Sample preparation method

• US 10,669,578 B2
• Filed: 02/19/2015
• Issued: 06/02/2020
• Est. Priority Date: 02/21/2014
• Status: Active Grant

• Current Assignee: Oxford Nanopore Technologies Limited
• Original Assignee: Oxford Nanopore Technologies Limited
• Inventors: Clarke, James Anthony, Crawford, Marion Louise, White, James
• Primary Examiner(s): Bhat, Narayan K
• Application Number:
  US15/120,186
• Publication Number:
  US 20170067101A1
• Time in Patent Office: 1,930 Days
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2525/113 incorporating modified backbone
• CPC Class Codes: C12Q 2525/117 incorporating modified base
• CPC Class Codes: C12Q 2565/518 characterised by the immobilisation of the nucleic acid sample or target
• CPC Class Codes: C12Q 2565/631 being a biochannel or pore

Abstract

The invention relates to an improved method for characterising a template polynucleotide. The method involves using a polymerase to prepare a modified polynucleotide which makes it easier to characterise than the template polynucleotide.

Claims

1. A method of characterising a template polynucleotide composed of different polymer units defining a sequence of template nucleotides, the method comprising:

a) contacting the template polynucleotide with a polymerase and a pool of free nucleotides under conditions in which the polymerase forms a modified polynucleotide using the template polynucleotide as a template for synthesizing the modified polynucleotide, wherein the pool of free nucleotides consists of;

i) at least two modified nucleotides that each base pair with at least one template nucleotide of the sequence of template nucleotides; and

ii) nucleotides that specifically base pair with each of the remaining template nucleotides of the sequence of nucleotides, but that do not specifically base pair with the at least one template nucleotide of i) wherein at least two of the two or more modified nucleotides in the pool base pair with one another;

b) contacting the modified polynucleotide with a transmembrane pore in the presence of an electrical potential across the transmembrane pore, such that the modified polynucleotide moves through the pore; and

c) taking one or more electrical measurements as the modified polynucleotide moves through the pore, wherein incorporation of the at least two modified nucleotides into the modified polynucleotide results in the one or more electrical measurements having an improved signal to noise ratio compared with electrical measurements taken as the template polynucleotide moves through the pore.

2. The method according to claim 1, wherein the polymerase does not form a complementary polynucleotide if the template polynucleotide is RNA.

3. The method according to claim 1, wherein the pool of free nucleotides includes, in i), two modified nucleotides, wherein each of the two modified nucleotides base pairs with a different template nucleotide.

4. The method according to claim 1, wherein:

(a) the template polynucleotide is DNA and the template nucleotides of the polymer units include deoxyadenosine monophosphate (dAMP), deoxyguanosine monophosphate (dGMP), deoxythymidine monophosphate (dTMP), deoxycytidine monophosphate (dCMP) and/or deoxymethylcytidine monophosphate;

or

(b) the template polynucleotide is RNA and the template nucleotides of the polymer units include adenosine monophosphate (AMP), guanosine monophosphate (GMP), uridine monophosphate (UMP), cytidine monophosphate (CMP) and/or 5-methylcytidine monophosphate.

5. The method according to claim 1, wherein:

(a) the template polynucleotide is DNA and the modified polynucleotide comprises a nucleobase which differs from adenine, guanine, thymine, cytosine or methylcytosine and/or comprises a nucleoside which differs from deoxyadenosine, deoxyguanosine, thymidine, deoxycytidine or deoxymethylcytidine;

or

(b) the template polynucleotide is RNA and the modified polynucleotide comprises a nucleobase which differs from adenine, guanine, uracil, cytosine or methylcytosine and/or comprises a nucleoside which differs from adenosine, guanosine, uridine, cytidine or methylcytidine.

6. The method according to claim 1, wherein each of the at least two modified nucleotides in i) is selected from the group consisting of hypoxanthine, 4-nitroindole, 5-nitroindole, 6-nitroindole, formylindole, 3-nitropyrrole, nitroimidazole, 4-nitropyrazole, 4-nitrobenzimidazole, 5-nitroindazole, 4-aminobenzimidazole, phenyl (C6-aromatic ring), 2′

-deoxyinosine, inosine, 7-deaza-2′

-deoxyinosine, 7-deaza-inosine, 2-aza-deoxyinosine, 2-aza-inosine, 2-O′

-methylinosine, 4-nitroindole 2′

-deoxyribonucleoside, 4-nitroindole ribonucleoside, 5-nitroindole 2′

-deoxyribonucleoside, 5-nitroindole ribonucleoside, 6-nitroindole 2′

-deoxyribonucleoside, 6-nitroindole ribonucleoside, 3-nitropyrrole 2′

-deoxyribonucleoside, 3-nitropyrrole ribonucleoside, an acyclic sugar analogue of hypoxanthine, nitroimidazole 2′

-deoxyribonucleoside, nitroimidazole ribonucleoside, 4-nitropyrazole 2′

-deoxyribonucleoside, 4-nitropyrazole ribonucleoside, 4-nitrobenzimidazole 2′

-deoxyribonucleoside, 4-nitrobenzimidazole ribonucleoside, 5-nitroindazole 2′

-deoxyribonucleoside, 5-nitroindazole ribonucleoside, 4-aminobenzimidazole 2′

-deoxyribonucleoside, 4-aminobenzimidazole ribonucleoside, phenyl C-ribonucleoside, phenyl C-2′

-deoxyribosyl nucleoside, 2′

-deoxynebularine, 2′

-deoxyisoguanosine, K-2′

-deoxyribose, P-2′

-deoxyribose and pyrrolidine.

7. The method according to claim 1, wherein the polymerase synthesizes a modified polynucleotide which comprises a chemical group or atom absent from the template polynucleotide.

8. The method according to claim 7, wherein the chemical group is selected from the group consisting of a propynyl group, a thio group, an oxo group, a methyl group, a hydroxymethyl group, a formyl group, a carboxy group, a carbonyl group, a benzyl group, a propargyl group or a propargylamine group.

9. The method according to claim 1, wherein the polymerase synthesizes a modified polynucleotide which lacks a chemical group or atom present in the template polynucleotide.

10. The method according to claim 1, wherein the polymerase synthesizes a modified polynucleotide having an altered electronegativity relative to the template polynucleotide.

11. The method according to claim 10, wherein the modified polynucleotide comprises a halogen atom.

12. The method according to claim 1, wherein step (a) further comprises selectively removing the nucleobases from one or more nucleotide species in the modified polynucleotide.

13. The method according to claim 1, wherein the template polynucleotide is single stranded.

14. The method according to claim 13, wherein the method further comprises before step (a) ligating a hairpin adaptor to one end of the template polynucleotide such that in step (a) the ligated hairpin adaptor acts as a primer for formation of the modified polynucleotide by the polymerase such that the modified and template polynucleotides are ligated by the hairpin adaptor.

15. The method according to claim 14, wherein the method comprises:

b) contacting the ligated modified and template polynucleotides with a transmembrane pore such that the polynucleotides move through the pore; and

c) taking one or more electrical measurements as the polynucleotides moves through the pore wherein the electrical measurements are indicative of one or more characteristics of the polynucleotides and thereby characterising the template polynucleotide.

16. The method according to claim 1, wherein the template polynucleotide is double stranded.