System for charge-based detection of nucleic acids

• US 20070178470A1
• Filed: 12/13/2004
• Published: 08/02/2007
• Est. Priority Date: 12/12/2003
• Status: Abandoned Application

• Current Assignee: Universite Laval
• Original Assignee: Infectio Recherche Inc.
• Inventors: Bissonnette, Luc, Peytavi, Regis, Raymond, Frederic
• Application Number:
  US10/596,397
• Publication Number:
  US 20070178470A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6818 involving interaction of two or more labels, e.g. resonant energy transfer
• CPC Class Codes: C12Q 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 2525/203 incorporating a composite nucleic acid containing a polypeptide sequence other than PNA
• CPC Class Codes: C12Q 2563/107 fluorescence
• CPC Class Codes: C12Q 2563/137 Metal/ion, e.g. metal label
• CPC Class Codes: C12Q 2565/501 being an array of oligonucleotides
• CPC Class Codes: C12Q 2565/519 characterised by the capture moiety being a single stranded oligonucleotide

Abstract

This present invention relates methods for detecting the presence of nucleic acids in a sample. In these methods, neutral capture probes are exposed to a sample possibly containing complementary nucleic acid targets. The foregoing mixture is submitted to conditions that provide for the nucleic acid targets to bind with the neutral probes thereby generating hybrids. These hybrids are submitted to positively charged reporters such as atoms, molecules or macromolecules, which electrostatically bind to the hybrids. The complexes formed between reporters and hybrids are detected by a variety of detection methods. Kits for detecting the presence of nucleic acids in a sample are also disclosed herein.

Claims

24-1. A method according to claim 1, wherein said reporters comprise enzymes.

27. A method for detecting the presence of nucleic acids in a sample, said method comprising:

(a) exposing uncomplexed neutral capture probes to a sample possibly containing complementary nucleic acid targets and containing positively charged reporters selected from group consisting of transition metal atoms, molecules and macromolecules, thereby generating a mixture;

(b) submitting said mixture to hybridization conditions which provide for said nucleic acids targets to bind specifically to complementary neutral capture probes, thereby generating negatively charged capture probe-nucleic acid target hybrids, said reporters being capable of electrostatically binding to said hybrids, thereby generating higher-order complexes; and

(c) detecting said higher-order complexes.

28. A method according to claim 27, wherein said nucleic acids targets are unlabeled.

39. A method according to claim 27, wherein said neutral capture probes are selected from the group consisting of peptide nucleic acids (PNA), and methylphosphonate.

40. A method according to claim 27, wherein said nucleic acid targets are selected from the group consisting of DNA and RNA molecules.

41. A method according to claim 27, wherein said nucleic acid targets are generated by methods selected from the group consisting of polymerase chain reaction (PCR), reverse transcriptase-PCR (RT-PCR), strand displacement amplification (SDA), ligase chain reaction (LCR), transcription-associated amplification, nucleic acid sequence-based amplification (NASBA), whole genome amplification (WGA), helicase-dependent isothermal amplification, and chemical synthesis.

42. A method according to claim 27, further comprising a washing step after step (b).

43. A method according to claim 27, wherein said reporters exhibit low affinity for uncharged probes.

44. A method according to claim 27, wherein said reporters are capable of electrostatically binding to the phosphate backbone of said hybrids.

45. A method according to claim 27, wherein said transition metal atoms are selected from the group consisting of Ag⁺ and Cd⁺⁺.

46. A method according to claim 27, wherein said transition metal atoms comprise ions that can be chemically modified to yield higher-order complexes using bound nucleic acids as a scaffold.

47. A method according to claim 27, wherein said detection includes a chemical reaction step rendering said transition metal cations detectable.

48. A method according to claim 27, wherein said reporters comprise polythiophenes.

49. A method according to claim 48, wherein said polythiophenes are water-soluble and cationic.

50. A method according to claim 27, wherein said reporters comprise enzymes.

52. A method according to claim 27, wherein said detection is selected from the group consisting of optical detection, fluorometric detection, colorimetric detection, electrochemical detection, chemiluminescent detection microscopy and spectrophotometric detection.

51. A method according to claim 51, wherein said enzymes comprise alkaline phosphatase having polystyrene beads conjugated thereto.

53. A kit for detecting the presence of nucleic acids in a sample, said kit comprising:

uncomplexed neutral capture probes;

a control sample possibly containing nucleic acid targets that are complementary to the neutral capture probes; and

one or more positively charged reporters selected from the group consisting of transition metal cations, molecules or macromolecules;

said reporters being capable of electrostatically binding to negatively charged capture probe-nucleic acid target hybrids.

54. A kit according to claim 53, wherein said neutral capture probes are selected from the group consisting of peptide nucleic acids (PNA) and methylphosphonate.

55. A kit according to claim 53, wherein said capture probes are immobilized on a support surface.

56. A kit according to claim 55, wherein said support surface is selected from the group consisting of a glass surface, a silicon surface, a gold surface, an electrode surface, a particle surface, a gel matrix, a membrane surface, a paper surface and a plastic surface.

57. A kit according to claim 55, wherein said support surface comprises a solid support surface 58, A kit according to claim 57, wherein said solid support surface comprises a probe array.