IP Library › Granted Patent US 12,371,743
Granted Patent B2
US 12,371,743 · App. 17/725,065 · Granted Jul 29, 2025

Double-stranded splint adaptors and methods of use

Inventors: William Light (Poway, CA); Samantha Snow (San Diego, CA); Junhua Zhao (San Diego, CA)
Assignee: Element Biosciences, Inc.
C12Q1/6869C12N15/1065C12N15/1068C12Q1/682C12Q1/6855
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Quick Facts
Patent No.
US 12,371,743
App. No.
17/725,065
Granted
Jul 29, 2025
Kind
B2
Abstract

The present disclosure provides compositions comprising nucleic acid double-stranded splint adaptors, including kits, and methods that employ the double-stranded splint adaptors. The double-stranded splint adaptors ( 200 ) can be used in a one-pot, multi-enzyme reaction to introduce one or more new adaptor sequences into a library molecule. The double-stranded splint adaptor ( 200 ) comprises a first splint strand (long splint strand ( 300 )) and a second splint strand (short splint strand ( 400 )), where the first and second splint strands are hybridized together to form the double-stranded splint adaptor ( 200 ) having a double-stranded region and two flanking single-stranded regions. The second splint strand ( 400 ) carries the new adaptor sequence(s) to be introduced, such as for example a universal binding sequence and/or an index sequence.

Claims (43)

1. A method for forming a plurality of library-splint complexes ( 500 ) on a support, the method comprising:

a) providing a plurality of double-stranded splint adaptors ( 200 ), wherein individual double-stranded splint adaptors ( 200 ) comprise a first splint strand ( 300 ) hybridized to a second splint strand ( 400 ), wherein the first splint strand ( 300 ) comprises regions arranged in a 5′ to 3′ order: (i) a first region ( 320 ), (ii) an internal region ( 310 ), and (iii) a second region ( 330 ), and wherein the internal region of the first splint strand ( 310 ) is hybridized to the second splint strand ( 400 ), wherein the second splint strand comprises regions arranged in a 5′ to 3′ order: (i) a second sub-region having a universal binding sequence for a first surface primer, and (ii) a first sub-region having a universal binding sequence for a second surface primer; and

b) hybridizing the plurality of double-stranded splint adaptors with a plurality of single-stranded nucleic acid library molecules ( 100 ), wherein individual library molecules comprise regions arranged in a 5′ to 3′ order: (i) a first left universal adaptor sequence ( 120 ) having a binding sequence for a third surface primer; (ii) a second left universal adaptor sequence ( 140 ) having a binding sequence for a first sequencing primer; (iii) a sequence of interest ( 110 ); (iv) a second right universal adaptor sequence ( 150 ) having a binding sequence for a second sequencing primer; and (v) a first right universal adaptor sequence ( 130 ) having a binding sequence for a fourth surface primer,

wherein portions of the first splint strand ( 300 ) hybridize to portions of the library molecule ( 100 ) thereby circularizing the library molecule to generate a library-splint complex ( 500 ), such that the first region ( 320 ) of the first splint strand is hybridized to the binding sequence for the third surface primer ( 120 ), and the second region ( 330 ) of the first splint strand is hybridized to the binding sequence for the fourth surface primer ( 130 ), wherein the library-splint complex ( 500 ) comprises a first nick between the 5′ end of the library molecule and the 3′ end of the second splint strand ( 400 ), wherein the library-splint complex ( 500 ) comprises a second nick between the 5′ end of the second splint strand ( 400 ) and the 3′ end of the library molecule ( 100 ), and wherein the first and second nicks are enzymatically ligated to generate a plurality of covalently closed circular library molecules ( 600 ); and

c) distributing the plurality of covalently closed circular library molecules ( 600 ) onto a support having a plurality of the second surface primers and first surface primers immobilized on the support, wherein individual covalently closed circular library molecules ( 600 ) hybridize to individual second surface primers and first surface primers thereby immobilizing the plurality of covalently closed circular library molecules ( 600 ) to the support,

and wherein the plurality of second surface primers and first surface primers on the support are located at pre-determined or random locations on the support.

2. The method of claim 1 , wherein the plurality of single-stranded nucleic acid library molecules ( 100 ) further comprises a first left index sequence ( 160 ) and/or a first right index sequence ( 170 ).

