Single molecule electronic multiplex snp assay and pcr analysis

Patent metadata

Field	Value
Publication number	US-2017058335-A1
Application number	US-201515118812-A
Country	US
Kind code	A1
Filing date	Feb 12, 2015
Priority date	Feb 12, 2014
Publication date	Mar 2, 2017
Grant date	—

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.

First claim

What is claimed is:

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.

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.

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 labe

Assignees

• Tao Chuanjuan
• Kumar Shiv
• Chien Minchen
• Ju Jingyue
• Univ Columbia

Inventors

• Tao Chuanjuan
• Kumar Shiv
• Chien Minchen
• Ju Jingyue

Classifications

• C12Q1/6858 Primary

  Allele-specific amplification · CPC title

• C12Q1/6853

  using modified primers or templates · CPC title

• C12Q2525/186

  incorporating a non-extendable or blocking moiety · CPC title

• C12Q2565/631

  being a biochannel or pore · CPC title