IP Library › Granted Patent US 12,195,777
Granted Patent B2
US 12,195,777 · App. 16/966,430 · Granted Jan 14, 2025

Polynucleotide synthesis method, kit and system

Inventor: Andrew John Heron (Oxford, GB)
Assignee: Oxford Nanopore Technologies PLC
C12P19/34
View Patent ↗
Loading inventors, assignments & file history…
Monitor This Case
Get email alerts when status or documents change.
Order Certified Copies
Most orders are placed with the USPTO same day — all within 24 business hours.
Order via The Patent Place →
Pre-filled with this patent's details
Quick Facts
Patent No.
US 12,195,777
App. No.
16/966,430
Granted
Jan 14, 2025
Kind
B2
Abstract

The invention relates to new in vitro methods for synthesising a polymer, particularly a polynucleotide molecule, having a pre-defined sequence of units such as nucleotides. For synthesising a polynucleotide molecule the methods involve a process of extending a polynucleotide synthesis molecule with a transfer nucleotide. The methods additionally involve repeating the extension process multiple times to iteratively extend the polynucleotide molecule with multiple transfer nucleotides to generate a new polynucleotide molecule having a pre-defined nucleotide sequence. The invention also relates to in vitro methods of joining multiple synthetic polynucleotides following synthesis to form larger synthetic polynucleotides, as well as devices and systems for performing the extension, synthesis and assembly methods of the invention.

Claims (35)

1. A method of extending a polynucleotide synthesis molecule with a transfer nucleotide, the method comprising an extension process comprising moving the transfer nucleotide through the channel of a nanopore disposed in a substrate from the cis side to the trans side of the substrate, and contacting the transfer nucleotide with an enzyme provided on the trans side of the substrate adjacent the nanopore whereupon the enzyme catalyses the transfer of the transfer nucleotide to the polynucleotide synthesis molecule thereby extending the polynucleotide synthesis molecule,

wherein the transfer nucleotide is attached to a feeder molecule and wherein the transfer nucleotide is cleaved from the feeder molecule prior to incorporation into the polynucleotide synthesis molecule.

2. The method according to claim 1 , wherein the transfer nucleotide is an unblocked nucleotide.

3. The method according to claim 1 , wherein the transfer nucleotide is cleaved by the enzyme.

4. The method of extending a polynucleotide synthesis molecule with a transfer nucleotide according to claim 1 , the method comprising an extension process comprising:

A. providing a substrate comprising a nanopore, wherein the nanopore comprises a channel allowing fluid flow from the cis side to the trans side of the substrate; providing a feeder molecule at the cis side of the substrate, the feeder molecule having an attached transfer nucleotide; providing an enzyme and the polynucleotide synthesis molecule in proximity to each other on the trans side of the substrate adjacent the nanopore; and

B. moving the feeder molecule through the nanopore to bring the attached transfer nucleotide into contact with the enzyme whereupon the enzyme catalyses the transfer of the transfer nucleotide to the polynucleotide synthesis molecule thereby extending the polynucleotide synthesis molecule.

5. The method according to claim 4 , further comprising:

C. moving the feeder molecule through the nanopore to the cis or trans side of the substrate following transfer of the transfer nucleotide to the polynucleotide synthesis molecule.

6. The method according to claim 1 , wherein the polynucleotide synthesis molecule is provided having a proximal terminus adjacent to the enzyme and a distal terminus, wherein the enzyme catalyses the addition of the transfer nucleotide to the proximal terminus of the polynucleotide synthesis molecule.

7. The method according to claim 1 , wherein feeder molecules are provided at the cis side of the substrate having differing attached transfer nucleotides.

8. The method according to claim 7 , wherein the cis side of the substrate comprises a mixture of feeder molecules in the same reaction volume, wherein the mixture comprises different populations of feeder molecules, wherein each feeder molecule of a population has the same transfer nucleotide attached, and wherein the different populations of feeder molecule have different transfer nucleotides attached.

9. The method according to claim 7 , wherein feeder molecules having differing attached transfer nucleotides are distinguishable.

10. The method according to claim 9 , wherein a feeder molecule is capable of providing an identifiable signal to uniquely identify the attached transfer nucleotide and/or to determine the integrity of the attached transfer nucleotide.