3. The method of claim 1 , where wherein the plurality of single-stranded nucleic acid library molecules ( 100 ) further comprises a first left unique identification sequence ( 180 ) and/or a first right unique identification sequence ( 190 ).

4. The method of claim 1 , wherein the plurality of covalently closed circular library molecules ( 600 ) of b) are each hybridized to the first splint strand ( 300 ).

5. The method of claim 4 , further comprising: contacting the plurality of covalently closed circular library molecules ( 600 ) of b) with at least one exonuclease enzyme to remove the plurality of first splint strands ( 300 ) and retaining the plurality of covalently closed circular library molecules ( 600 ).

6. The method of claim 1 , further comprising: d) contacting the plurality of covalently closed circular library molecules ( 600 ) immobilized on the support with a plurality of strand-displacing polymerases and a plurality of nucleotides, and conducting a rolling circle amplification reaction on the support using the plurality of second surface primers as immobilized amplification primers and the plurality of covalently closed circular library molecules ( 600 ) as template molecules, thereby generating a plurality of immobilized nucleic acid concatemer molecules.

7. The method of claim 6 , wherein the plurality of nucleotides comprises dATP, dGTP, dCTP, dTTP and/or dUTP.

8. The method of claim 6 , wherein the plurality of second surface primers and first surface primers on the support are in fluid communication with each other to permit flowing a solution of reagents onto the support so that the plurality of second surface primers can be simultaneously reacted with the reagents in a massively parallel manner, optionally wherein the solution of reagents comprises enzymes, nucleotides and divalent cations.

9. The method of claim 6 , wherein the density of the plurality of immobilized nucleic acid concatemer molecules on the support is 10 4 -10 8 per mm 2 .

10. The method of claim 6 , further comprising: sequencing the plurality of immobilized nucleic acid concatemer molecules, wherein the sequencing comprises:

a) contacting the plurality of immobilized nucleic acid concatemer molecules with (i) a plurality of sequencing polymerases and (ii) a plurality of the soluble sequencing primers, thereby forming a plurality of complexed polymerases comprising a sequencing polymerase bound to a nucleic acid duplex, wherein the nucleic acid duplex comprises a concatemer molecule hybridized to a soluble sequencing primer;

b) contacting the plurality of complexed sequencing polymerases with a plurality of nucleotides and binding at least one nucleotide to a complexed sequencing polymerase, wherein the plurality of nucleotides comprises at least one nucleotide analog labeled with a fluorophore and having a removable chain terminating moiety at the sugar 3′ position;

c) incorporating at least one nucleotide into the 3′ end of the hybridized sequencing primers thereby generating a plurality of nascent extended sequencing primers; and

d) detecting the incorporated nucleotide and identifying the nucleo-base of the incorporated nucleotide.

11. The method of claim 10 , wherein the plurality of nucleotides comprises a removable chain terminating moiety at the 3′ sugar group, wherein the removable chain terminating moiety comprises an alkyl group, alkenyl group, alkynyl group, allyl group, aryl group, benzyl group, azide group, azido group, O-azidomethyl group, amine group, amide group, keto group, isocyanate group, phosphate group, thio group, disulfide group, carbonate group, urea group, or silyl group, and wherein the removable chain terminating moiety is cleavable with a chemical compound to generate an extendible 3′OH moiety on the sugar group.

12. The method of claim 10 , wherein the plurality of nucleotides comprises one type of nucleotide selected from a group consisting of dATP, dGTP, dCTP, dTTP and dUTP.

13. The method of claim 10 , wherein the plurality of nucleotides comprises a mixture of any combination of two or more types of nucleotides selected from a group consisting of dATP, dGTP, dCTP, dTTP and/or dUTP.

14. The method of claim 6 , further comprising: sequencing the plurality of immobilized nucleic acid concatemer molecules, wherein the sequencing comprises:

a) contacting the plurality of immobilized nucleic acid concatemer molecules with (i) a plurality of sequencing polymerases and (ii) a plurality of soluble universal sequencing primers, thereby forming a plurality of first complexed polymerases comprising a sequencing polymerase bound to a nucleic acid duplex, wherein the nucleic acid duplex comprises a concatemer molecule hybridized to a soluble sequencing primer;

b) contacting the plurality of first complexed sequencing polymerases with a plurality of detectably labeled multivalent molecules to form a plurality of multivalent-complexed polymerases, wherein complementary nucleotide units of the multivalent molecules are bound to at least two of the plurality of first complexed polymerases, thereby forming a plurality of multivalent-complexed polymerases, wherein incorporation of the complementary nucleotide units into the sequencing primers of the plurality of multivalent-complexed polymerases is inhibited, and wherein individual multivalent molecules in the plurality of multivalent molecules comprise a core attached to multiple nucleotide arms and each nucleotide arm is attached to a nucleotide unit; and

c) detecting the plurality of multivalent-complexed polymerases.

