Methods and compositions for identifying nucleic acid molecules using nucleolytic activities and hybridization

• US 6,900,013 B1
• Filed: 08/25/2000
• Issued: 05/31/2005
• Est. Priority Date: 08/25/2000
• Status: Expired due to Fees

• Current Assignee: AVIVA Biosciences Corporation
• Original Assignee: AVIVA Biosciences Corporation
• Inventors: Wang, Guoqing, Yang, WeiPing, Wang, Xiaobo, Wu, Lei, Cheng, Jing
• Primary Examiner(s): Whisenant, Ethan
• Assistant Examiner(s): LU, FRANK WEI MIN
• Application Number:
  US09/648,081
• Time in Patent Office: 1,740 Days
• Field of Search: 435/6, 435/91.1, 435/91.51, 435/7.5, 435/183, 435/287.2, 436/94, 536/23.1, 536/24.3, 536/24.33, 536/25.3, 536/25.32, 536/25.4
• US Class Current: 435/6.14
• CPC Class Codes: C07H 21/04 with deoxyribosyl as saccharide radical
• CPC Class Codes: C12Q 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• 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 2521/101 DNA polymerase
• CPC Class Codes: C12Q 2521/327 RNAse, e.g. RNAseH
• CPC Class Codes: C12Q 2533/101 Primer extension
• 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
• CPC Class Codes: C12Q 2563/107 fluorescence
• CPC Class Codes: C12Q 2565/501 being an array of oligonucleotides

Abstract

The present invention recognizes that identifying genes expressed during developmental processes, stress responses, and disease states can advance understanding of these biological functions, and can contribute to identifying targets for therapeutic drugs. In addition, the present invention recognizes that rapid and reliable profiling of genetic variations, such as mutations and SNPs, is of increasing importance to diagnostics, prognostics, forensics, heredity determinations, and pharmacogenetics. One aspect of the present invention provides a method of identifying one or more nucleic acid molecules that are expressed under a given set of conditions based on their complementarity to known sequences, or one or more mutations or SNPs in a population of nucleic acid molecules. The method includes: contacting at least one probe nucleic acid molecule with a survey population of nucleic acid molecules under conditions that promote nucleic acid hybridization to generate a probe-survey population mixture of nucleic acid molecules, treating the probe-survey population mixture of nucleic acid molecules with a nucleolytic activity, such that nucleolytic activity-sensitive nucleic acid molecules are digested, and contacting the resulting mixture of nucleolytic activity-protected nucleic acid molecules with a solid support comprising one or more attached nucleic acid molecules to generate attached nucleic acid molecule/nucleolytic activity-protected nucleic acid molecule complexes, and identifying one or more of the attached nucleic acid molecules or one or more of the nucleolytic activity-protected nucleic acid molecules in one or more attached nucleic acid molecule/nucleolytic activity-protected nucleic acid molecule complexes.

Claims

1. A method of detecting a mutation or single nucleotide polymorphism (SNP), comprising:

a) contacting one or more probes that comprise DNA with a survey population of RNA molecules under conditions that promote hybridization between complementary nucleic acid molecules to generate a probe-survey population mixture of nucleic acid molecules that comprises at least one of said one or more probes hybridized to one or more survey population RNA molecules, wherein said one or more probes are at least partially complementary to one or more RNA molecules known to be or suspected of being present in the survey population, further wherein terminal nucleotide of said one or more probes is a SNP or mutation;

b) treating said probe-survey population mixture of nucleic acid molecules with at least one nuclease that digests single-stranded DNA molecules, such that non-base-paired deoxynucleotides are digested, to generate a population of nucleic acid molecules comprising one or more nuclease-protected probes;

c) contacting said population of nucleic acid molecules comprising one or more nuclease-protected probes with a solid support that comprises one or more attached nucleic acid molecules under conditions that promote hybridization between complementary nucleic acid molecules, wherein said one or more attached nucleic acid molecules are at least partially complementary to said one or more probes, to generate one or more attached nucleic acid molecule/nuclease-protected probe complexes; and

d) detecting at least one of said one or more attached nucleic acid molecule/probe complexes to detect said SNP or mutation.

