Assays for single molecule detection and use thereof

• US 10,626,450 B2
• Filed: 08/11/2017
• Issued: 04/21/2020
• Est. Priority Date: 08/19/2013
• Status: Active Grant

• Current Assignee: Invitae Corporation
• Original Assignee: Singular Bio, Inc. (Invitae Corporation)
• Inventors: Fehr, Adrian Nielsen, Collins, Patrick James, Herschleb, Jill Lyndon, Jones, Hywel Bowden
• Primary Examiner(s): Wilder, Cynthia B
• Application Number:
  US15/675,234
• Publication Number:
  US 20180179586A1
• Time in Patent Office: 984 Days
• CPC Class Codes: C12Q 1/6809 Methods for determination or identification of nucleic acids involving differential detection
• CPC Class Codes: C12Q 1/6825 Nucleic acid detection involving sensors
• 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 1/6876 Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
• CPC Class Codes: C12Q 2521/101 DNA polymerase
• CPC Class Codes: C12Q 2525/155 incorporating/generating a new priming site
• CPC Class Codes: C12Q 2525/161 incorporating target specific and non-target specific sites
• CPC Class Codes: C12Q 2525/185 incorporating bases where the precise position of the bases in the nucleic acid string is important
• CPC Class Codes: C12Q 2531/137 Ligase Chain Reaction [LCR]
• CPC Class Codes: C12Q 2533/107 Probe or oligonucleotide ligation
• CPC Class Codes: C12Q 2535/125 Allele specific primer extension
• CPC Class Codes: C12Q 2535/131 Allele specific probes
• 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 2545/101 with an internal standard/control
• CPC Class Codes: C12Q 2563/107 fluorescence
• CPC Class Codes: C12Q 2565/102 Multiple non-interacting labels
• CPC Class Codes: C12Q 2565/519 characterised by the capture moiety being a single stranded oligonucleotide
• CPC Class Codes: C12Q 2565/601 being a microscope, e.g. atomic force microscopy [AFM]
• CPC Class Codes: C12Q 2600/156 Polymorphic or mutational markers
• CPC Class Codes: C12Q 2600/158 : Expression markers
• CPC Class Codes: C12Q 2600/16 : Primer sets for multiplex assays

Abstract

The invention relates to methods of detecting a genetic variation in a genetic sample from a subject using labeled probes and counting the number of labels in the probes.

Claims

1. A method of detecting nucleic acid copy numbers in a genetic sample obtained from a subject, comprising

contacting first and second probe sets to the genetic sample, wherein the first probe set comprises a first labeling probe and a first tagging probe, and the second probe set comprises a second labeling probe and a second tagging probe;

hybridizing at least parts of probes of the first and second probe sets to first and second nucleic acid regions of interest in nucleotide molecules present in the genetic sample to produce first and second probe products, respectively, wherein the first and second probe products are circular molecules;

amplifying the first and second probe products to form first and second amplified probe products;

labeling the first and second amplified probe products with first and second labels to produce first and second labeled amplified probe products;

immobilizing at least parts of probes of the first and second labeled amplified probe products to a substrate at a density in which the first and second labeled probe products are optically resolvable after immobilization; and

detecting (i) a first number of the first labeled amplified probe products, and (ii) a second number of the second labeled amplified probe products to detect the nucleic acid copy numbers.

2. The method according to claim 1, wherein the first probe set further comprises a first gap probe, and the second probe set further comprises a second gap probe.

3. The method according to claim 2, wherein each of the first and second gap probes comprises an artificial nucleotide sequence.

4. The method according to claim 2, wherein

in the first probe set, the first gap probe, the first labeling probe, and the first tagging probe are adjacent to each other, and

in the second probe set, the second gap probe, the second labeling probe, and the second tagging probe are adjacent to each other.

5. The method according to claim 2, further comprising, after the hybridizing before the amplifying, (i) ligating the first gap probe, the first labeling probe, and the first tagging probe in the first probe set, and (ii) ligating the second gap probe, the second labeling probe, and the second tagging probe in the second probe set.