15. The method of claim 14 , further comprising:

d) dissociating the plurality of multivalent-complexed polymerases and removing the plurality of first sequencing polymerases and their bound multivalent molecules, and retaining the plurality of nucleic acid duplexes;

e) contacting the plurality of the retained nucleic acid duplexes of step d) with a plurality of second sequencing polymerases, and binding the plurality of second sequencing polymerases to the plurality of the retained nucleic acid duplexes, thereby forming a plurality of second complexed polymerases comprising a second sequencing polymerase bound to a nucleic acid duplex;

f) contacting the plurality of second complexed polymerases with a plurality of nucleotides comprising at least one nucleotide analog having a removable chain terminating moiety at the sugar 3′ position, wherein complementary nucleotides from the plurality of nucleotides are bound to at least two of the second complexed polymerases of step e) thereby forming a plurality of nucleotide-complexed polymerases, and wherein the complementary nucleotides are incorporated into the sequencing primers of the nucleotide-complexed polymerases.

16. The method of claim 15 , further comprising:

g) detecting the complementary nucleotides which are incorporated into the sequencing primers of the nucleotide-complexed polymerases.

17. The method of claim 15 , further comprising:

g) detecting the complementary nucleotides which are incorporated into the sequencing primers of the nucleotide-complexed polymerases; and

h) identifying the nucleo-bases of the complementary nucleotides which are incorporated into the sequencing primers of the nucleotide-complexed polymerases.

18. The method of claim 15 , wherein the plurality of nucleotides comprises a removable chain terminating moiety at the 3′ sugar group, wherein the removable chain terminating moiety comprises an alkyl group, alkenyl group, alkynyl group, allyl group, aryl group, benzyl group, azide group, azido group, O-azidomethyl group, amine group, amide group, keto group, isocyanate group, phosphate group, thio group, disulfide group, carbonate group, urea group, or silyl group, and wherein the removable chain terminating moiety is cleavable with a chemical compound to generate an extendible 3′OH moiety on the sugar group.

19. The method of claim 15 , wherein the plurality of nucleotides comprises one type of nucleotide selected from the group consisting of dATP, dGTP, dCTP, dTTP and dUTP.

20. The method of claim 15 , wherein the plurality of nucleotides comprises a mixture of any combination of two or more types of nucleotides selected from the group consisting of dATP, dGTP, dCTP, dTTP and dUTP.

21. The method of claim 14 , wherein the plurality of nucleotide arms attached to cores of the individual multivalent molecules has the same type of a nucleotide unit, and wherein the type of nucleotide unit is selected from the group consisting of dATP, dGTP, dCTP, dTTP and dUTP.

22. The method of claim 14 , wherein the plurality of multivalent molecules comprises a mixture of any combination of two or more types of multivalent molecules, each type having nucleotide units selected from the group consisting of dATP, dGTP, dCTP, dTTP and dUTP.

23. The method of claim 1 , wherein the support comprises a glass or plastic substrate.

24. The method of claim 1 , wherein the support is passivated with at least one hydrophilic polymer coating having a water contact angle of no more than 45 degrees.

25. The method of claim 24 , wherein the at least one hydrophilic polymer coating comprises a molecule selected from the group consisting of polyethylene glycol (PEG), poly(vinyl alcohol) (PVA), poly(vinyl pyridine), poly(vinyl pyrrolidone) (PVP), poly(acrylic acid) (PAA), polyacrylamide, poly(N-isopropylacrylamide) (PNIPAM), poly(methyl methacrylate) (PMA), poly(2-hydroxylethyl methacrylate) (PHEMA), poly(oligo(ethylene glycol) methyl ether methacrylate) (POEGMA), polyglutamic acid (PGA), poly-lysine, poly-glucoside, streptavidin, and dextran.

