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WO2017139669A1 - Processus de modification d'un acide nucléique par voie enzymatique - Google Patents

Processus de modification d'un acide nucléique par voie enzymatique Download PDF

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Publication number
WO2017139669A1
WO2017139669A1 PCT/US2017/017508 US2017017508W WO2017139669A1 WO 2017139669 A1 WO2017139669 A1 WO 2017139669A1 US 2017017508 W US2017017508 W US 2017017508W WO 2017139669 A1 WO2017139669 A1 WO 2017139669A1
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WIPO (PCT)
Prior art keywords
reagent
nucleic acid
protease
subsequent
inhibitor
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PCT/US2017/017508
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English (en)
Inventor
Ezra Abrams
Danny YUN
T. Christian Boles
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Sage Science, Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
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Publication date
Application filed by Sage Science, Inc. filed Critical Sage Science, Inc.
Publication of WO2017139669A1 publication Critical patent/WO2017139669A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/99—Enzyme inactivation by chemical treatment
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • C12Q1/37—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving peptidase or proteinase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay

Definitions

  • Embodiments of the present disclosure seek to simplify multi-step molecular biology workflows by further reducing the number of DNA purification steps to improve reproducibility and enable the process more amenable to automation.
  • Methods disclosed herein may include providing a nucleic acid sample.
  • the sample may be treated by applying a first enzymatic nucleic acid modifying reagent to produce a first nucleic acid solution.
  • a first protease reagent may be applied to the first nucleic acid solution to produce a second nucleic acid solution.
  • Application of the first protease reagent may completely or substantially inactivate the first enzymatic nucleic acid modifying agent.
  • a first inhibitor reagent may be applied to produce a third nucleic acid solution, and the first inhibitor reagent may completely or substantially inactivate the first protease reagent.
  • the first nucleic acid may not be purified. In other embodiments, the nucleic acid sample, the first nucleic acid solution, and the second nucleic acid solution are not purified.
  • Chemicals may be added to improve accessibility of the enzyme reagent and the protease reagent.
  • the chemicals may be detergents and/or chaotropes.
  • the reaction caused by the enzymatic reagent may be terminated.
  • This termination may include adding a second protease to digest the enzymatic reagents to produce a reaction.
  • the reaction may be incubated for a time and at a temperature, after which an amount of protease inhibitor may be added.
  • the amount of protease may be determined such the activity of the protease is terminated.
  • the time and temperature of the incubation of the reaction may be determined based on digestion of the enzymatic reagents.
  • Temperature may be varied at various stages.
  • the temperature of the nucleic acid sample may be varied when applying the first modifying reagent.
  • the temperature of the first nucleic acid solution may be varied when the second modifying reagent is applied.
  • the method may further comprise applying at least one subsequent enzymatic nucleic acid modifying reagent. At least one subsequent protease reagent may also be applied, and the application of the subsequent protease reagent(s) may completely or substantially inactivate the subsequent enzymatic nucleic acid modifying reagent(s). At least one subsequent inhibitor reagent may also be applied. Each subsequent inhibitor reagent may be orthogonal to the applied subsequent protease(s), such that the subsequent inhibitor reagent completely or substantially inactivates the subsequent protease agent. The application of each subsequent inhibitor may not inhibit a new dose of protease that is used subsequently.
  • FIGURE 1 shows an image of ethidium stained gel according to Example 1.
  • FIGURE 2 shows an image of ethidium stained gel according to Example 2.
  • the nucleic acid modifications are carried out in a defined order to obtain the desired final molecular construction.
  • a first modifying reagent enzyme or chemical reagent
  • the workflow can be regulated by staged addition of reagents.
  • T4 DNA ligase binds tightly to DNA at ends and nicks, and will block DNA processing by exonucleases and other enzymes.
  • Tn5 transposases used in molecular biology, such as Tn5 (Nextera reagent, Illumina, Inc.) remain tightly bound to DNA after their reaction and can block the action of DNA polymerases. Tn5 transposase needs heat, detergent, or protease treatment to be released from its transposition target DNA.
  • Adey et al. Genome Res., 24(12):2041-2049, Dec 2014.
  • PMC4248320 [DOI: 10.1101/gr. l78319.114]
  • an enzymatic modification step such as, for instance, adapter ligation
  • a purified protease e.g., trypsin
  • protease e.g., trypsin
  • a specific inhibitor e.g., bovine trypsin inhibitor or the Kunitz soybean trypsin inhibitor in the case of trypsin.
  • the same protease inhibitor combination may be used in both steps provided that (in some embodiments) the protease used in the earlier step is completely inactivated (and in some embodiments, substantially inactivated) by the inhibitor added to terminate that step, and provided that the amount of protease used to terminate the later step is sufficiently large to overcome any residual inhibitor used in the earlier step.
  • protease-inhibitor combination may be used at each process step. For instance, trypsin and bovine trypsin inhibitor might be used to terminate one reaction step, while papain and antipain might be used to terminate a different reaction step.
  • protease inhibitor reagents may include protease inhibitors of many types, such as inhibitory peptides, small molecule inhibitors, naturally occurring inhibitory proteins, antibodies, recombinant protein inhibitors, and nucleic acid aptamers.