11. The method according to claim 10 , comprising performing a first verification process to determine the identity and/or integrity of the transfer nucleotide of the feeder molecule, wherein:

a) if the feeder molecule is determined to have the desired transfer nucleotide attached, moving the feeder molecule to bring the transfer nucleotide into contact with the enzyme; or

b) if the feeder molecule is determined not to have the desired transfer nucleotide attached:

i. moving the feeder molecule to the cis or trans side of the substrate;

ii. moving a feeder molecule from the mixture of feeder molecules at the cis side of the substrate into the nanopore towards the trans side; and

iii. repeating the first verification process until the feeder molecule is determined to have the desired transfer nucleotide attached, following which the feeder molecule is moved to bring the transfer nucleotide into contact with the enzyme;

optionally wherein the first verification process is performed whilst the feeder molecule is at least partially within the channel of the nanopore.

12. The method according to claim 10 , wherein a feeder molecule is capable of providing a different identifiable signal when the transfer nucleotide is no longer attached to the feeder molecule.

13. The method according to claim 12 , comprising a step (C) of moving the feeder molecule through the nanopore to the cis or trans side of the substrate following transfer of the transfer nucleotide to the polynucleotide synthesis molecule,

wherein step (C) is performed only following a second verification process performed to verify that the enzyme has catalysed the transfer of the transfer nucleotide from the feeder molecule to the polynucleotide synthesis molecule, wherein the second verification process comprises:

I. moving the feeder molecule through the nanopore in the cis direction, and determining the presence or absence of the nucleobase of the transfer nucleotide;

II. moving the feeder molecule back in the trans direction to bring the nucleotide into contact with the enzyme if the nucleobase of the transfer nucleotide is determined to be attached to the feeder molecule; and

III. repeating steps (I) and (II) until the desired transfer nucleotide is determined to have been removed from the feeder molecule.

14. The method according to claim 10 , wherein the identity and/or integrity of the nucleobase and/or the presence or absence of the transfer nucleotide attached to the feeder molecule is determined by measurement of the feeder molecule.

15. The method according to claim 14 , wherein measurement of the feeder molecule is with respect to the nanopore.

16. The method according to claim 15 , wherein the feeder molecule is measured by measuring ion current flow through the nanopore under the action of a potential difference applied across the substrate; optionally wherein a change in the ion current flowing through the nanopore is dependent upon the presence and/or structure of the nucleobase of the nucleotide and thereby provides the identifiable signal to uniquely identify the transfer nucleotide and/or to determine the absence, presence and/or integrity of the transfer nucleotide.

17. The method according to claim 16 , wherein changes in the ion current flowing through the nanopore are dependent upon the presence of a pre-defined sequence of nucleobases integral to the feeder molecule (barcode) and which thereby provides the identifiable signal to uniquely identify the transfer nucleotide.

18. The method according to claim 1 wherein the identity and/or structure of the transfer nucleotide is pre-defined.

19. The method according to claim 1 , wherein the nanopore is a biological nanopore; a synthetic nanopore; a solid state nanopore; or a hybrid nanopore comprising a biological or synthetic nanopore disposed within a solid state substrate.

20. The method according to claim 1 wherein the feeder molecule comprises a sequence of nucleotides (barcode) capable of uniquely identifying a feeder molecule and the type of transfer nucleotide attached thereto.

21. The method according to claim 1 , wherein the feeder molecule comprises one or more blocking moieties, wherein a blocking moiety is provided at a position on the feeder molecule so that when the feeder molecule is moved into a nanopore to a position of interest in the nanopore, the blocking moiety acts to inhibit further translocation of the feeder molecule in the trans direction; optionally wherein one or more blocking moieties comprises a reversible blocking moiety; wherein the one or more blocking moieties are provided along the length of the feeder molecule and/or are provided at a terminal end of the feeder molecule and/or are provided as an integral part of the feeder molecule; optionally wherein a blocking moiety is a molecule attached to the feeder molecule; further optionally wherein a blocking moiety is a peptide, oligopeptide, polypeptide, protein or other polymer.

