WO2024073034A1 - Préparation de banque de séquençage simplifiée pour adn - Google Patents

Préparation de banque de séquençage simplifiée pour adn Download PDF

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WO2024073034A1
WO2024073034A1 PCT/US2023/034095 US2023034095W WO2024073034A1 WO 2024073034 A1 WO2024073034 A1 WO 2024073034A1 US 2023034095 W US2023034095 W US 2023034095W WO 2024073034 A1 WO2024073034 A1 WO 2024073034A1
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dna
adapter
uracil
reaction mixture
glycosylase
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PCT/US2023/034095
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English (en)
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Joseph W. Foley
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The Board Of Trustees Of The Leland Stanford Junior University
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    • 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

  • the present invention relates to compositions and methods for DNA sequencing and sequencing library preparation.
  • DNA sequencing is a large and growing field of biotechnology with uses in both research and clinical practice.
  • the throughput and cost of sequencing have decreased so much in recent years that the labor time of preparing sequencing libraries from DNA may be a more substantial concern than the sequencing itself.
  • the preparation of a DNA library suitable for sequencing generally requires multiple enzymatic modifications of the DNA molecules performed sequentially, often requiring intervening “cleanup” steps to remove the reagents and/or byproducts of a previous reaction.
  • genomic DNA or other template DNA molecules must undergo a series of modifications including fragmentation into smaller sizes suitable for amplification or cloning, optional “end repair”, A-tailing to incorporate or leave a single adenine overhang at the 3' ends of the DNA molecules, ligation of adapter oligonucleotides to the ends of the DNA molecules, degradation or removal of excess adapter molecules, and amplification of the DNA, typically using a polymerase chain reaction ("PCR”) based method.
  • PCR polymerase chain reaction
  • the present invention provides methods and related compositions that advantageously simplify the preparation of DNA for sequencing by providing a single reaction mixture and a single set of reaction conditions for ligation of adapter oligonucleotides, degradation of unligated adapter molecules, and amplification of DNA-adapter molecules.
  • the invention provides a method for producing a sequencing ready DNA library, the method including forming a reaction mixture by contacting a plurality of DNA fragments with an enzyme mixture that includes a DNA ligase, a uracil-DNA glycosylase, a DNA polymerase, a set of double-stranded adapter oligonucleotides such as stem loop adapter oligonucleotides, a set of polymerase amplification primers complementary to at least a portion of the adapter oligonucleotides, deoxynucleoside triphosphates (dNTPs), adenosine triphosphate (ATP) or nicotinamide adenine dinucleotide (NAD), and a buffer suitable for activity of the polymerase, glycosylase, and ligase, where the DNA fragments of the plurality consist of double-stranded DNA molecules having terminal 5’phosphate and 3’hydroxyl groups, optionally with a single enzyme mixture that includes a DNA
  • the method may also include where the conditions suitable for activity of the DNA ligase include a temperature of from 4-50°C or from 4-37°C. In embodiments, the conditions suitable for activity of the DNA ligase include a temperature of about 4°C, about 16°C, about 25°C, or about 37°C.
  • the method may also include where the conditions suitable for activity of the uracil- DNA glycosylase include a temperature above 50°C, or from 65-98°C.
  • the method may also include where the uracil-DNA glycosylase is active only at temperatures higher than a temperature at which the DNA ligase is active.
  • the method may also include where the conditions suitable for activity of the DNA polymerase include a series of extensions at a temperature of from 37-72°C and denaturations a temperature of from 95-98°C.
  • the method may also include where the double stranded or stem loop adapter oligonucleotide includes deoxyuridine residues.
  • the double stranded or stem loop adapter oligonucleotide includes a plurality of deoxyuridine residues located in the 5' stem region and/or in the loop region.
  • the method may also include where there is no intervening post-ligation manipulation prior to amplifying the adapter-linked DNA fragments.
  • the method may also further include, in the enzyme mixture, a solid support having attached thereto a plurality of oligonucleotides including a 3' end complementary to a region of the adapter oligonucleotides.
  • the method may also further include, before the step of forming a reaction mixture, forming a plurality of DNA fragments from the double stranded template DNA.
  • the double stranded template DNA is selected from genomic DNA, cDNA, cell- free DNA, amplicons, e.g., PCR amplicons, and DNA selected by an enrichment method such as immunoprecipitation.