Assignments (1)
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Jul 26, 2023
From: LIGHT, WILLIAM; SNOW, SAMANTHA; ZHAO, JUNHUA
To: ELEMENT BIOSCIENCES, INC.
Reel/Frame 064385/0948 →
Continuity (2)
Provisional Application 63316784 · Mar 4, 2022
Related Publication 20230279483A1 · Sep 7, 2023
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Anderson, J.P. et al.; Fluorescent Structural DNA Nanoballs Functionalized with Phosphate- Linked Nucleotide Triphosphates. Nano Letters 10(3):788-792 (2010). [cited by applicant]
Balakrishnan, L., et al.; “Flap Endonuclease 1,” Annual Review Biochemistry 82:119-138 (2013). [cited by applicant]
Chen, X., et al.; “Efficient in situ barcode sequencing using padlock probe-based BaristaSeq,” Nucleic Acids Research; 46(4):e22 pp. 1-10 (2018). [cited by applicant]
Ericsson, O., et al.; “A dual-tag microarray platform for high-performance nucleic acid and protein analyses,” Nucleic Acids Research, 36(8):e45, pp. 1-9 (2008). [cited by applicant]
Friedrich-Heineken, E., et al.; “The Fen1 extrahelical 3'-flap pocket is conserved from archaea to human and regulates DNA substrate specificity,” Nucleic Acids Research, 32(8):2520-2528 (2004). [cited by applicant]
GenBank Accession AAB52611.1; “Dna polymerase | [Geobacillus stearothermophilus],” Apr. 21, 1997; [retrieved online Sep. 23, 2024] URL: https://www.ncbi.nlm.nih.gov/protein/AAB52611.1, 2 pages. [cited by applicant]
GenBank Accession KUO42443.1; “MAG: hypothetical protein APZ16_03045 [Candidatus Hadarchaeum yellowstonense],” Jan. 14, 2016; [retrieved online Sep. 23, 2024] URL: https://www.ncbi.nlm.nih.gov/protein/KUO42443.1, 2 page… [cited by applicant]
GenBank Accession MBC7218772.1; “MAG: DNA polymerase [Hadesarchaea archaeon],” Sep. 1, 2020; [retrieved online Sep. 23, 2024] URL: https://www.ncbi.nlm.nih.gov/protein/mbc7218772.1, 2 pages. [cited by applicant]
GenBank Accession NOZ58130.1; “MAG: DNA polymerase [Euryarchaeota archaeon],” Mar. 17, 2023 [retrieved online Sep. 23, 2024] URL: https://www.ncbi.nlm.nih.gov/protein/NOZ58130, 2 pages. [cited by applicant]
GenBank Accession NOZ77387.1; “MAG: DNA polymerase, partial [Euryarchaeota archaeon],” Mar. 17, 2023 [retrieved online Sep. 23, 2024] URL: https://www.ncbi.nlm.nih.gov/protein/NOZ77387.1, 2 pages. [cited by applicant]
GenBank Accession RLF78286.1; “MAG: DNA polymerase [Thermococci archaeon],” Oct. 15, 2018; URL: https://www.ncbi.nlm.nih.gov/protein/RLF78286.1, 2 pages. [cited by applicant]
GenBank Accession RLF89458.1; “MAG: DNA polymerase [Thermococci archaeon],” Oct. 15, 2018; [retrieved online Sep. 23, 2024] URL: https://www.ncbi.nlm.nih.gov/protein/RLF89458.1, 2 pages. [cited by applicant]
GenBank Accession RLI89578.1; “MAG: DNA polymerase [Candidatus Altiarchaeales archaeon],” Oct. 15, 2018; [retrieved online Sep. 23, 2024] URL: https://www.ncbi.nlm.nih.gov/protein/RLI89578.1, 2 pages. [cited by applicant]
GenBank Accession RMF90817.1; “MAG: DNA polymerase [Euryarchaeota archaeon],” Oct. 29, 2018; [retrieved online Sep. 23, 2024] URL: https://www.ncbi.nlm.nih.gov/protein/RMF90817.1, 2 pages. [cited by applicant]
Greenough, L., et al.; “Adapting capillary gel electrophoresis as a sensitive, high- throughput method to accelerate characterization of nucleic acid metabolic enzymes,” Nucleic Acids Research, 44(2):e15, pp. 1-11 (2016… [cited by applicant]