Assignments (2)
CHANGE OF NAME Recorded Jan 14, 2022
From: OXFORD NANOPORE TECHNOLOGIES LIMITED
To: OXFORD NANOPORE TECHNOLOGIES PLC
Reel/Frame 058737/0664 →
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Nov 16, 2020
From: HERON, ANDREW JOHN
To: OXFORD NANOPORE TECHNOLOGIES LIMITED
Reel/Frame 054372/0182 →
Priority Claims (1)
GB 1801768 · Feb 2, 2018 · national
Continuity (1)
Related Publication 20220177937A1 · Jun 9, 2022
References Cited (155)
US 5795782A · Church et al. · 1998 [cited by applicant]
US 6015714A · Baldarelli et al. · 2000 [cited by applicant]
US 6267872B1 · Akeson et al. · 2001 [cited by applicant]
US 6362002B1 · Denison et al. · 2002 [cited by applicant]
US 6464842B1 · Golovchenko et al. · 2002 [cited by applicant]
US 6627067B1 · Branton et al. · 2003 [cited by applicant]
US 6673615B2 · Denison et al. · 2004 [cited by applicant]
US 6746594B2 · Akeson et al. · 2004 [cited by applicant]
US 7060440B1 · Kless · 2006 [cited by applicant]
US 7253434B2 · Golovchenko et al. · 2007 [cited by applicant]
US 7258838B2 · Li et al. · 2007 [cited by applicant]
US 7466069B2 · Golovchenko et al. · 2008 [cited by applicant]
US 7468271B2 · Golovchenko et al. · 2008 [cited by applicant]
US 8653832B2 · Hadwen et al. · 2014 [cited by applicant]
US 8828336B2 · Hadwen et al. · 2014 [cited by applicant]
US 9222082B2 · Jayasinghe et al. · 2015 [cited by applicant]
US 20030104428A1 · Branton et al. · 2003 [cited by applicant]
US 20040001371A1 · Mansuripur et al. · 2004 [cited by applicant]
US 20040121525A1 · Chopra et al. · 2004 [cited by applicant]
US 20070004047A1 · Lee et al. · 2007 [cited by applicant]
US 20140197028A1 · Jacobs et al. · 2014 [cited by applicant]
US 20140202863A1 · Hadwen · 2014 [cited by applicant]
US 20140363852A1 · Efcavitch et al. · 2014 [cited by applicant]
US 20150344944A1 · Reid et al. · 2015 [cited by applicant]
US 20160046973A1 · Efcavitch et al. · 2016 [cited by applicant]
US 20160108382A1 · Efcavitch et al. · 2016 [cited by applicant]
US 20160168611A1 · Efcavitch et al. · 2016 [cited by applicant]
US 20220042967A1 · Brown et al. · 2022 [cited by applicant]
WO 2000028312A1 · 2000 [cited by applicant]
WO 2000079257A1 · 2000 [cited by applicant]
WO 2003003446A2 · 2003 [cited by applicant]
WO 2005061373A1 · 2005 [cited by applicant]
WO 2005124888A1 · 2005 [cited by applicant]
WO 2006028508A2 · 2006 [cited by applicant]
WO 2006100484A2 · 2006 [cited by applicant]
WO 2008102120A1 · 2008 [cited by applicant]
WO 2008102121A1 · 2008 [cited by applicant]
WO 2009020682A2 · 2009 [cited by applicant]
WO 2009035647A1 · 2009 [cited by applicant]
WO 2009077734A2 · 2009 [cited by applicant]
WO 2010004265A1 · 2010 [cited by applicant]
WO 2010004273A1 · 2010 [cited by applicant]
WO 2010086602A1 · 2010 [cited by applicant]
WO 2010086603A1 · 2010 [cited by applicant]
WO 2010109197A2 · 2010 [cited by applicant]
WO 2010122293A1 · 2010 [cited by applicant]
WO 2011046706A1 · 2011 [cited by applicant]
WO 2011067559A1 · 2011 [cited by applicant]
WO 2012005857A1 · 2012 [cited by applicant]
WO 2012033524A2 · 2012 [cited by applicant]
WO 2012042226A2 · 2012 [cited by applicant]
WO 2012107778A2 · 2012 [cited by applicant]
WO 2012164270A1 · 2012 [cited by applicant]
WO 2013057495A2 · 2013 [cited by applicant]
WO 2013083983A1 · 2013 [cited by applicant]
WO 2013098561A1 · 2013 [cited by applicant]
WO 2013098562A2 · 2013 [cited by applicant]
WO 2013153359A1 · 2013 [cited by applicant]