  • the method may also include where the forming a plurality of DNA fragments from template DNA includes subjecting template DNA to digestion by one or more endonucleases, including restriction endonucleases.
  • endonucleases including restriction endonucleases.
  • the invention provides a kit of parts including a first container that includes an enzyme mixture, the enzyme mixture including a DNA ligase, a uracil-DNA glycosylase, and a DNA polymerase; a second container including a reactant mixture, the reactant mixture includes deoxynucleoside triphosphates, adenosine triphosphate (ATP) or nicotinamide adenine dinucleotide (NAD), and a source of divalent cation such as magnesium; and an optional third container including double stranded or stem loop adapter oligonucleotide each having a plurality of deoxyuridine residues, optionally located in the 5' stem region and/or in the loop region, and optionally a set of polymerase amplification primers complementary to at least a portion of the adapter oligonucleotides.
  • an enzyme mixture including a DNA ligase, a uracil-DNA glycosylase, and a DNA polymerase
  • FIG. 1 is a flow chart illustrating the steps in a standard protocol for making a sequencing ready library from genomic DNA.
  • FIG. 2 is a flow chart illustrating the steps in a protocol for making a sequencing ready library from genomic DNA with reduced steps compared to the standard protocol.
  • FIG. 3 is a flow chart illustrating the steps in a protocol for making a sequencing ready library from genomic DNA in accordance with one embodiment.
  • FIG. 4 is a flow chart illustrating the steps in a protocol for making a sequencing ready library from genomic DNA in accordance with a second embodiment.
  • FIG. 5 shows comparative results of amplification of a DNA library using a uracil- DNA glycosylase (UDG) from either Escherichia coli (“low temp”, triangles) or Archaeoglobus fulgidus (“high temp”, circles) under a “two step” or “separate steps” protocol (left panel) and a “single step” or “combined steps” protocol (right panel).
  • UDG uracil- DNA glycosylase
  • the present invention provides new methods and related compositions for adapter ligation and amplification of DNA molecules to produce a sequencing ready DNA library.
  • the disclosure provides methods and related kits for producing a sequencing ready DNA library by performing ligation and amplification in a single combined step, rather than in two separate steps.
  • the methods described here advantageously provide for streamlining library production by performing adapter ligation and library amplification in a single reaction mixture, without the need for one or more intervening steps to remove unreacted materials, enzymes, buffers, etc., referred to herein as “cleanup” steps.
  • the methods comprise contacting a plurality of double stranded template DNA fragments with a single mixture of three enzymes in a buffer suitable for activity of each of the three enzymes, the three enzymes including a DNA ligase, a uracil-DNA glycosylase, and a DNA polymerase.
  • the methods comprises providing, in a single container, a plurality of double stranded template DNA fragments and contacting the plurality of DNA fragments with a single mixture of three enzymes in a buffer suitable for activity of the three enzymes, the three enzymes including a DNA ligase, a uracil-DNA glycosylase, and a DNA polymerase.
  • the DNA fragments of the plurality consist of double-stranded DNA molecules having terminal 5’phosphate and 3 ’hydroxyl groups, optionally with a single adenine overhang at each 3' end.
  • Suitable DNA fragments may be produced, for example, by random chemical fragmentation, by sonication, or by digestion with endonucleases, including restriction endonucleases.
  • Terminal 5’phosphate and 3 ’hydroxyl groups which may be generated simultaneously with fragmentation, for example by restriction endonuclease digestion, or may be generated in a separate step, for example using end repair.
  • End repair may involve for example using a DNA polymerase to fill in or digest overhangs and using a polynucleotide kinase to phosphorylate 5’ ends and dephosphorylate 3’ ends.
  • the termini of the DNA fragments may be either blunt ends, with or without A-tailing, or overhangs, including single 3' adenine overhangs, or other “sticky ends” including short overhangs from one to several bases in length, for example from 1 -15 bases, or from 1-10 bases, or from 1-5 bases in length.
  • a reaction mixture is formed when the plurality of DNA fragments is contacted with the single enzyme mixture comprising a DNA ligase, a uracil-DNA glycosylase, and a DNA polymerase.
