumor suppressor genes or oncogenes.

33. The method of claim 25, where the genes selected are genes known to be overexpressed in a malignant cell, wherein overexpression is determined by comparison to the gene'"'"'s expression in a corresponding non-malignant cell.

34. The method of claim 25, wherein the array is a chip or a microsphere.

35. The method of claim 25, further comprising determining whether the mismatch is a mutation or polymorphism.

2. A method of identifying alteration(s), including polymorphisms, mutations, and methylation, in a target nucleic acid, which comprises:

(a) preparing the target nucleic acid and a control nucleic acid;

(b) hybridizing the target nucleic acid with a control nucleic acid to create a heteroduplex, wherein the control nucleic acid is the wild type nucleic acid corresponding to the target nucleic acid;

(c) removing any 5′

phosphate groups or 3′

hydroxyl groups from the DNA ends;

(d) cleaving the nucleic acid at the site of any mismatch or methylation in said heteroduplex using any agent that generates a single strand breaks at the sites of mismatch or methylation;

(e) denaturing the heteroduplex into two separate nucleic acid stands and synthesizing a second strand for each nucleic acid strand;

(f) ligating a linker at the 5′

phosphate group at the newly generated 5′

phosphate group(s) at the site of cleavage if 5′

phosphate groups have been removed, or ligating a linker at the newly generated 3′

hydroxyl group(s) at the site of cleavage if 3′

hydroxyl groups have been removed; and

(g) selectively amplifying the nucleic acid fragments which have a linker ligated thereto using polymerase chain reaction.

16. A method of identifying alteration(s), including polymorphisms and mutations, in a target nucleic acid, which comprises:

(a) preparing a target nucleic acid that in a wild-type allele lacks a specific recognition sequence for a restriction endonuclease enzyme;

(b) removing any 5′

phosphate groups or 3′

hydroxyl from the DNA ends;

(c) cleaving the nucleic acid to create a double strand break at the site of any mutation which alters the sequence so that it generates a restriction site for the restriction-enzyme;

(d) ligating a linker at the newly generated 5′

phosphate group(s) at the site(s) of cleavage if 5′

phosphate groups have been removed, or ligating a linker at the newly generated 3′

hydroxyl groups at the site of cleavage, if 3′

hydroxyl groups have been removed;

(e) selectively amplifying the nucleic acid fragments which have a linker ligated thereto using polymerase chain reaction; and

(f) detecting the nucleic acid fragments amplified in step (e) to identify alterations.

17. The method of claim 16, wherein the detection of the nucleic acid fragments is performed via nested-PCR using primers internal to the nucleic acid fragments.

18. The method of claim 17, wherein DNA ends are removed by circularizing DNA, and any non-circularized DNA is selectively degraded.

19. The method of claim 16, wherein the restriction endonuclease is a four-base cutter enzyme.

20. The method of claim 19, wherein the restriction endonuclease is selected from the group consisting of N1AIII, SAU3A1, TaqI, MaeII, MaeI and MseI.

21. The method of claim 16, wherein the target nucleic acid is selected from the group consisting of cDNA, genomic DNA, PCR-amplification product and mRNA.

22. A method of identifying alteration(s), including polymorphisms, mutations, and methylation, in a target nucleic acid, which comprises:

(a) preparing the target nucleic acid and a control nucleic acid;

(b) hybridizing the target nucleic acid with a control nucleic acid to create a heteroduplex, wherein the control nucleic acid is the wild type nucleic acid corresponding to the target nucleic acid;

(c) removing any 5′

phosphate groups or 3′

hydroxyl groups from the DNA ends;

(d) cleaving the nucleic acid at the site of any mismatch or methylation in said heteroduplex using any combination of agents that generate blunt-ended double strand breaks at the sites of mismatch or methylation;

(e) ligating a linker at the 5′

phosphate group at the newly generated 5′

phosphate group(s) at the site of cleavage if 5′

phosphate groups have been removed, or ligating a linker at the newly generated 3′

hydroxyl group(s) at the site of cleavage if 3′

hydroxyl groups have been removed; and

(f) selectively amplifying the nucleic acid fragments which have a linker ligated thereto using polymerase chain reaction.

23. The method of claim 22, wherein following cleavage of the nucleic acid at sites of mismatch, S1 nuclease is used to convert any single strand breaks to blunt-ended double strand breaks.