Hatch, A., et al.; “Rolling circle amplification of DNA immobilized on solid surfaces and its application to multiplex mutation detection,” Genetic Analysis: Biomolecular Engineering, 15(2):35-40 (1999). [cited by applicant]
Illumina: Illumina Adapter Sequences guide (Oct. 2015), 34 pages. [cited by applicant]
Kao, H-I, et al.; “Cleavage specificity of Saccharomyces cerevisiae flap endonuclease 1 suggests a double-flap structure as the cellular substrate,” Journal of Biological Chemistry, 277(17):14379-14389 (2002). [cited by applicant]
Konry, T., et al.; “Microsphere-based rolling circle amplification microarray for the detection of DNA and proteins in a single assay,” Analytical Chemistry, 81(14):5777-5782 (2009). [cited by applicant]
Lee, B-I., et al.; “The RAD2 domain of human exonuclease 1 exhibits 5' to 3' exonuclease and flap structure-specific endonuclease activities,” Journal of Biological Chemistry, 274(53):37763-37769 (1999). [cited by applicant]
Lee, J., et al.; “Diffractometric detection of proteins using microbead-based rolling circle amplification,” Analytical Chemistry, 82(1):197-202 (2010). [cited by applicant]
Lin, T., et al.; “Biochemical characterization and mutational analysis of a novel flap endonuclease 1 from Thermococcus barophilus Ch5,” International Journal of Biochemistry and Cell Biology, 143:106154, pp. 1-11 (2022… [cited by applicant]
Lu, M., et al.; “A surface invasive cleavage assay for highly parallel SNP analysis,” Human Mutation, 19(4):416-422 (2002). [cited by applicant]
Mignardi, M., et al.; “Fourth-generation sequencing in the cell and the clinic,” Genome Med.; 6(4):31; pp. 1-4 (2014). [cited by applicant]
NCBI Reference Sequence: NP_041963.1; Accession NC_001604.1; “DNA ligase [Escherichia phage T7],” Jan. 7, 2023; [retrieved online Sep. 23, 2024] URL: https://www.ncbi.nlm.nih.gov/protein/9627435, 3 pages. [cited by applicant]
NCBI Reference Sequence: NP_049813.1; Accession NC_000866.4; “DNA ligase [Escherichia phage T4],” Jan. 11, 2023; [retrieved online Sep. 23, 2024] URL: https://www.ncbi.nlm.nih.gov/protein/9632609, 2 pages. [cited by applicant]
NCBI Reference Sequence: NP_523305.1; Accession NC_003298.1; “DNA ligase [Enterobacteria phage T3],” Jan. 7, 2023; [retrieved online Sep. 23, 2024] URL: https://www.ncbi.nlm.nih.gov/protein/17570796, 2 pages. [cited by applicant]
NCBI Reference Sequence: WP_042693257.1; Accession WP_042693257; “ATP-dependent DNA ligase [Thermococcus nautili],” Jun. 2, 2024; [retrieved online Sep. 23, 2024] URL: https://www.ncbi.nlm.nih.gov/protein/757139009, 1 p… [cited by applicant]
NCBI Reference Sequence: WP_175059460.1; Accession WP_175059460; “DNA-directed DNA polymerase [Thermococcus sp. 2319x1],” May 21, 2021; [retrieved online Sep. 23, 2024] URL: https://www.ncbi.nlm.nih.gov/protein/WP_17505… [cited by applicant]
Russell, C., et al.; “Gold nanowire based electrical DNA detection using rolling circle amplification,” ACS Nano, 8(2):1147-1153 (2014). [cited by applicant]
Stougaard, M., et al.; “In situ detection of non-polyadenylated RNA molecules using Turtle Probes and target primed rolling circle PRINS,” BMC Biotechnology, 7:69, pp. 1-10 (2007). [cited by applicant]
Tsutakawa, S.E., et al.; “Human flap endonuclease structures, DNA double-base flipping, and a unified understanding of the FEN1 superfamily,” Cell., 145(2):198-211, with supplemental pages S1-S7, 21 pages (2011). [cited by applicant]
Tsutakawa, S.E., et al.; “Phosphate steering by Flap Endonuclease 1 promotes 5'-flap specificity and incision to prevent genome instability,” Nature Communications, 8:15855, pp. 1-15 (2017). [cited by applicant]