  • the reaction mixture also contains appropriate primers and adapter oligonucleotides containing deoxyuridine, for example, stem loop adapter oligonucleotides containing deoxyuridine located, for example, in the 5' stem region and/or in the loop region of the adapter, and polymerase amplification primers complementary to at least a portion of the adapter oligonucleotides.
  • the reaction mixture also contains appropriate reagents for performing an adapter ligation reaction and a DNA amplification reaction, for example deoxynucleoside triphosphates (dNTPs), polyethylene glycol, a source of magnesium such as magnesium chloride, and either adenosine triphosphate (ATP) or nicotinamide adenine dinucleotide (NAD).
  • dNTPs deoxynucleoside triphosphates
  • ATP adenosine triphosphate
  • NAD nicotinamide adenine dinucleotide
  • the reaction mixture is subjected to conditions suitable for activity of the DNA ligase, thereby producing adapter-linked DNA molecules; followed by conditions suitable for activity of the uracil-DNA glycosylase, thereby excising uracil and generating abasic sites in the adapters which function as replication stops; and conditions suitable for activity of the DNA polymerase, thereby producing and amplifying the adapter-linked DNA molecules in a single reaction mixture without an intervening postligation cleanup step prior to amplification.
  • the activities of the uracil-DNA glycosylase and the DNA polymerase are concurrent or sequential.
  • adapter oligonucleotides are incorporated at each terminal end of the fragmented DNA molecules to provide a target site for the amplification primers and, optionally, for sequencing primers.
  • the adapter oligonucleotides are stem-loop oligonucleotides.
  • the stem-loop structure also referred to as a hairpin, is formed by the intramolecular base pairing between two complementary regions of the oligonucleotide, which may also be referred to as inverted repeat or palindromic regions, i.e., a region that is the same as its reverse complement.
  • the stem-loop adapters contain one or more deoxyuridine residues which are excised by the uracil-DNA glycosylase after adapter ligation and before amplification. This base excision produces abasic sites along the phosphate backbone of the DNA strand.
  • An abasic site which may also be referred to as an apurinic/apyrimidinic site, is a location in DNA that lacks a purine or a pyrimidine base.
  • these abasic sites are cleaved by a second enzyme with apurinic/apyrimidinic lyase activity such as endonuclease VIII or by spontaneous hydrolysis at high temperature, e.g., a denaturation temperature such as a temperature above 90 or 95°C.
  • a denaturation temperature such as a temperature above 90 or 95°C.
  • the one or more deoxyuridine residues are utilized to introduce at least one replication stop into the adapter molecule during polymerase chain reaction amplification of the library via conversion of deoxyuridine residues into abasic sites through the activity of the uracil-DNA glycosylase.
  • the reaction proceeds through ligation of the stem-loop adapter oligonucleotides to the ends of the DNA fragments.
  • Adapter degradation and fill-in are performed by the uracil-DNA glycosylase and DNA polymerase enzymes, respectively.
  • the ligase enzyme attaches a 3' end of a stem-loop oligonucleotide to a 5' phosphate of a DNA fragment producing an adaptor-linked DNA fragment comprising a nick having a 3' hydroxyl group.
  • the polymerase displaces the 5' portion of the adapter from the nicked site and extends the 3' end of the DNA fragment until it reaches a non-replicable abasic gap, e.g., within the adapter loop region.
  • the denaturation step of the PCR cycle creates breaks at the abasic sites and eliminates the 5' portion of the stem-loop oligonucleotide.
  • a hot denaturation step may be introduced for this purpose. This provides a mechanism for elimination of the inverted repeat of the hairpin structure from the ends of the ligated DNA- adapter molecules and prevents the formation of amplifiable adapter dimers.
  • the adapter-linked DNA fragments are attached to a solid support.
  • a solid support For example, following formation of adapter-linked DNA fragments comprising a nick having a 3' hydroxyl group, polymerization of the adapter-linked DNA fragment to the abasic gap generates a 5' overhang which hybridizes to a complementary oligonucleotide covalently attached to the solid support, thereby forming adapter-linked DNA fragments attached to the solid support.
  • stem-loop adapter oligonucleotides examples include, for example, those disclosed in US 7,803,550.
  • the uracil-DNA glycosylase is selected to be active only at temperatures higher than those required for the DNA ligase activity, for example higher than 16°C, or higher than 25°C, or higher than 37°C, or higher than 50°C.