  • the temperature of the protease reaction may be varied to allow better digestion of the reagent enzyme by the protease.
  • higher temperatures loosen up the structure of proteins and make them more accessible to protease cleavage.
  • useful temperatures can range up to 95°C, but the optimum temperature for each protease and reagent enzyme pair may be determined empirically under the specific process conditions to be employed.
  • a temperature range of 25°C to 60°C is adequate for accelerating digestion of many enzyme reagents, while avoiding denaturation of the protease.
  • reagents such as detergents or chaotropes
  • the addition of such reagents may be optimized in order to prevent adverse effects on the enzyme reagents to be used in later process steps.
  • reagent kits may comprise enzymatic reagents optimized for a specific molecular biology process. Such kits may also include protease/inhibitor reagent combinations, where the protease/inhibitor reagents are used to terminate specific enzymatic steps in the process.
  • termination of specific enzymatic steps are carried out by:
  • An advantage of some of the embodiments disclosed herein is that they enable many types of multi-step enzymatic processes to be performed by addition of reagents to a reaction mixture without the need to purify nucleic acid products between steps. Such embodiments may be extremely useful for automating complex molecular biology processes (like next-generation library preparation) on liquid handling robots or in microfluidic processors.
  • T4 DNA ligase inhibits exonuclease digestion of linear DNA.
  • T4 DNA ligase binds avidly to DNA ends and nicks and prevents many nucleases and polymerases from processing double-stranded DNA.
  • Linear T7 genomic DNA (New England Biolabs, NEB) was diluted into restriction buffer as follows:
  • Tube 1 add 5 microliters stop mix (29 mM EDTA, 2% SDS, 22% Ficoll 400)
  • Tube 2 Add 1 microliters of exo mix (1 microliters NEB Exonuclease III, 1 microliters NEB Exonuclease I, 6 microliters of NEB Diluent B). Incubate for 30 minutes at 37°C and then add stop mix
  • Tube 3 Add 0.5 microliters of NEB restriction enzyme Bst Z171 (5 units/ microliter); incubate at 37°C for one hour, then add stop mix
  • Tube 4 Treat with Bst Z171 as tube 3, then add Exo mix as tube 2, then stop mix
  • Tube 5 Treat with Bst Z171, then add 0.5 microliters of NEB T4 DNA ligase, 400 unit/ microliter; incubate at 16°C for 2.5 hours, then add stop mix
  • Tube 6 Treat with Bst Z171, then add 0.5 microliters of NEB T4 DNA ligase, 400 unit/ microliter; incubate at 16°C for 2.5 hours, then add Exo mix as tube 2; then add stop mix
  • Lane 3 shows that undigested T7 is completely degraded by the exonuclease mix
  • Lane 4 and 5 show T7 digested with BstZ171, and that digested DNA is completely degraded by the exo mix
  • Lane 6 shows DNA treated with ligase; the BstZ171 remains active in this reaction
  • Lane 7 shows that DNA treated with ligase is resistant to the exonuclease mix.
  • T4 DNA ligase binds avidly to DNA ends and nicks and prevents many nucleases and polymerases from processing double- stranded DNA.
  • Fraction One store at 4°C with no further treatment (no ligation control).
  • Fraction Two add 3.5 microliters of T4 DNA ligase (400 units/ microliter, NEB), 1400 units total.
  • Fraction Three add 3.5 microliters of T4 DNA ligase as in #2 above, plus 4 microliters of a 20 micromolar solution of a hairpin adapter (which includes a PacBio (for example) 2 loop sequence) sequencing primer binding site.
  • a hairpin adapter which includes a PacBio (for example) 2 loop sequence
  • Trypsin was prepared as follows: Trypsin (e.g., Sigma Chemical, catalog T1426) was dissolved in 50mM Acetic Acid at 10 micrograms/microliter, and stored at -20°C in 20 microliter aliquots. An aliquot of trypsin was thawed just before use, and 1.2 microliters of 1M Tris base was added; after addition of Tris base, the pH was approximately 8, as determined by pH paper.
  • Lane 1 contains the starting supercoiled pBR322 DNA.
  • Lane 2 is the digested control.
  • Lane 3 is Fraction 1, the no ligase control.
  • Lane 4 shows fraction 2 after ligation without the hairpin sequencing adapter. Notice shift of the DNA into a smear of higher molecular weight species, indicating ligation of the blunt ended Hae III restriction fragments to each other.
  • Lane 5 shows fraction 3 after ligation in the presence of the hairpin sequencing adapter.
  • restriction bands are preserved but shifted to a higher size, consistent with the addition of the large hairpin adapters to each end of the restriction fragments. There is also a smear of fragments of higher size, indicating some additional fragment to fragment ligation also occurred.
  • Lane 6 and 7 show the same DNA samples of lanes 3 and 4 after combined digestion by exonucleases I and III. Both of those samples are completely degraded by the exonucleases, as expected if they have blunt ends.
  • Lane 8 shows the same DNA of lane 5 after exonuclease digestion.
  • the shifted restriction ladder is resistant to exonuclease treatment, as expected if they have been successfully ligated to the hairpin adapters (such molecules have no single-stranded or double-stranded ends for attack by the exonucleases III/I).
  • the smear of higher molecular weight DNA from intermolecular ligation is largely removed, because most of that DNA has at least one blunt end.
  • Lanes 9 and 10 are NEB low MW markers and NEB lkb ladder markers, respectively.
  • embodiments of the subject disclosure may include methods, compositions, systems and apparatuses/devices which may further include any and all elements from any other disclosed methods, compositions, systems, and devices.
  • elements from one or another disclosed embodiments may be interchangeable with elements from other disclosed embodiments.
  • some further embodiments may be realized by combining one and/or another feature disclosed herein with methods, compositions, systems and devices, and one or more features thereof, disclosed in materials incorporated by reference.

