SINGLE MOLECULE ELECTRONIC MULTIPLEX SNP ASSAY AND PCR ANALYSIS

• US 20170058335A1
• Filed: 02/12/2015
• Published: 03/02/2017
• Est. Priority Date: 02/12/2014
• Status: Active Grant

• Current Assignee: Trustees Of Columbia University In The City Of New York (Columbia University)
• Original Assignee: Trustees Of Columbia University In The City Of New York (Columbia University)
• Inventors: Tao, Chuanjuan, Kumar, Shiv, Chien, Minchen, Ju, Jingyue
• Granted Patent: US 10,718,011 B2
• Time in Patent Office: Days
• US Class Current: 1/1
• CPC Class Codes: C12Q 1/6853 using modified primers or templates
• CPC Class Codes: C12Q 1/6858 Allele-specific amplification
• CPC Class Codes: C12Q 2525/186 incorporating a non-extendable or blocking moiety
• CPC Class Codes: C12Q 2535/113 Cycle sequencing
• CPC Class Codes: C12Q 2565/631 being a biochannel or pore

Abstract

This invention provides methods of using labeled primers or probes for nucleic acid target detection and to detect the identity or presence of a nucleotide at certain positions in nucleic acid sequences with single molecule sensitivity using nanopore detection, and sets of oligonucleotide primers for use in such methods, as well as methods of quantitative PCR coupled with nanopore detection.

Claims

1. A method for identifying a single nucleotide residue of interest at a position within a stretch of consecutive nucleotide residues in a DNA, comprising the steps of:

(a) incubating the DNA with

(1) at least one oligonucleotide primer, each primer comprising a removably attached label (i) corresponding to a particular primer sequence, and (ii) having a unique signature detectable by a nanopore, wherein the nucleotides in the primer that are 5′

to the nucleotide at the 3′

-terminus of the primer are substantially fully complementary to the nucleotides in the DNA immediately 3′

to the single nucleotide of interest,

(2) terminating nucleotides, and

(3) DNA polymerase,

 

so as to perform a single base extension of a primer whose 3′

terminal nucleotide hybridized to the single nucleotide residue of interest in the DNA, if such a primer was present, using the terminating nucleotide, thereby forming an extension product of the primer which had a 3′

nucleotide complementary to the nucleotide of interest in the DNA;

(b) removing the label from the extension product, if present;

(c) detecting by nanopore the signature of the label of the primer whose 3′

terminal nucleotide hybridized to the single nucleotide residue of interest, so as to identify the label and primer, if present;

thereby identifying the single nucleotide residue of interest.

2. The method of claim 1, wherein the DNA is a single-stranded DNA, or is a double-stranded DNA.

3. The method of any one of claims 1-2, wherein in step (a) the DNA is incubated with a plurality of oligonucleotide primers, wherein the plurality of oligonucleotide primers comprises at least two primers, at least three primers, or at least four primers having (i) identical nucleotide sequences except for having a different nucleotide at the 3′

terminus of each of the primers, wherein the identical nucleotides in each of the primers are substantially fully complementary to the nucleotides in the DNA immediately 3′

to the single nucleotide residue of interest.

4. The method of any one of claims 1-3, wherein the terminating nucleotides are dideoxynucleotides, wherein the terminating nucleotides comprise a hook, wherein the terminating nucleotides comprise a hook which is a biotin moiety, or wherein the terminating nucleotides comprise a hook which is a phenylboronic acid (PBA) moiety.

5. The method of any one of claims 1-4, wherein prior to removing the label, the extension product is separated from the unextended primers, or wherein prior to removing the label, the extension product is separated from the unextended primers by capturing the extension product on streptavidin-coated magnetic beads, or wherein prior to removing the label, the extension product is separated from the unextended primers by capturing the extension product on salicylhydroxamic acid (SHA)-coated magnetic beads.

6. The method of any one of claims 1-5, wherein the label is removably attached via a cleavable linker, or is removably attached via a cleavable linker which is an azido linker, or is removably attached via a cleavable linker which is cleaved in step (b) by a phosphine-containing moiety or by tris-(2-carboxyethyl)phosphine (TCEP), or is removably attached via a cleavable linker which is attached to the 5′

-terminus of the oligonucleotide primer between the label and the oligonucleotide.