6. The method according to claim 1, further ligating probes in each of the first and second probe sets after the hybridizing before the amplifying.

7. The method according to claim 1, wherein the amplifying comprises linear amplification.

8. The method according to claim 1, further immobilizing probe products to a well.

9. The method according to claim 1, wherein each of the first and second labeling probes and the first and second tagging probes independently comprises a naturally-occurring nucleotide sequence.

10. The method according to claim 1, wherein the amplifying and labeling comprise amplifying and labeling the first and second probe products with first and second labels by using labelled first and second primers.

11. The method according to claim 1, wherein the labeling further comprises labeling the first and second amplified probe products with first and second affinity tags.

12. The method according to claim 1, wherein the labeling further comprises labeling the first and second amplified probe products with third and fourth labels, respectively.

13. The method according to claim 1, wherein each of the circular molecules comprises a naturally-occurring oligonucleotide and a non-naturally occurring oligonucleotide.

14. The method according to claim 1, wherein the said at least parts of probes of the first and second labeled amplified probe products are covalently immobilized to the substrate via biotin-Strepatavidin.

15. A method of detecting nucleic acid copy numbers in a genetic sample obtained from a subject, comprising

contacting first and second probe sets to the genetic sample, wherein the first probe set comprises a first labeling probe and a first tagging probe, and the second probe set comprises a second labeling probe and a second tagging probe;

hybridizing at least parts of probes of the first and second probe sets to first and second nucleic acid regions of interest in nucleotide molecules present in the genetic sample to produce first and second probe products, respectively, wherein the first and second probe products are circular molecules;

amplifying the first and second probe products to form first and second amplified probe products;

immobilizing at least parts of probes of the first and second amplified probe products to a substrate at a density in which the first and second probe products are optically resolvable after immobilization to produce first and second immobilized amplified probe products;

labeling the first and second immobilized amplified probe products with first and second labels to produce first and second labeled amplified probe products; and

detecting (i) a first number of the first labeled amplified probe products, and (ii) a second number of the second labeled amplified probe products to detect the nucleic acid copy numbers.

16. The method according to claim 15, wherein the first probe set further comprises a first gap probe, and the second probe set further comprises a second gap probe.

17. The method according to claim 16, wherein each of the first and second gap probes comprises an artificial nucleotide sequence.

18. The method according to claim 16, wherein

in the first probe set, the first gap probe, the first labeling probe, and the first tagging probe are adjacent to each other, and

in the second probe set, the second gap probe, the second labeling probe, and the second tagging probe are adjacent to each other.

19. The method according to claim 16, further comprising, after the hybridizing before the amplifying, (i) ligating the first gap probe, the first labeling probe, and the first tagging probe in the first probe set, and (ii) ligating the second gap probe, the second labeling probe, and the second tagging probe in the second probe set.

20. The method according to claim 15, further ligating probes in each of the first and second probe sets after the hybridizing before the amplifying.

21. The method according to claim 15, wherein the amplifying comprises linear amplification.

22. The method according to claim 15, further immobilizing probe products to a well.

23. The method according to claim 15, wherein each of the first and second labeling probes and the first and second tagging probes independently comprises a naturally-occurring nucleotide sequence.

24. The method according to claim 15, wherein the amplifying and labeling comprise amplifying and labeling the first and second probe products with first and second labels by using labelled first and second primers.

25. The method according to claim 15, wherein the labeling further comprises labeling the first and second amplified probe products with first and second affinity tags.

26. The method according to claim 15, wherein the labeling further comprises labeling the first and second amplified probe products with third and fourth labels, respectively.

27. The method according to claim 15, wherein each of the circular molecules comprises a naturally-occurring oligonucleotide and a non-naturally occurring oligonucleotide.

28. The method according to claim 15, wherein the said at least parts of probes of the first and second labeled amplified probe products are covalently immobilized to the substrate via biotin-Strepatavidin.