WO 2014013259A1 · 2014 [cited by applicant]
WO 2014013260A1 · 2014 [cited by applicant]
WO 2014013262A1 · 2014 [cited by applicant]
WO 2014064443A2 · 2014 [cited by applicant]
WO 2014064444A1 · 2014 [cited by applicant]
WO 2015055981A2 · 2015 [cited by applicant]
WO 2015110813A1 · 2015 [cited by applicant]
WO 2016028843A2 · 2016 [cited by applicant]
WO 2016034591A2 · 2016 [cited by applicant]
WO 2016034807A1 · 2016 [cited by applicant]
WO 2016055777A2 · 2016 [cited by applicant]
WO 2016059375A1 · 2016 [cited by applicant]
WO 2016059427A1 · 2016 [cited by applicant]
WO 2016128731A1 · 2016 [cited by applicant]
WO 2016139477A1 · 2016 [cited by applicant]
WO 2017009663A1 · 2017 [cited by applicant]
WO 2017149293A1 · 2017 [cited by applicant]
WO 2017149316A1 · 2017 [cited by applicant]
WO 2017149317A1 · 2017 [cited by applicant]
WO 2017149318A1 · 2017 [cited by applicant]
WO 2017151680A2 · 2017 [cited by applicant]
WO WO2017184677A1 · 2017 [cited by examiner]
WO 2017203268A1 · 2017 [cited by applicant]
WO 2017203269A1 · 2017 [cited by applicant]
WO 2018081745A1 · 2018 [cited by applicant]
WO 2018146491A1 · 2018 [cited by applicant]
WO 2018211241A1 · 2018 [cited by applicant]
WO 2018222853A1 · 2018 [cited by applicant]
WO 2019002893A1 · 2019 [cited by applicant]
WO 2019213437A1 · 2019 [cited by applicant]
International Preliminary Report on Patentability for Application No. PCT/GB2019/050296, mailed Aug. 13, 2020. [cited by applicant]
International Search Report and Written Opinion for Application No. PCT/GB2019/053669, mailed Apr. 14, 2020. [cited by applicant]
International Preliminary Report on Patentability for Application No. PCT/GB2019/053669, mailed Jul. 1, 2021. [cited by applicant]
Altschul et al., Basic local alignment search tool. J Mol Biol. Oct. 5, 1990;215(3):403-10. doi: 10.1016/S0022-2836(05)80360-2. [cited by applicant]
Altschul, A protein alignment scoring system sensitive at all evolutionary distances. J Mol Evol. Mar. 1993;36(3):290-300. doi: 10.1007/BF00160485. [cited by applicant]
Chari et al., Beyond editing to writing large genomes. Nat Rev Genet. Dec. 2017;18(12):749-760. doi: 10.1038/nrg.2017.59. Epub Aug. 30, 2017. Author Manuscript, 30 pages. [cited by applicant]
Chavis et al., Single Molecule Nanopore Spectrometry for Peptide Detection. ACS Sens. Sep. 22, 2017;2(9):1319-1328. doi: 10.1021/acssensors.7b00362. Epub Aug. 16, 2017. [cited by applicant]
Chen et al., Digital data storage using DNA nanostructures and solid-state nanopores. Nano letters. Dec. 26, 2018;19(2):1210-5. doi: 10.1021/acs.nanolett.8b04715. [cited by applicant]
Colas et al., Microscopical investigations of nisin-loaded nanoliposomes prepared by Mozafari method and their bacterial targeting. Micron. 2007;38(8):841-7. doi: 10.1016/j.micron.2007.06.013. Epub Jul. 3, 2007. [cited by applicant]
Crnkovic et al., Biological Nanopores: Engineering on Demand. Life (Basel). Jan. 5, 2021;11(1):27. doi: 10.3390/life11010027. [cited by applicant]
Devereux et al., A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res. Jan. 11, 1984; 12(1 Pt 1):387-95. doi: 10.1093/nar/12.1part1.387. [cited by applicant]
Eliseev et al., Molecular recognition of nucleotides, nucleosides, and sugars by aminocyclodextrins. Journal of the American Chemical Society. Jul. 1994;116(14):6081-8. [cited by applicant]
Haugland et al., Synthetically Diversified Protein Nanopores: Resolving Click Reaction Mechanisms. ACS Nano. Apr. 23, 2019;13(4):4101-4110. doi: 10.1021/acsnano.8b08691. Epub Mar. 18, 2019. [cited by applicant]