7. The method of any one of claims 1-6, wherein the label comprises one or more of ethylene glycol, an amino acid, a carbohydrate, a peptide, a dye, a chemiluminiscent compound, a mononucleotide, a dinucleotide, a trinucleotide, a tetranucleotide, a pentanucleotide, a hexanucleotide, an aliphatic acid, an aromatic acid, an alcohol, a thiol group, a cyano group, a nitro group, an alkyl group, an alkenyl group, an alkynyl group, an azido group, or a combination thereof.

8. The method of any one of claims 1-7, wherein the labels are polyethylene glycol (PEG) labels, or are PEG labels which each have a different length from each other.

9. The method of any one of claims 1-8, wherein the DNA is incubated with a plurality of primers, or at least 20 different oligonucleotide primers, each primer comprising an attached label having a unique signature detectable by a nanopore.

10. The method of any one of claims 1-9, wherein the nanopore is biological, is proteinaceous, comprises alpha hemolysin, is a solid-state nanopore, or is in a solid-state membrane.

11. The method of any one of claims 1-10, wherein the signature is an electronic signature, or is an electrical current blockade signature.

12. The method of any one of claims 1-11, wherein the nucleotide sequence of the portion of each primer which is 5′

to the 3′

nucleotide of the primer is at least 85% complementary, is at least 90% complementary, is at least 95% complementary, or is 100% complementary to the sequence of the DNA which is 3′

to the single nucleotide residue of interest.

13. The method of any one of claims 1-12, wherein the sequence of the primer is 10-40 nucleotides long, or is 18-24 nucleotides long.

14. The method of any one of claims 1-13, wherein the single nucleotide of interest is at the site of a single nucleotide polymorphism (SNP).

15. An assay for performing the method of any one of claims 1-14.

16. A set of oligonucleotide primers, wherein each primer comprises a removably attached label (1) corresponding to a particular primer sequence, and (2) having a unique signature detectable by a nanopore, wherein the set comprises at least two primers, at least three primers, or at least four primers having (i) identical nucleotide sequences except for having a different nucleotide at the 3′

terminus of each of the primers, and (ii) a different label corresponding to the different nucleotide at the 3′

terminus, wherein the identical nucleotides in each of the primers are substantially fully complementary to the nucleotides in a strand of DNA immediately 3′

to a single nucleotide residue of interest.

18. The set of oligonucleotide primers of any one of claims 16-17, wherein the single nucleotide residue of interest is at a site of a single nucleotide polymorphism (SNP).

19. The set of oligonucleotide primers of any one of claims 16-18, wherein the sequence of each primer is 15-40 nucleotides long, or is 18-24 nucleotides long.

20. The set of oligonucleotide primers of any one of claims 16-19, wherein each removably attached label comprises one or more of ethylene glycol, an amino acid, a carbohydrate, a peptide, a dye, a chemiluminiscent compound, a mononucleotide, a dinucleotide, a trinucleotide, a tetranucleotide, a pentanucleotide, a hexanucleotide, an aliphatic acid, an aromatic acid, an alcohol, a thiol group, a cyano group, a nitro group, an alkyl group, an alkenyl group, an alkynyl group, an azido group, or a combination thereof.

21. The set of oligonucleotide primers of any one of claims 16-20, wherein the removably attached labels are PEG labels, each having different lengths from each other.

17. A set of oligonucleotide primers, wherein each primer comprises a removably attached label (1) corresponding to a particular primer sequence, and (2) having a unique signature detectable by a nanopore;

and wherein the 3′

nucleotide of each primer is complementary to a single nucleotide residue of interest in a strand of DNA and the other nucleotides in that primer are substantially fully complementary to the nucleotides in the DNA immediately 3′

of the single nucleotide residue of interest.

22. A method for simultaneously detecting in a sample the presence of one or more of a plurality of different target nucleic acids comprising the steps of:

(a) contacting the sample with a plurality of nucleic acid primers simultaneously and under conditions permitting, and for a time sufficient for, primer extension to occur, wherein (i) for each target nucleic acid at least one predetermined primer is used which corresponds to that target nucleic acid, and (ii) each primer has a removably attached label having a unique signature detectable by a nanopore;

(b) separating any unextended primers from any extended primers;

(c) simultaneously removing the labels from any extended primers; and

(d) detecting the presence of any labels so removed;

wherein the presence of a removed label having a signature identical to the label removably attached to a predetermined primer indicates the presence in the sample of the target nucleic acid specifically recognized by that predetermined primer.

