WO2016145039A1 - Fragmentation et réparation améliorées d'acides nucléiques - Google Patents

Fragmentation et réparation améliorées d'acides nucléiques Download PDF

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WO2016145039A1
WO2016145039A1 PCT/US2016/021503 US2016021503W WO2016145039A1 WO 2016145039 A1 WO2016145039 A1 WO 2016145039A1 US 2016021503 W US2016021503 W US 2016021503W WO 2016145039 A1 WO2016145039 A1 WO 2016145039A1
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nucleic acid
dna
acid molecule
adaptor
particle
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PCT/US2016/021503
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English (en)
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John LANGMORE
Emmanuel Kamberov
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Rubicon Genomics, Inc.
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Publication of WO2016145039A1 publication Critical patent/WO2016145039A1/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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology

Definitions

  • the present invention relates generally to the field of molecular biology. More particularly, it concerns methods of shearing and repair of nucleic acids. 2. Description of Related Art
  • Hydrodynamic shearing is usually used to fragment DNA or RNA to an optimal size (e.g., 500 bp) for applications, such as amplification and/or sequencing.
  • Current hydrodynamic shearing methods require high-power ultrasonic devices able to provide the strong shearing forces necessary to break purified nucleic acid molecules.
  • the high shearing forces are able to break nucleic acids through a combination of inducing velocity gradients in the liquid and inducing cavitation when gas bubbles are created and collapse rapidly. In the beginning of the reaction, the cleavage of any high molecular weight nucleic acids occurs very quickly because of the large force exerted on long molecules.
  • Extended shearing time also allows thermal convection and cavitation to mix the bulk liquid so as to more uniformly subject the nucleic acid molecules to the shearing forces.
  • longer and stronger hydrodynamic shearing also increases damage to the ends of the nucleic acid molecules (e.g. , terminal damage such as 3' or 5' overhangs, 3'-P, or 5'-OH), which prevent ligation to oligonucleotide adaptors necessary for sequencing or amplification by PCR or other methods.
  • a method for fragmenting a nucleic acid molecule comprising obtaining a nucleic acid molecule; adding to the nucleic acid molecule at least one particle that selectively binds to a terminus of the nucleic acid molecule to generate a particle-bound nucleic acid molecule; and exposing the particle-bound nucleic acid molecule to a shear force, thereby producing nucleic acid fragments.
  • the shear force may be a hydrodynamic shear force, such as those generated by acoustic or mechanical means.
  • a nucleic acid fragment may have a size of about 100 bp, about 200 bp, about 300 bp, about 400 bp, about 500 bp, about 1000 bp, or about 2000 bp. In certain aspects, the nucleic acid fragments may have an average size of about 100 bp, about 200 bp, about 300 bp, about 400 bp, about 500 bp, about 1000 bp, or about 2000 bp.
  • a nucleic acid molecule may have a size of about 2000 bp, 5000 bp, 7500 bp, 10,000 bp, 20,000 bp, 30,000 bp, 40,000 bp, 50,000 bp, 60,000 bp, 70,000 bp, 80,000 bp, 90,000 bp, or 100,000 bp.
  • Nucleic acids may be, for example, RNA or DNA. Modified forms of RNA or DNA may also be used.
  • a nucleic acid molecule may be a purified nucleic acid molecule. In certain aspects, a nucleic acid molecule may be essentially free of chromatin proteins. In various aspects, a particle-bound nucleic acid molecule does not comprise any particles covalently bound to the nucleic acid molecule. In various aspects, a particle-bound nucleic acid molecule does not contain any particles covalently bound to the nucleic acid molecule. [0008] In certain aspects, a particle that selectively binds to a terminus of the nucleic acid molecule may be a protein.
  • the protein may be a nucleic acid repair enzyme (e.g., T4 Polymerase, Klenow, and/or T4 polynucleotide kinase).
  • the nucleic acid end-repair enzyme may be bound to a second particle, such as, for example, a protein (e.g., an enzymatically inert protein), a high-molecular weight polymer (e.g.
  • polyethylene glycol PEG and derivatives thereof, polyvinylpirrolidone (PVP), polyvinyl alcohol (PVA), polyacrylic acid (PAA), polyacrylamide (PAM), polyacrylic maleic acid (PAMA), PEG-based copolymers, such as poloxamers, poloxamines and polysorbates, or any combination thereof), a nanoparticle (e.g., a gold nanoparticle, a silver nanoparticle, a gelatin nanoparticle, a silica nanoparticle, etc.), or any such high molecular-weight particle.
  • the second particle may be bound to the nucleic acid repair enzyme either covalently or non- covalently.
  • the second particle is an inert particle.
  • an “inert particle” is a particle that lacks enzymatic activity, such as, for example, the protein albumin.
  • a nucleic acid repair enzyme may be bound to a bead (e.g., a plastic bead or a glass bead). It is preferable that a nucleic acid repair enzyme bound to a second particle or a bead retains its activity and specificity as an end-repair protein.
  • the method may comprise incubating the nucleic acid fragments under conditions to allow for repair of the ends of the nucleic acid fragments.
  • a method of fragmentation comprising the use of nucleic acid repair enzymes as particles that selectively bind to a terminus of a nucleic acid molecule may be a method of simultaneous fragmentation and repair.
  • a method of fragmentation comprising the use of nucleic acid repair enzymes as particles that selectively bind to a terminus of a nucleic acid molecule may be a method of sequential fragmentation and repair performed in the absence of exogenous manipulation.