2. The method of claim 1, wherein the terminal nucleotide is labeled.

3. The method of claim 2, wherein said label is a radioisotope, a fluorochrome, an enzyme, or a specific binding member.

4. The method of claim 3, wherein said label is a fluorochrome.

5. The method of claim 1, wherein said one or more probes are from 10 to 250 bases in length.

6. The method of claim 5, wherein said one or more probes have one or more nuclease-resistant linkages.

7. The method of claim 1, wherein said probes are two or more probe.

8. The method of claim 1, wherein said survey population of RNA molecules is isolated from at least one cell, at least one tissue, at least one biological sample, at least one organism, or at least one environmental sample.

9. The method of claim 8, wherein said survey population of RNA molecules is isolated from at least one biological sample or at least one environmental sample.

10. The method of claim 9, wherein said survey population of RNA molecules is isolated from at least one biological sample.

11. The method of claim 1, wherein said nuclease that digests single-stranded DNA is S1 nuclease, Mung Bean nuclease, or Exonuclease III.

12. The method of claim 1, wherein solid support comprises glass, silicon, nylon, one or more polymers, one more plastics;

one or more ceramics, or one or more metals.

13. The method of claim 12, wherein said solid support is an array.

14. The method of claim 12, wherein said solid support is a paramagnetic particle.

15. The method of claim 1, wherein said one or more attached nucleic acid molecules are between 10 and 250 nucleotides in length.

16. The method of claim 15, wherein said one or more attached nucleic acid molecules comprised DNA or peptide nucleic acids.

17. The method of claim 13, wherein said attached nucleic acid molecules are two or more attached nucleic acid molecules.

18. A method of detecting at least one RNA molecule in a survey population of RNA molecules, comprising:

a) contacting one or more probes that comprise DNA with a survey population of RNA molecules under conditions that promote hybridization between complementary nucleic acid molecules to generate a probe-survey population mixture of nucleic acid molecules that comprises one or more probes hybridized to one or more survey population RNA molecules;

b) treating said probe-survey population mixture of nucleic acid molecules with a nuclease that digests single-stranded DNA molecules, to generate a population of nuclease-resistant nucleic acid molecules comprising one or more nuclease-protected probes hybridized to one or more survey population RNA molecules;

c) contacting said population of nuclease-resistant nucleic acid molecules with a solid support comprising one or more attached nucleic acid molecules, wherein said one or more attached nucleic acid molecules are at least partially complementary to said one or more probes, under conditions that promote hybridization between complementary nucleic acid molecules, to generate one or more attached nucleic acid molecule/nuclease-protected probe complexes;

wherein said one or more attached nucleic acid molecule/nuclease-protected probe complexes comprise single-stranded overhangs having a uniform number of bases;

d) labeling said one or more attached nucleic acid molecule/nuclease-protected probe complexes using at least one polymerase and at least one labeled nucleotide; and

e) detecting label incorporated into at least one of said one or more attached nucleic acid molecule/nuclease-protected probe complexes, thereby detecting one or more RNA molecules of a survey population of RNA molecules.

19. The method of claim 18, wherein said one or more probes are from 10 to 250 bases in length.

20. The method of claim 18, wherein said probes are more than one probe.

21. The method of claim 18, wherein said survey population of RNA molecules is isolated from at least one cell, at least one tissue, at least one biological sample, at least one organism, or at least one environmental sample.

22. The method of claim 21, wherein said survey population of RNA molecules is isolated from at least one biological sample.

23. The method of claim 18, wherein said nuclease that digests single-stranded DNA molecules is S1 nuclease, Mung Bean nuclease, or Exonuclease III.

24. The method of claim 18, wherein said solid support comprises glass, silicon, nylon, one or more polymers, one or more plastics, one or more ceramics, or one or more metals.

25. The method of claim 24, wherein said solid support is an array.

26. The method of claim 18, wherein said one or more attached nucleic acid molecules are from 10 to 250 nucleotides in length.

27. The method of claim 18, wherein said one or more attached nucleic acid molecules comprise DNA.

28. The method of claim 25, wherein said attached nucleic acid molecules are two or more attached nucleic acid molecules.

29. The method of claim 18, in which said at least one polymerase is one selected from the group consisting of T4 DNA polymerase, T. aquaticus polymerase, Klenow fragment, T7 RNA polymerase, DNA polymerase 1, and SP6 RNA polymerase.

30. The method of claim 18, wherein said at least one labeled nucleotide comprises a radiosotope, a fluorochrome, an enzyme, or a specific binding member.

31. The method of claim 30, wherein said at least one labeled nucleotide comprises a fluorochrome.

32. The method of claim 18, wherein said at least one labeled nucleotide is four labeled nucleotides.