  • An exemplary uracil-DNA glycosylase for use in the claimed methods is produced by the hyperthermophile Archaeoglobus fulgidus and is commercially available, for example from New England Biolabs.
  • any suitable DNA polymerase may be used to perform DNA amplification in accordance with the methods described here.
  • the polymerase may be a thermostable DNA polymerase, or one suitable for isothermal amplification, including loop- mediated isothermal amplification (LAMP).
  • Exemplary thermostable DNA polymerases suitable for use in the methods described here include Taq polymerase, Tfl polymerase, Tth polymerase, Pfu polymerase, and Pfx polymerase, and modified versions of the foregoing.
  • Other exemplary polymerases include Bst polymerase and phi29 polymerase, and modified versions thereof.
  • a suitable DNA polymerase for performance of DNA amplification in accordance with the methods described here is not required to have exonuclease activity, since nick extension proceeds by strand displacement.
  • Any suitable DNA ligase may be used to perform ligation of the adapter oligonucleotides to the DNA fragments in accordance with the methods described here.
  • An exemplary DNA ligase for use in the methods described here is a T4 DNA ligase.
  • Other exemplary DNA ligases include T3 DNA ligase, T7 DNA ligase, E. Coli DNA ligase, etc.
  • the methods and compositions described here can be incorporated into a workflow to provide a DNA library suitable for sequencing from genomic DNA.
  • the methods and compositions described here may also be incorporated into other methods, for example a method of DNA methylation profiling using a restriction endonuclease that specifically cuts at methylated DNA; and a three-step RAD-seq protocol, in which restriction digest products are sequenced for narrowly targeted genome coverage.
  • the library protocol specifies the use of UDG from the hyperthermophile Archae globus fulgidus in a combined ligation/loop-breaking/PCR master mix.
  • the traditional Escherichia coli UDG interferes with the low-temperature ligation and would require an additional step between ligation and PCR, whereas the hyperthermophilic UDG is inert at ligation temperatures.
  • FIG. 1 depicts nine sequential steps forming an exemplary standard protocol in the prior art for making a sequencing ready library starting from genomic DNA.
  • genomic DNA is broken into a range of smaller fragments by any one of several methods.
  • genomic DNA may be broken into fragments having a range of size distributions, for example less than 3 kilobasepairs (kb), less than 2 kb, or less than 1 kb, for example in a range of from 100-250 bp, from 150-350 bp, from 200-450 bp, from 300- 700 bp, or from 500 bp to 1 kb.
  • kb kilobasepairs
  • Suitable DNA fragments may be produced, for example, by random chemical fragmentation, by sonication, or by digestion with endonucleases using standard protocols such as those described in Molecular Cloning: A l aboratory Manual (Fourth Edition) 2012 Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
  • End repair 104 the ends of the DNA fragments are modified to generate terminal 5’phosphate and 3’hydroxyl groups. This step is optional where endonucleases are used to fragment the DNA.
  • the termini of the DNA fragments may be either blunt ends or single or multiple base overhangs, depending on the type of endonuclease used in the first or optional second step.
  • the ends of the fragmented DNA are further modified to include a single adenine overhang at each 3' end to facilitate ligation of adapter oligonucleotides using a process referred to as A-tailing 106 or dA-tailing.
  • Standard protocols for end repair and A-tailing may also be found, for example, in Molecular Cloning: A Laboratory Manual (Fourth Edition) 2012 Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
  • a cleanup 108 step is performed to remove reagents and enzymes from the fragmentation, end repair, and A-tailing steps.
  • a futher cleanup 112 step is performed after adapter ligation 110 in order to remove unligated adapter oligonucleotides prior to amplification 114.
  • Amplification may by a polymerase chain reaction (PCR) or by an isothermal amplification method, including loop-mediated isothermal amplification (LAMP).
  • LAMP loop-mediated isothermal amplification
  • a final cleanup 116 follows amplification.
  • FIG. 2 depicts the six sequential steps forming an exemplary prior art protocol in which DNA fragmentation, end repair, and A-tailing are performed 202 in a single container in a single incubation with one reaction mixture containing the necessary enzymes and reagents.