Holden et al., Direct introduction of single protein channels and pores into lipid bilayers. J Am Chem Soc. May 11, 2005;127(18):6502-3. doi: 10.1021/ja042470p. [cited by applicant]
Langecker et al., Synthetic lipid membrane channels formed by designed DNA nanostructures. Science. Nov. 16, 2012;338(6109):932-6. doi: 10.1126/science.1225624. [cited by applicant]
Mayer et al., An Epigenetics-Inspired DNA-Based Data Storage System. Angew Chem Int Ed Engl. Sep. 5, 2016;55(37):11144-8. doi: 10.1002/anie.201605531. Epub Jul. 21, 2016. [cited by applicant]
Nani et al., Near-IR Light-Mediated Cleavage of Antibody-Drug Conjugates Using Cyanine Photocages. Angew Chem Int Ed Engl. Nov. 9, 2015;54(46):13635-8. doi: 10.1002/anie.201507391. Epub Sep. 25, 2015. Author Manuscript,… [cited by applicant]
Reiner et al., Temperature sculpting in yoctoliter vols. J Am Chem Soc. Feb. 27, 2013;135(8):3087-94. doi: 10.1021/ja309892e. Epub Feb. 14, 2013. [cited by applicant]
Wang et al., The evolution of nanopore sequencing. Front Genet. Jan. 7, 2015;5:449. doi: 10.3389/fgene.2014.00449. [cited by applicant]
Yamazaki et al., Label-Free Single-Molecule Thermoscopy Using a Laser-Heated Nanopore. Nano Lett. Nov. 8, 2017;17(11):7067-7074. doi: 10.1021/acs.nanolett.7b03752. Epub Oct. 10, 2017. [cited by applicant]
PCT/GB2019/050296, Date of Mailing Apr. 15, 2019, International Search Report and Written Opinion. [cited by applicant]
GB 1801768.1, Date of Mailing Oct. 31, 2018, Combined Search and Examination Report. [cited by applicant]
Combined Search and Examination Report for Application No. GB1801768.1, mailed Oct. 31, 2018. [cited by applicant]
Astier et al., Toward single molecule DNA sequencing: direct identification of ribonucleoside and deoxyribonucleoside 5'-monophosphates by using an engineered protein nanopore equipped with a molecular adapter. J Am Che… [cited by applicant]
Bayley et al., Droplet interface bilayers. Mol Biosyst. Dec. 2008;4(12):1191-208. doi: 10.1039/b808893d. Epub Sep. 5, 2008. [cited by applicant]
Bayley et al., Stochastic sensors inspired by biology. Nature. Sep. 13, 2001;413(6852):226-30. doi: 10.1038/35093038. [cited by applicant]
Braha et al., Designed protein pores as components for biosensors. Chem Biol. Jul. 1997;4(7):497-505. doi: 10.1016/s1074-5521(97)90321-5. [cited by applicant]
Butler et al., Single-molecule DNA detection with an engineered MspA protein nanopore. Proc Natl Acad Sci U S A. Dec. 30, 2008;105(52):20647-52. doi: 10.1073/pnas.0807514106. Epub Dec. 19, 2008. [cited by applicant]
Chou et al., Recent Advances in Application of Droplet Microfluidics. Micromachines. 2015;6:1249-1271. doi: 10.3390/mi6091249. [cited by applicant]
Clarke et al., Continuous base identification for single-molecule nanopore DNA sequencing. Nat Nanotechnol. Apr. 2009;4(4):265-70. doi: 10.1038/nnano.2009.12. Epub Feb. 22, 2009. [cited by applicant]
David et al., Chemistry of Glycosylases and Endonucleases Involved in Base-Excision Repair. Chem Rev. May 7, 1998;98(3):1221-1262. doi: 10.1021/cr980321h. [cited by applicant]
Derrington et al., Nanopore DNA sequencing with MspA. Proc Natl Acad Sci U S A. Sep. 14, 2010;107(37):16060-5. doi: 10.1073/pnas.1001831107. Epub Aug. 26, 2010. [cited by applicant]
Farimani et al., DNA Origami-Graphene Hybrid Nanopore for DNA Detection. ACS Appl Mater Interfaces. Jan. 11, 2017;9(1):92-100. doi: 10.1021/acsami.6b11001. Epub Dec. 22, 2016. [cited by applicant]