UniProtKB: POCL77—Dpol_PYRAB; “DNA polymerase 1,” Last Updated: Apr. 5, 2011; [retrieved online Sep. 23, 2024] URL: https://www.uniprot.org/uniprotkb/P0CL77/entry, 5 pages. [cited by applicant]
UniProtKB: P30317—DPOL_THELI; “DNA polymerase,” Last Updated: Apr. 1, 1993 [retrieved online Sep. 23, 2024] URL: https://www.uniprot.org/uniprotkb/P30317/entry, 6 pages. [cited by applicant]
UniProtKB: P61875—DPOL_PYRFU; “DNA polymerase,” Last Updated: Jun. 7, 2004 [retrieved online Sep. 23, 2024] URL: https://www.uniprot.org/uniprotkb/P61875/entry, 6 pages. [cited by applicant]
UniProtKB: Q38087—DPOL_BPR69; “DNA-directed DNA polymerase,” Last Updated: Nov. 1, 1996 [retrieved online Sep. 23, 2024] URL: https://www.uniprot.org/uniprotkb/Q38087/entry, 6 pages. [cited by applicant]
UniProtKB: Q51334—DPOL_PYRSD; “DNA polymerase,” Last Updated: Nov. 1, 1996 [retrieved online Sep. 23, 2024] URL: https://www.uniprot.org/uniprotkb/Q51334/entry, 6 pages. [cited by applicant]
UniProtKB: Q56366—DPOL_THES9; “DNA polymerase,” Last Updated: Nov. 1, 1996 [retrieved online Sep. 23, 2024] URL: https://www.uniprot.org/uniprotkb/Q56366/entry, 7 pages. [cited by applicant]
UniProtKB/Swiss-Prot: P03680.1—DPOL_BPPH2; “DNA polymerase,” Last Updated: Jul. 21, 1986; [retrieved online Sep. 23, 2024] URL: https://www.uniprot.org/uniprotkb/P03680/entry, 7 pages. [cited by applicant]
UniProtKB/Swiss-Prot: Q9HH07.1; “RecName: Full=DNA ligase; AltName: Full=Polydeoxyribonucleotide synthase [ATP/NAD(+)],” Date updated: Mar. 1, 2001; [retrieved online Sep. 20, 2024] URL: https://www.ncbi.nlm.nih.gov/pro… [cited by applicant]
U.S. Appl. No. 17/554,396, filed Dec. 17, 2021, by Sinan Arslan, et al. [cited by applicant]
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U.S. Appl. No. 18/824,527, filed Sep. 4, 2024, by Sinan Arslan, et al. [cited by applicant]
Gao, H., et al.; “Rolling circle amplification for single cell analysis and in situ sequencing,” TrAC Trends in Analytical Chemistry; 121:115700; pp. 1-13 (2019). [cited by applicant]
Hu, T., et al.; “Next-generation sequencing technologies: An overview,” Hum Immunol .; 82(11):801-811 (2021). [cited by applicant]
Illumina “Overview of Illumina Sequencing by Synthesis Workflow,” Oct. 5, 2016 (Oct. 5, 2016) [retrieved online Oct. 9, 2024] https://www.youtube.com/watch?v=fCd6B5HRaZ8, 2 pages. [cited by applicant]
Pettersson, E., et al.; “Generations of sequencing technologies,” Genomics 93(2):105-111 (2009). [cited by applicant]
Schlecht, U., et al.; “ConcatSeq: A method for increasing throughput of single molecule sequencing by concatenating short DNA fragments,” Sci Rep; 7(1):5252; pp. 1-10 (2017). [cited by applicant]
Ulahannan, N., et al.; “Nanopore sequencing of DNA concatemers reveals higher- order features of chromatin structure,” bioRxiv, Nov. 7, 2019 [retrieved on Oct. 9, 2024] https://www.biorxiv.org/content/10.1101/833590v1.f… [cited by applicant]
Ohtsubo, Y., et al.; “Efficient N-tailing of blunt DNA ends by Moloney murine leukemia virus reverse transcriptase,” Sci Rep.; 7:41769; pp. 1-10; doi: 10.1038/srep41769 (2017). [cited by applicant]
Ohtsubo, Y., et al.; “Optimization of single strand DNA incorporation reaction by Moloney murine leukaemia virus reverse transcriptase,” DNA Res.; 25(5):477-487 (2018). [cited by applicant]
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US 12,606,819