  • the next step of adapter ligation 204 is performed in a second incubation in the same container after addition of a mixture containing ligase, adapter oligonucleotides, and other required reagents, such as salts.
  • a mixture containing ligase, adapter oligonucleotides, and other required reagents, such as salts Following adapter ligation, unligated adapter oligonucleotides are degraded 206 in a third incubation followed by a cleanup 208 step to remove degraded oligos, enzymes and other materials from the earlier steps, leaving the DNA fragments ligated to adapter oligonucleotides.
  • the DNA fragments are then subjected to amplification 210 by a polymerase chain reaction, followed by a final cleanup 212 step.
  • An exemplary prior art protocol of this type is described in the New England Biolabs NEBNEXT Ultra II FS kit.
  • FIG. 3 depicts one exemplary protocol for preparing a sequencing ready DNA library utilizing the methods and compositions described here.
  • the input fragmented DNA having terminal 5 ’phosphate and 3 ’hydroxyl groups and a single adenine overhang at each 3' terminal end is prepared using a prior art protocol for performing DNA fragmentation, end repair, and A-tailing in a single incubation 302.
  • Suitable enzymes and protocols for performing this step 302 are commercially available, for example from New England Biolabs.
  • a reaction mixture containing a DNA ligase, a uracil-DNA glycosylase, and a DNA polymerase in a buffer suitable for activity of all three enzymes is added to the container of fragmented DNA, without any intervening cleanup step.
  • the reaction mixture also contains appropriate amplification primers and adapter oligonucleotides, for example, stem loop adapter oligonucleotides containing deoxyuridine and amplification primers complementary to at least a portion of the adapter oligonucleotides.
  • the reaction mixture also contains appropriate reagents for performing the ligation and amplification reactions, for example deoxynucleoside triphosphates (dNTPs), adenosine triphosphate (ATP), and a source of magnesium.
  • dNTPs deoxynucleoside triphosphates
  • ATP adenosine triphosphate
  • DNA amplification is followed by a final cleanup 306.
  • DNA amplification may by a polymerase chain reaction (PCR) or by an isothermal amplification method, including loop-mediated isothermal amplification (LAMP).
  • PCR polymerase chain reaction
  • LAMP loop-mediated isothermal amplification
  • FIG. 4 depicts another exemplary protocol for preparing a sequencing ready DNA library utilizing the methods and compositions described here.
  • the input fragmented DNA having terminal 5 ’phosphate and 3 ’hydroxyl groups and a single adenine overhang at each 3' terminal end is prepared using a Restriction Enzyme Digestion 402.
  • a reaction mixture containing a DNA ligase, a uracil-DNA glycosylase, and a DNA polymerase in a buffer suitable for activity of all three enzymes is added to the container of fragmented DNA, without any intervening cleanup step.
  • the reaction mixture also contains appropriate amplification primers and adapter oligonucleotides, for example, stem loop adapter oligonucleotides containing deoxyuridine and amplification primers complementary to at least a portion of the adapter oligonucleotides.
  • the reaction mixture also contains appropriate reagents for performing the ligation and amplification reactions, for example deoxynucleoside triphosphates (dNTPs), adenosine triphosphate (ATP), and a source of magnesium.
  • dNTPs deoxynucleoside triphosphates
  • ATP adenosine triphosphate
  • DNA amplification is followed by a final cleanup 406.
  • DNA amplification may by a polymerase chain reaction (PCR) or by an isothermal amplification method, including loop-mediated isothermal amplification (LAMP).
  • PCR polymerase chain reaction
  • LAMP loop-mediated isothermal amplification
  • FIG. 5 shows the results of a comparative example of an embodiment of the present invention and a standard “two step” protocol.
  • Human genomic DNA from the Jurkat cell line was digested with MspJI restriction endonuclease (New England Biolabs) according to the manufacturer’s instructions.
  • a second reaction mix was added, containing the ligation reagents T4 DNA ligase, PEG 4000, and ATP (Thermo Fisher); PCR buffer, hot-start polymerase, and dNTPs (Roche); PicoGreen and ROX dyes (Thermo Fisher); a pair of stem-loop adapters with a 5’ 4 nucleotide overhang and uracil replacing thymine in the 5’ portion of the stem and a pair of PCR primers matching the adapter sequences (Integrated DNA Technologies); and uracil-DNA glycosylase (UDG) from either Escherichia coli (“low temp”) or Archaeoglobus fulgidus (“high temp”) (New England Biolabs).