Fuller et al., Real-time single-molecule electronic DNA sequencing by synthesis using polymer-tagged nucleotides on a nanopore array. Proc Natl Acad Sci U S A. May 10, 2016;113(19):5233-8. doi: 10.1073/pnas.1601782113. … [cited by applicant]
Georges et al., Novel Approach to the Ligation of Single-Stranded DNA Fragments by T4 Dna Ligase—DNA Mobile Multiple-Restriction Fragmens: “UNI-LINKERS” for Cloning of Genes. Nucleosides & Nucleotides. 1989;8(8):1427-14… [cited by applicant]
González-Pérez et al., Biomimetic triblock copolymer membrane arrays: a stable template for functional membrane proteins. Langmuir. Sep. 15, 2009;25(18):10447-50. doi: 10.1021/la902417m. [cited by applicant]
Haque et al., Solid-State and Biological Nanopore for Real-Time Sensing of Single Chemical and Sequencing of DNA. Nano Today. Feb. 2013;8(1):56-74. doi: 10.1016/j.nantod.2012.12.008. [cited by applicant]
Heron et al., Simultaneous measurement of ionic current and fluorescence from single protein pores. J Am Chem Soc. Feb. 11, 2009;131(5):1652-3. doi: 10.1021/ja808128s. [cited by applicant]
Howorka et al., Building membrane nanopores. Nat Nanotechnol. Jul. 6, 2017;12(7):619-630. doi: 10.1038/nnano.2017.99. [cited by applicant]
Ivanov et al., DNA tunneling detector embedded in a nanopore. Nano Lett. Jan. 12, 2011;11(1):279-85. doi: 10.1021/nl103873a. Epub Dec. 6, 2010. [cited by applicant]
Kocalka et al., Rapid and efficient DNA strand cross-linking by click chemistry. Chembiochem. May 23, 2008;9(8):1280-5. doi: 10.1002/cbic.200800006. [cited by applicant]
Kolb et al., Click Chemistry: Diverse Chemical Function from a Few Good Reactions. Angew Chem Int Ed Engl. Jun. 1, 2001;40(11):2004-2021. doi: 10.1002/1521-3773(20010601)40:11<2004::aid-anie2004>3.3.co;2-x. [cited by applicant]
Kuhn et al., Template-independent ligation of single-stranded DNA by T4 DNA ligase. FEBS J. Dec. 2005;272(23):5991-6000. doi: 10.1111/j.1742-4658.2005.04954.x. [cited by applicant]
Kumar et al., PEG-labeled nucleotides and nanopore detection for single molecule DNA sequencing by synthesis. Sci Rep. 2012;2:684. doi: 10.1038/srep00684. Epub Sep. 21, 2012. [cited by applicant]
Lieberman et al., Processive replication of single DNA molecules in a nanopore catalyzed by phi29 DNA polymerase. J Am Chem Soc. Dec. 22, 2010;132(50):17961-72. doi: 10.1021/ja1087612. Epub Dec. 1, 2010. [cited by applicant]
Luan et al., Base-by-base ratcheting of single stranded DNA through a solid-state nanopore. Phys Rev Lett. Jun. 11, 2010;104(23):238103. doi: 10.1103/PhysRevLett.104.238103. Epub Jun. 10, 2010. [cited by applicant]
Magierowski et al., Nanopore-CMOS Interfaces for DNA Sequencing. Biosensors (Basel). Aug. 6, 2016;6(3):42. doi: 10.3390/bios6030042. [cited by applicant]
Minhaz Ud-Dean, A theoretical model for template-free synthesis of long DNA sequence. Syst Synth Biol. Dec. 2008;2(3-4):67-73. doi: 10.1007/s11693-009-9023-x. Epub Apr. 3, 2009. [cited by applicant]
Montal et al., Formation of bimolecular membranes from lipid monolayers and a study of their electrical properties. Proc Natl Acad Sci U S A. Dec. 1972;69(12):3561-6. doi: 10.1073/pnas.69.12.3561. [cited by applicant]
Motea et al., Terminal deoxynucleotidyl transferase: the story of a misguided DNA polymerase. Biochim Biophys Acta. May 2010;1804(5):1151-66. doi: 10.1016/j.bbapap.2009.06.030. Epub Jul. 29, 2009. [cited by applicant]