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  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

Cette invention a pour objet de pourvoir à un échantillon d'acide nucléique, et de traiter ledit échantillon par application d'un premier réactif enzymatique de modification d'acide nucléique pour obtenir une première solution d'acide nucléique. Un premier réactif de type protéase peut être appliqué à la première solution d'acide nucléique pour obtenir une deuxième solution d'acide nucléique. L'application du premier réactif de type protéase peut complètement ou sensiblement inactiver le premier réactif enzymatique de modification d'acide nucléique. Un premier réactif inhibiteur peut également être appliqué pour obtenir une troisième solution d'acide nucléique, et ce premier réactif inhibiteur peut complètement ou sensiblement inactiver le premier réactif de type protéase.
PCT/US2017/017508 2016-02-11 2017-02-10 Processus de modification d'un acide nucléique par voie enzymatique WO2017139669A1 (fr)

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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10131901B2 (en) 2014-10-15 2018-11-20 Sage Science, Inc. Apparatuses, methods and systems for automated processing of nucleic acids and electrophoretic sample preparation
CN109504676A (zh) * 2018-11-30 2019-03-22 厦门胜芨科技有限公司 一种dna大片段筛选回收试剂盒及其使用方法
US10473619B2 (en) 2012-10-12 2019-11-12 Sage Science, Inc. Side-eluting molecular fractionator
CN114045319A (zh) * 2019-10-18 2022-02-15 武汉爱博泰克生物科技有限公司 使用蛋白酶控制限制酶活性
US11542495B2 (en) 2015-11-20 2023-01-03 Sage Science, Inc. Preparative electrophoretic method for targeted purification of genomic DNA fragments
US11867661B2 (en) 2017-04-07 2024-01-09 Sage Science, Inc. Systems and methods for detection of genetic structural variation using integrated electrophoretic DNA purification

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1986006743A1 (fr) * 1985-05-10 1986-11-20 A/S Alfred Benzon Enzymes bacteriens
US20030190634A1 (en) * 1999-01-06 2003-10-09 Francis Barany Accelerating identification of single nucleotide polymorphisms and alignment of clones in genomic sequencing
US20090241216A1 (en) * 2006-10-11 2009-09-24 Gefu Wang-Pruski Proteins Involved in After-Cooking Darkening in Potatoes
US20100048412A1 (en) * 2006-05-03 2010-02-25 The Regents Of The University Of California Detection of protease and protease activity using a single nanoscrescent sers probe
US20110287436A1 (en) * 2006-08-01 2011-11-24 Applied Biosystems, Llc Detection Of Analytes And Nucleic Acids
US20140271602A1 (en) * 2011-11-30 2014-09-18 The Broad Institute Inc. Nucleotide-specific recognition sequences for designer tal effectors

