Methods, kits and compositions for the identification of nucleic acids electrostatically bound to matrices

• US 6,441,152 B1
• Filed: 12/08/1999
• Issued: 08/27/2002
• Est. Priority Date: 12/08/1998
• Status: Expired due to Term

• Current Assignee: Applied Biosystems LLC (Thermo Fisher Scientific Incorporated)
• Original Assignee: Boston Probes, Inc. (Thermo Fisher Scientific Incorporated)
• Inventors: Fiandaca, Mark J., Johansen, Jack T., Coull, James M., Hyldig-Nielsen, Jens J.
• Primary Examiner(s): Whisenant, Ethan C.
• Assistant Examiner(s): LU, FRANK WEI MIN
• Application Number:
  US09/456,773
• Time in Patent Office: 993 Days
• Field of Search: 435/6, 435/91.1, 435/91.2, 436/94, 536/23.1, 536/24.3, 536/24.33, 536/25.3, 536/25.32
• US Class Current: 536/23.1
• CPC Class Codes: C12Q 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6834 Enzymatic or biochemical coupling of nucleic acids to a solid phase
• CPC Class Codes: C12Q 2525/107 incorporating a peptide nucleic acid
• CPC Class Codes: C12Q 2527/107 Temperature of melting, i.e. Tm
• CPC Class Codes: C12Q 2545/114 involving a quantitation step
• CPC Class Codes: C12Q 2563/131 the label being a member of a cognate binding pair, i.e. extends to antibodies, haptens, avidin
• CPC Class Codes: C12Q 2565/1015 labels being on the same oligonucleotide
• CPC Class Codes: C12Q 2565/627 being a mass spectrometer

Abstract

This invention pertains to methods, kits and compositions suitable for the detection, identification and/or quantitation of nucleic acids which are electrostatically immobilized to matrices using non-nucleotide probes which sequence specifically hybridize to one or more target sequences of the nucleic acid but do not otherwise substantially interact with the matrix. Once the nucleic acid is immobilized, the detectable non-nucleotide probe/target sequence complex, formed before or after the immobilization of the nucleic acid, can be detected, identified or quantitated under a wide range of assay conditions as a means to detect, identify or quantitate the target sequence in the sample. Because it is reversibly bound, the non-nucleotide probe/target sequence can optionally be removed from the matrix for detecting, identifying or quantitating the target sequence in the sample. Because the non-nucleotide probe/target sequence is protected against degradation, it is another advantage of this invention that the sample can be treated with enzymes which degrade sample components, either before or after the nucleic acid is bound to the matrix, in order to “clean up” the sample (e.g. a complex biological sample such as a cell lysate) and thereby improve the detection, identification or quantitation of the target sequence in the sample. The methods, kits and compositions of this invention are therefore particularly well suited for the analysis, and particularly single point mutation analysis, in a particle assay, in an array assay, in a nuclease digestion/protection assay and/or in a line assay format. When utilized in combination with non-nucleotide “Beacon” probes, the invention is particularly well suited for use in a self-indicating assay format.

Claims

1. A composition comprising:

a) a matrix;

b) at least one nucleic acid molecule, comprising at least one target sequence, that is electrostatically bound to said matrix under suitable electrostatic binding conditions; and

c) at least one non-nucleotide probe comprising a probing nucleobase sequence that is sequence specifically hybridized to at least a portion of the one or more target sequences to thereby form a non-nucleotide probe/target sequence complex and wherein the backbone of the non-nucleotide probe or probes is sufficiently neutral or positively charged, under electrostatic binding conditions that it exhibits little or no affinity for the matrix.

2. The composition of claim 1, wherein said matrix is selected from the group consisting of:

a) a solution insoluble polymer;

b) a surface;

c) a beaded support;

d) a porous beaded support;

e) a cast polymer;

f) a co-polymeric material; and

g) a gel;

wherein said matrix comprises charged functional groups that are suitable for binding nucleic acid under electrostatic binding conditions.

3. The composition of claim 2, wherein said matrix is beaded anion exchange media.

4. The composition of claim 1, wherein the non-nucleotide probe or probes are unlabeled.

5. The composition of claim 4, wherein to the non-nucleotide probe/target sequence complex is directly or indirectly linked a detectable antibody.

6. The composition of claim 5, wherein the non-nucleotide probe or probes are peptide nucleic acid.

7. The composition of claim 1, wherein the composition is formed at one or more positions on an array.

8. The composition of claim 1, wherein the one or more nucleic acid molecules have been degraded except for that portion of the molecule or molecules that are protected from degradation by hybridization to the non-nucleotide probe.

9. The composition of claim 1, wherein the matrix is formulated into a line or shape on a support.

10. The composition of claim 9, wherein composition is formed in a lateral flow assay device.

11. The composition of claim 1, wherein said target sequence or sequences are characteristic for the detection of an organism, virus, fungi or pathogen sought to be detected in an assay.

12. The composition of claim 1, wherein said target sequence or sequences are characteristic for the detection of a genetically-based disease or is characteristic for the detection of a predisposition to a genetically-based disease.

13. The composition of claim 1, wherein electrostatic binding occurs by salt pair formation between charged groups of the nucleic acid backbone and charged groups of the matrix.

14. The composition of claim 1, wherein the non-nucleotide probe or probes comprises a completely neutral backbone under electrostatic binding conditions.