  • the samples were incubated 5 min at 16 C then transferred to a qPCR thermal cycler and incubated 3 min at 72 C, 2 min at 85 C, and 2 min at 98 C before a standard PCR program according to the manufacturer’s instructions, with fluorescence readings taken each cycle.
  • Two protocols were performed. In the first protocol, only the ligation reagents and adapters were added before the 16 C incubation, followed by the remaining reagents before the qPCR program. In the second protocol, all of the reagents were added at the same time, before the 16 C incubation. The results of the first protocol are depicted in the left panel of FIG.
  • an “embodiment” may refer to an illustrative representation of a process or article or component in which a disclosed concept or feature may be provided or embodied, or to the representation of a manner in which just the concept or feature may be provided or embodied.
  • illustrated embodiments are to be understood as examples (unless otherwise stated), and other manners of embodying the described concepts or features, such as may be understood by one of ordinary skill in the art upon learning the concepts or features from the present disclosure, are within the scope of the disclosure.
  • the present subject matter covers such modifications and variations as come within the scope of the appended claims and their equivalents.
  • nucleotide refers to a nucleotide (e.g., RNA or DNA) or a sequence of nucleotides capable of base pairing with a complementary nucleotide or sequence of nucleotides.
  • Complementarity is determined by the ability of an associated nitrogenous base of a nucleotide, also referred to as a “nucleobase” or simply a “base”, to hydrogen bond with the nitrogenous base of a different nucleotide, e.g., a nucleotide on a different nucleic acid. This interaction may also be referred to as “base pairing”.
  • the base adenine binds to thymine or uracil and the base guanine binds to cytosine.
  • Adenine may therefore be referred to as the complement of thymine or uracil and guanine may be referred to as the complement of cytosine, and vice versa.
  • a complement may include a sequence of nucleotides that base pair with corresponding complementary nucleotides of a second nucleic acid sequence.
  • the term “contacting” is used in accordance with its plain ordinary meaning and refers to the process of allowing at least two distinct species such as chemical compounds, biomolecules, and enzymes, to become sufficiently proximal to react, interact or physically touch.
  • the term “endonuclease” refers to an enzyme which possesses endonucleolytic catalytic activity for polynucleotide cleavage. For example, an endonuclease can cleave a phosphodiester bond of an oligonucleotide or polynucleotide. An endonuclease cleaves at a phosphodiester bond within or adjacent to its recognition site sequence, which spans at least 4 base pairs in length.
  • Types of endonucleases include, but are not limited to restriction enzymes, AP endonuclease, T7 endonuclease, T4 endonuclease, Bal 31 endonuclease, Endonuclease I, etc.
  • nucleic acid refers to a polymer of nucleotides, e.g., deoxyribonucleotides or ribonucleotides, and may be used herein as shorthand for deoxyribonucleic acid (DNA) or ribonucleic acid (RNA).
  • DNA deoxyribonucleic acid
  • RNA ribonucleic acid
  • nucleoside refers, in the usual and customary sense, to a glycosylamine including a nitrogenous base, also referred to as a “nucleobase”, and a five-carbon sugar, z.e., ribose or deoxyribose.
  • nucleosides include cytidine, uridine, adenosine, guanosine, thymidine and inosine.
  • nucleotide refers, in the usual and customary sense, to the monomeric units of nucleic acids, each unit consisting of a nucleoside and a phosphate.
  • base refers to the nucleobase moiety of the nucleoside, e.g., cytosine, adenine, guanine, thymine, and uracil.
  • oligonucleotide refers to the alphabetical representation of a polynucleotide molecule; alternatively, the term may be applied to the polynucleotide molecule itself.

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Abstract

La présente invention concerne des procédés et des compositions connexes permettant de produire une banque d'ADN prête pour le séquençage en trois étapes seulement, par combinaison de ligature d'adaptateur, de dégradation, de remplissage et d'amplification de fragments d'adaptateur-ADN en une seule réaction.
PCT/US2023/034095 2022-09-29 2023-09-29 Préparation de banque de séquençage simplifiée pour adn WO2024073034A1 (fr)

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