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1986006743A1 (fr) * 1985-05-10 1986-11-20 A/S Alfred Benzon Enzymes bacteriens
US20030190634A1 (en) * 1999-01-06 2003-10-09 Francis Barany Accelerating identification of single nucleotide polymorphisms and alignment of clones in genomic sequencing
US20100048412A1 (en) * 2006-05-03 2010-02-25 The Regents Of The University Of California Detection of protease and protease activity using a single nanoscrescent sers probe
US20110287436A1 (en) * 2006-08-01 2011-11-24 Applied Biosystems, Llc Detection Of Analytes And Nucleic Acids
US20090241216A1 (en) * 2006-10-11 2009-09-24 Gefu Wang-Pruski Proteins Involved in After-Cooking Darkening in Potatoes
US20140271602A1 (en) * 2011-11-30 2014-09-18 The Broad Institute Inc. Nucleotide-specific recognition sequences for designer tal effectors

Non-Patent Citations (11)

* Cited by examiner, † Cited by third party
Title
"NewEngland_Restriction_Buffer", NEBUFFER PERFORMANCE CHART WITH RESTRICTION ENZYMES, 2013, pages 1 - 9, XP055409321, Retrieved from the Internet <URL:https://www.neb.com/~/media/NebUs/Files/nebuffer-performance-chart-with-restriction-enzymes.pdf> [retrieved on 20170523] *
"SIGMA_P8340, Protease Inhibitor Cocktail for use with mammalian cell and tissue extracts", CATALOG NUMBER P8340. SIGMA-ALDRICH, 2010, XP055409305, Retrieved from the Internet <URL:https://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma/Datasheet/5/p8340dat.pdf> [retrieved on 20170320] *
COST ET AL.: "Directed assembly of DNA molecules via simultaneous ligation and digestion", BIOTECHNIQUES, vol. 42, no. 1, 2007, pages 84 , 86 - 9, XP008145964 *
HOLLAND ET AL.: "Isolation and Characterization of a Small Catalytic Domain Released from the Adenylate Cyclase from Escherichia coli by Digestion with Trypsin", J BIOL CHEM., vol. 263, no. 29, 1988, pages 14661 - 8, XP055409303 *
MEYER ET AL.: "Expanding Proteome Coverage with Orthogonal-specificity Alpha-Lytic Proteases", MOL CELL PROTEOMICS, vol. 13, no. 3, 2014, pages 823 - 35 *
OLSEN ET AL.: "Trypsin Cleaves Exclusively C-terminal to Arginine and Lysine Residues", MOL CELL PROTEOMICS, vol. 3, no. 6, 2004, pages 608 - 14, XP055409307 *
REN ET AL.: "A Simplified Method to Prepare PCR Template DNA for Screening of Transgenic and Knockout Mice", J BIOL CHEM., vol. 290, no. 45, 2015, pages 27248 - 60 *
RITTIE ET AL.: "Enzymes used in molecular biology: a useful guide", J CELL COMMUN SIGNAL, vol. 2, no. 1-2, 2008, pages 25 - 45, XP055409312 *
TOMKINSON ET AL.: "Location of the active site for enzyme-adenylate formation in DNA ligases", PROC NATL ACAD SCI U S A., vol. 88, no. 2, 1991, pages 400 - 4, XP055409307 *
WANG ET AL.: "IRDL Cloning: A One-Tube, Zero-Background, Easy-to-Use, Directional Cloning Method Improves Throughput in Recombinant DNA Preparation", PLOS ONE, vol. 9, no. 9, 2014, pages e107907, XP055409315 *
WILSON ET AL.: "Engineered DNA ligases with improved activities in vitro", PROTEIN ENG DES SEL, vol. 26, no. 7, 2013, pages 471 - 8, XP055297919 *

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10473619B2 (en) 2012-10-12 2019-11-12 Sage Science, Inc. Side-eluting molecular fractionator
US10131901B2 (en) 2014-10-15 2018-11-20 Sage Science, Inc. Apparatuses, methods and systems for automated processing of nucleic acids and electrophoretic sample preparation
US10738298B2 (en) 2014-10-15 2020-08-11 Sage Science, Inc. Apparatuses, methods and systems for automated processing of nucleic acids and electrophoretic sample preparation
US11542495B2 (en) 2015-11-20 2023-01-03 Sage Science, Inc. Preparative electrophoretic method for targeted purification of genomic DNA fragments
US11867661B2 (en) 2017-04-07 2024-01-09 Sage Science, Inc. Systems and methods for detection of genetic structural variation using integrated electrophoretic DNA purification
CN109504676A (zh) * 2018-11-30 2019-03-22 厦门胜芨科技有限公司 一种dna大片段筛选回收试剂盒及其使用方法
CN114045319A (zh) * 2019-10-18 2022-02-15 武汉爱博泰克生物科技有限公司 使用蛋白酶控制限制酶活性
CN114045319B (zh) * 2019-10-18 2023-10-17 武汉爱博泰克生物科技有限公司 使用蛋白酶控制限制酶活性

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