Nichols et al., RNA ligases. Curr Protoc Mol Biol. Oct. 2008;Chapter 3:Unit3.15. doi: 10.1002/0471142727.mb0315s84. [cited by applicant]
Pavelka et al., A new technology for the synthesis of long DNA strings. Int J Nano Med & Eng. 2017;2(7):127-130. doi: 10.25141/2474-8811-2017-7.0127. [cited by applicant]
Ponferrada-Marín et al., ROS1 5-methylcytosine DNA glycosylase is a slow-turnover catalyst that initiates DNA demethylation in a distributive fashion. Nucleic Acids Res. Jul. 2009;37(13):4264-74. doi: 10.1093/nar/gkp390… [cited by applicant]
Ramadan et al., De novo DNA synthesis by human DNA polymerase λ, DNA polymerase μ and terminal deoxyribonucleotidyl transferase. J Mol Biol. May 28, 2004;339(2):395-404. doi: 10.1016/j.jmb.2004.03.056. [cited by applicant]
Roy et al., Synthesis of DNA/RNA and their analogs via phosphoramidite and H-phosphonate chemistries. Molecules. Nov. 18, 2013;18(11):14268-84. doi: 10.3390/molecules181114268. [cited by applicant]
Soni et al., Synchronous optical and electrical detection of biomolecules traversing through solid-state nanopores. Rev Sci Instrum. Jan. 2010;81(1):014301. doi: 10.1063/1.3277116. [cited by applicant]
Stoddart et al., Multiple base-recognition sites in a biological nanopore: two heads are better than one. Angew Chem Int Ed Engl. 2010;49(3):556-9. doi: 10.1002/anie.200905483. [cited by applicant]
Stoddart et al., Nucleobase recognition in ssDNA at the central constriction of the alpha-hemolysin pore. Nano Lett. Sep. 8, 2010;10(9):3633-7. doi: 10.1021/nl101955a. [cited by applicant]
Stoddart et al., Single-nucleotide discrimination in immobilized DNA oligonucleotides with a biological nanopore. Proc Natl Acad Sci U S A. May 12, 2009;106(19):7702-7. doi: 10.1073/pnas.0901054106. Epub Apr. 20, 2009. [cited by applicant]
Stranges et al., Design and characterization of a nanopore-coupled polymerase for single-molecule DNA sequencing by synthesis on an electrode array. Proc Natl Acad Sci U S A. Nov. 1, 2016;113(44):E6749-E6756. doi: 10.10… [cited by applicant]
International Search Report and Written Opinion for Application No. PCT/GB2019/050296, mailed Apr. 15, 2019. [cited by applicant]
Biswas et al., Click Addition of a DNA Thread to the N-termini of Peptides for Their Translocation through Solid-State Nanopores. ACS Nano. Oct. 27, 2015; 9(10): 9652-9664. EPub Sep. 16, 2015. doi: 10.1021/acsnano.5b049… [cited by applicant]
Carson et al., Challenges in DNA motion control and sequence readout using nanopore devices. Nanotechnology. Feb. 20, 2015;26(7):074004. doi: 10.1088/0957-4484/26/7/074004. Epub Feb. 2, 2015. [cited by applicant]
Feng et al., Nanopore-based fourth-generation DNA sequencing technology. Genomics Proteomics Bioinformatics. Feb. 2015;13(1):4-16. doi: 10.1016/j.gpb.2015.01.009. Epub Mar. 2, 2015. Erratum in: Genomics Proteomics Bioin… [cited by applicant]
Kosuri et al., Large-scale de novo DNA synthesis: technologies and applications. Nat Methods. May 2014;11(5):499-507. doi: 10.1038/nmeth.2918. [cited by applicant]
Korlach et al., Long, processive enzymatic DNA synthesis using 100% dye-labeled terminal phosphate-linked nucleotides. Nucleosides Nucleotides Nucleic Acids. Sep. 2008;27(9):1072-83. doi: 10.1080/15257770802260741. [cited by applicant]
Sun et al., Synthesis of Fluorescent Metal Nanoclusters and Their Biomedical Applications. Journal of Instrumental Analysis. Oct. 2018;37(10):1119-1129. [cited by applicant]