Detection of Nucleic Acids

• US 20120052498A1
• Filed: 07/01/2011
• Published: 03/01/2012
• Est. Priority Date: 07/01/2010
• Status: Abandoned Application

• Current Assignee: Affymetrix, Inc. (Thermo Fisher Scientific Incorporated)
• Original Assignee: Affymetrix, Inc. (Thermo Fisher Scientific Incorporated)
• Inventors: Nguyen, Quan N., McMaster, Gary K., Ma, Yunqing, Nguyen, Cung-Tuong, Wu, Yen-Chieh, Lin, Audrey, Lai, Chunfai
• Application Number:
  US13/175,582
• Publication Number:
  US 20120052498A1
• Time in Patent Office: Days
• US Class Current: 435/6.11
• CPC Class Codes: C12Q 1/682 Signal amplification
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 2525/313 Branched oligonucleotides
• 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 2563/149 Particles, e.g. beads
• CPC Class Codes: C12Q 2565/101 Interaction between at least two labels

Abstract

Methods of detecting various types of nucleic acids, including methods of detecting two or more nucleic acids in multiplex branched-chain DNA assays, are provided. Detection assays may be conducted at least in vitro, in cellulo, and in situ. Nucleic acids which are optionally captured on a solid support are detected, for example, through cooperative hybridization events that result in specific association of a label probe system with the nucleic acids. Various label probe system embodiments are provided. Compositions, kits, and systems related to the methods are also described.

Claims

1. A method of genotyping a single nucleotide polymorphism (SNP), which comprises:

providing a sample comprising or suspected of comprising a target nucleic acid comprising at least one SNP;

incubating at least two label extender probes each comprising a different L-1 sequence, at least two capture extender probes each comprising a different C-1 sequence, and a label probe system with the sample comprising or suspected of comprising the target nucleic acid comprising the at least one SNP, wherein at least one L-1 sequence is perfectly complementary to the at least one allele of a SNP;

adding one or more subsets of particles comprising covalently bound thereto at least two capture probes, under conditions in which the particles bind to the capture extender probes, wherein the capture probes are covalently bound to the particles, wherein one or more particles within a subset of particles is distinguishable from one or more particles in any other subset of particles;

detecting whether the particles have bound thereto the label probe system, thereby genotyping the at least one allele of the SNP.

2. The method according to claim 1, wherein the at least one L-1 sequence comprises one or more locked nucleic acids.

3. The method according to claim 2, wherein the one or more locked nucleic acid(s) is/are constrained ethyl nucleic acid(s) (cEt).

4. The method according to claim 1, wherein the target is selected from one or more of the group consisting essentially of:

double-stranded DNA, miRNA, siRNA, mRNA, and single-stranded DNA.

5. The method according to claim 1, wherein the method is performed in situ.

6. The method according to claim 1, wherein the sample is purified chromosomes.

7. The method according to claim 1, wherein the sample is cells obtained from a cell culture.

8. The method according to claim 1, wherein the sample comprises or is suspected of comprising a target nucleic acid comprising at least two single nucleotide polymorphisms.

9. The method according to claim 1, wherein the label extenders are designed in the cruciform orientation.

10. The method according to claim 1, wherein the particles are distinguishable based on color.

11. The method according to claim 1, wherein the particles are microparticles.

12. The method according to claim 11, wherein the microparticles are encoded microparticles comprising a visually detectable spatial code.

13. A method of genotyping a single nucleotide polymorphism (SNP), which comprises:

providing a sample comprising or suspected of comprising a target nucleic acid comprising at least one SNP;

incubating at least two label extender probes each comprising a different L-1 sequence, and a label probe system with the sample comprising or suspected of comprising the target nucleic acid comprising the at least one SNP, wherein at least one L-1 sequence is perfectly complementary to the at least one SNP allele;

detecting whether the label probe system is associated with the sample.

14. The method according to claim 13, wherein the sample is purified chromosomes.

15. The method according to claim 13, wherein the sample comprises or is suspected of comprising a target nucleic acid comprising at least two single nucleotide polymorphisms.

16. The method according to claim 13, wherein the sample is a tissue.

17. The method according to claim 13, wherein the at least one L-1 sequence comprises one or more locked nucleic acids.

18. The method according to claim 17, wherein the one or more locked nucleic acid(s) is/are constrained ethyl nucleic acid(s) (cEt).

19. The method according to claim 13, wherein the label extenders are designed in the cruciform orientation.

20. A method of detecting a microRNA or siRNA, which comprises:

providing a sample comprising or suspected of comprising a microRNA or siRNA;

incubating at least two label extender probes each comprising a different L-1 sequence, and a label probe system with the sample comprising or suspected of comprising the microRNA or siRNA, wherein at least one L-1 sequence is complementary to at least part of the microRNA or siRNA sequence;

detecting whether the label probe system is associated with the sample.

21. The method according to claim 20, wherein the sample is a tissue.

22. The method according to claim 20, wherein the sample is cells from a cell culture.

23. The method according to claim 20, wherein the cells are human cells.

24. The method according to claim 20, wherein the at least one L-1 sequence comprises one or more locked nucleic acids.

25. The method according to claim 24, wherein the one or more locked nucleic acid(s) is/are constrained ethyl nucleic acid(s) (cEt).

26. The method according to claim 20, wherein the sample comprises both mature miRNA and pre-miRNA and wherein two differently labeled label probe systems are present, thereby detecting whether the sample comprises mature miRNA, immature miRNA or both.

27. The method according to claim 20, wherein at least two different miRNA or siRNA sequences are in the sample.

28. The method according to claim 20, wherein the label extenders are designed in the cruciform orientation.

29. A method of detecting a translocation event or fusion event, which comprises:

providing a sample comprising or suspected of comprising a translocation event or fusion event at a translocation site;

incubating at least two label extender probes each comprising a different L-1 sequence, and a label probe system with the sample comprising or suspected of comprising the translocation event or fusion event, wherein at least one L-1 sequence is perfectly complementary to a translocation sequence, wherein the translocation sequence is centered at the translocation site;

detecting whether the label probe system is associated with the sample.

30. The method according to claim 29, wherein the sample is a tissue.

31. The method according to claim 29, wherein the sample is cells from a cell culture.

32. The method according to claim 29, wherein the cells are human cells.

33. The method according to claim 29, wherein the at least one L-1 sequence comprises one or more locked nucleic acids.

34. The method according to claim 33, wherein the one or more locked nucleic acid(s) is/are constrained ethyl nucleic acid(s) (cEt).

35. The method according to claim 29, wherein one L-1 sequence is complementary to one half of the translocation or fusion sequence and a second L-1 sequence is complementary to the second half of the translocation or fusion sequence.

36. The method according to claim 29, wherein the label extenders are designed in the cruciform orientation.