15. The composition of claim 14, wherein the non-nucleotide probe or probes are peptide nucleic acids.

16. The composition of claim 1, wherein the non-nucleotide probe or probes are labeled with at least one detectable moiety.

17. The composition of claim 16, wherein the labeled non-nucleotide probe or probes are non-nucleotide “

Beacon”

probes.

18. The composition of claim 16, wherein one or more detectable moieties are selected from the group consisting a chromophore, a fluorochrome, a spin label, a radioisotope, an enzyme, a hapten and a chemiluminescent compound.

19. The composition of claim 18, wherein the enzyme is selected from the group consisting of alkaline phosphatase, soybean peroxidase and horseradish peroxidase.

20. The composition of claim 18, wherein the hapten is selected from the group consisting of fluorescein, biotin, 2,4-dinitrophenyl and digoxigenin.

21. The composition of claim 1, wherein said probing nucleobase sequence is substantially complementary to the target sequence.

22. The composition of claim 1, wherein said probing nucleobase sequence is exactly complementary to the target sequence.

23. A composition comprising:

a) a matrix;

b) at least one nucleic acid molecule, comprising at least one target sequence, that is electrostatically bound to said matrix under suitable electrostatic binding conditions; and

c) at least one self-indicating non-nucleotide probe comprising a probing nucleobase sequence that is sequence specifically hybridized to at least a portion of the one or more target sequences to thereby form a non-nucleotide probe/target sequence complex and wherein the backbone of the self-indicating non-nucleotide probe or probes is sufficiently neutral or positively charged, under electrostatic binding conditions, that it exhibits little or no affinity for the matrix.

24. A composition comprising:

a) a matrix array of at least two nucleic acid molecules, at least one of which comprises a target sequence, that are electrostatically bound at unique locations to said matrix under suitable electrostatic binding conditions; and

b) at least one non-nucleotide probe comprising a probing nucleobase sequence that is sequence specifically hybridized to at least a portion of any one or more target sequences electrostatically immobilized to the matrix to thereby form non-nucleotide probe/target sequence complexes at said unique locations and wherein the backbone of the non-nucleotide probe or probes is sufficiently neutral or positively charged, under electrostatic binding conditions, that it exhibits little or no affinity for the matrix.

25. The composition of claim 24, wherein the non-nucleotide probe is a non-nucleotide “

Beacon”

probe.

26. The composition of claim 24, wherein the non-nucleotide probe or probes are unlabeled.

27. The composition of claim 26, wherein to the non-nucleotide probe/target sequence complex is directly or indirectly linked a detectable antibody.

28. The composition of claim 27, wherein the non-nucleotide probe or probes are peptide nucleic acid.

29. The composition of claim 24, wherein the non-nucleotide probe or probes are labeled with at least one detectable moiety.

30. The composition of claim 29, wherein one or more detectable moieties are selected from the group consisting a chromophore, a fluorochrome, a spin label, a radioisotope, an enzyme, a hapten and a chemiluminescent compound.

31. The composition of claim 30, wherein the enzyme is selected from the group consisting of alkaline phosphatase, soybean peroxidase and horseradish peroxidase.

32. The composition of claim 30, wherein the hapten is selected from the group consisting of fluorescein, biotin, 2,4-dinitrophenyl and digoxigenin.

33. A kit for the analysis of a sample containing a nucleic acid molecule comprising a target sequence, said kit comprising a matrix “

at least one nucleic acid molecule having at least one target sequence that is electrostatically bound to said matrix under suitable electrostatic binding conditions,”

between “

a matrix” and

“

and at least one non-nucleotide probe” and

at least one non-nucleotide probe having a probing nucleobase sequence that sequence specifically hybridizes, under suitable hybridization conditions, to at least a portion of the target sequence sought to be detected in said sample and wherein the backbone of the non-nucleotide probe or probes is sufficiently neutral or positively charged, under electrostatic binding conditions that it exhibits little or no affinity for the matrix.

34. The kit of claim 33, further comprising one or more reagents suitable for modulating the electrostatic binding conditions of the assay.

35. The kit of claim 33, further comprising enzymes that degrade sample contaminants but not a non-nucleotide probe/target sequence complex.

36. The kit of claim 33, wherein the kit is used to detect organisms in food, beverages, water, pharmaceutical products, personal care products, dairy products or environmental samples.

37. The kit of claim 33, wherein the kit is used to test raw materials, products or processes.

38. The kit of claim 33, wherein the kit is used to examine clinical samples such as clinical specimens or equipment, fixtures and products used to treat humans or animals.

39. The kit of claim 33, wherein the kit is used to detect a target sequence that is specific-for a genetically-based disease or is specific for a predisposition to a genetically-based disease.

40. The kit of claim 33, wherein the kit is used to detect a target sequence in a forensic technique such as prenatal screening, paternity testing, identity confirmation or crime investigation.

41. The kit of claim 33, wherein the kit is used to perform a self-indicating assay.

42. The kit of claim 33, wherein the kit comprises two or more independently detectable non-nucleotide probes and is designed to perform a multiplex assay.

43. The kit of claim 42, wherein the two or more independently detectable non-nucleotide probes are non-nucleotide “

Beacon”

probes.

44. The kit of claim 33, wherein the non-nucleotide probe is a non-nucleotide “

Beacon”

probe.