23. The method of claim 22, wherein at least two different primers correspond to the same target nucleic acid.

24. The method of claim 23, wherein a first primer is a forward primer for the target nucleic acid and a second primer is a reverse primer for the same target nucleic acid.

25. The method of claim 24, wherein the labels attached to the first and second primers have the same signature, or have different signatures.

26. The method of any one of claims 22-25, wherein the method detects the presence in the sample of 10 or more different target nucleic acids, 50 or more different target nucleic acids, 100 or more different target nucleic acids, or 200 or more different target nucleic acids.

27. The method of any one of claims 22-26, wherein the sample is contacted with 1 or more different primers, 10 or more different primers, 50 or more different primers, 100 or more different primers, or 200 or more different primers.

28. The method of any one of claims 22-27, wherein the label is removed in step (d) by photocleaving, is removed in step (d) by photocleaving with ultraviolet light, or is removed in step (d) by chemical cleaving.

29. The method of any one of claims 22-28, wherein each removably attached label comprises one or more of ethylene glycol, an amino acid, a carbohydrate, a peptide, a dye, a chemiluminiscent compound, a mononucleotide, a dinucleotide, a trinucleotide, a tetranucleotide, a pentanucleotide, a hexanucleotide, an aliphatic acid, an aromatic acid, an alcohol, a thiol group, a cyano group, a nitro group, an alkyl group, an alkenyl group, an alkynyl group, an azido group, or a combination thereof, or the labels are polyethylene glycol (PEG) labels, or are PEG labels which each have a different length from each other.

30. The method of any one of claims 22-29, wherein the nanopore is biological, is proteinaceous, comprises alpha hemolysin, is a solid-state nanopore, or is in a solid-state membrane.

31. The method of any one of claims 22-30, wherein the signature is an electronic signature, or is an electrical current blockade signature.

32. A method of identifying or quantifying a target nucleic acid, comprising the steps of:

(a) incubating the target nucleic acid with a probe, wherein the probe comprises (i) a nucleotide sequence region that is fully substantially complementary to the target nucleic acid, and (ii) an attached label having a unique signature detectable by a nanopore, in conditions permitting the probe to hybridize to the target nucleic acid;

(b) performing PCR;

(c) releasing the label from the probe;

(d) detecting by nanopore an electronic change caused by the label; and

(e) correlating the amplitude of the electronic change determined in step (d) with the quantity of label,

thereby identifying or quantifying the target nucleic acid.

33. The method of claim 32, wherein the probe comprises a 5′

-terminal region of nucleotides that does not hybridize to the target nucleic acid, or wherein the probe comprises a 5′

-terminal region of nucleotides that does not hybridize to the target nucleic acid which is 3-15 nucleotides, or the probe comprises a 5′

-terminal region of nucleotides that does not hybridize to the target nucleic acid which is 5-10 nucleotides.

34. The method of any one of claims 32-33, wherein the nucleotide sequence region that is fully substantially complementary to the target nucleic acid is 10-40 nucleotides, or is 18-24 nucleotides.

35. The method of any one of claims 32-34, wherein the label is attached to the 5′

-terminus of the probe.

36. The method of any one of claims 32-35, wherein the label comprises one or more of ethylene glycol, an amino acid, a carbohydrate, a peptide, a dye, a chemiluminiscent compound, a mononucleotide, a dinucleotide, a trinucleotide, a tetranucleotide, a pentanucleotide, a hexanucleotide, an aliphatic acid, an aromatic acid, an alcohol, a thiol group, a cyano group, a nitro group, an alkyl group, an alkenyl group, an alkynyl group, an azido group, or a combination thereof, or is a polyethylene glycol (PEG) label.

37. The method of any one of claims 32-36, wherein the PCR is performed using Taq polymerase.

38. The method of any one of claims 32-37, wherein the polymerase activity of the polymerase during the PCR reaction causes the release of the label.

39. The method of any one of claims 32-38, wherein the nanopore is biological, is proteinaceous, comprises alpha hemolysin, is a solid-state nanopore, or is in a solid-state membrane.

40. The method of any one of claims 32-39, wherein the signature is an electronic signature, or is an electrical current blockade signature.