  • binding of the repair proteins may first occur under conditions (e.g. , temperature, pH, salt concentrations) that increase shearing forces and the protective functions while not allowing for enzymatic activity, followed by changes in conditions to enable repair at a later time.
  • a nucleic acid fragment produced by the method may be a ligation-competent DNA fragment.
  • a ligation-competent DNA fragment may comprise a blunt end.
  • a method of fragmentation may be performed in the presence of one or more of the following: a nucleic acid end-repair enzyme, an adaptor, a ligase, polynucleotide kinase, reverse transcriptase, one or more DNA polymerases, RNA polymerase, ATP, rNTPs, dNTPs, and one or more primers.
  • the method may be carried out in a single solution. In certain aspect, the process may occur in the absence of exogenous manipulation.
  • a method of fragmentation may be performed in the presence of an oligonucleotide adaptor, such as, for example, a stem-loop adaptor.
  • the oligonucleotide adaptor may lack a phosphate on its 5' end.
  • the adaptor may comprise a known sequence.
  • the method may comprise attaching one strand of the oligonucleotide adaptor to the nucleic acid molecule to produce an oligonucleotide-attached nucleic acid fragment.
  • attaching may be further defined as ligating.
  • ligating may comprise providing T4 DNA ligase to the adaptor and the nucleic acid fragment.
  • attaching may produce a nick in the oligonucleotide-attached nucleic acid fragment.
  • the method may be carried out in a single solution. In certain aspect, the process may occur in the absence of exogenous manipulation.
  • a nucleic acid molecule may be a double-stranded DNA, such as, for example, human genomic DNA.
  • the method may further comprise preparing a library of nucleic acid fragments. In certain aspects, the method may further comprise sequencing a plurality of the library of nucleic acid fragments. [0014] In some aspects, the method may further comprise amplifying a plurality of the nucleic acid fragments. In certain aspects, the method may further comprise determining at least a partial sequence of at least one or more of the nucleic acid fragments.
  • Ligating embodiments may be further defined as comprising: generating ligatable ends on a first double-stranded nucleic acid molecule, and covalently linking both strands to a second double-stranded nucleic acid molecule with two ligatable ends.
  • ligation of only the 5' end of the first double-stranded nucleic acid molecule can be to the 3' end of the second double-stranded nucleic acid molecule, leaving a non- covalent junction, such as a nick, gap or flap on the opposite strand of the oligonucleotide- attached nucleic acid molecule.
  • the double-stranded nucleic acid molecules are comprised of complementary or partially complementary single strands.
  • first and second double-stranded molecules are products of the shearing reaction.
  • first double-stranded molecule is a product of the shearing reaction and the second double-stranded molecule is a double-stranded synthetic oligonucleotide composed of two or more complementary or partially complementary oligonucleotides.
  • first double-stranded molecule is a product of the shearing reaction and the second double-stranded molecules is a stem-loop oligonucleotide.
  • the methods comprise generating blunt dsDNA ends on the nucleic acid molecule, adding single-base extensions to the 3' ends, producing hydroxyl groups at the 3' ends, adding phosphate groups to the 5' ends, etc.
  • kit housed in a suitable container that comprises one or more compositions of the invention and/or comprises one or more compositions suitable for at least one method of the invention.
  • Additional embodiments of the invention include a library of DNA molecules prepared by the methods of the invention.
  • essentially free in terms of a specified component, is used herein to mean the specified component has not been purposefully formulated into a composition and/or is present only as a contaminant or in trace amounts.
  • the total amount of the specified component resulting from any unintended contamination of a composition is therefore well below 0.05%, preferably below 0.01%.
  • Most preferred is a composition in which no amount of the specified component can be detected with standard analytical methods.
  • FIGS. 1A-C show the effects of hydrodynamic force on DNA with and without protein or other molecules at the ends.
  • FIG. 1A shows naked DNA in the absence of hydrodynamic shear.
  • FIG. IB shows naked DNA in the presence of hydrodynamic shear.
  • the arrow represents low tension at the center of the DNA molecule.
  • FIG. 1C shows DNA with terminal binding proteins (or other entities non-covalently bound to the ends) in the presence of hydrodynamic shear.
  • the arrow represents high tension at the center of the DNA molecule owing to the high frictional coefficient of the globular protein(s) at the end(s).
  • FIG. 2 shows a schematic diagram of shearing in the presence of terminal proteins bound to DNA.
  • Condition (i) shows DNA with terminal proteins before hydrodynamic shear.
  • Condition (ii) shows DNA with terminal proteins with hydrodynamic shear.
  • Condition (iii) shows DNA after double-strand breakage and subsequent binding of proteins to the new ends.
  • the present invention provides methods to prepare a nucleic acid molecule or a library of nucleic acid molecules, or both.
  • the preparation of the nucleic acid molecule comprises random fragmentation of the molecule and, optionally, amplification of at least one fragment of the molecule.
  • the prepared nucleic acid molecule may be used for any purpose known in the art, in a specific embodiment it is used for sequencing of at least a portion of the molecule.
  • the present invention is also directed to libraries of nucleic acid molecules, particularly fragments of the molecules generated by random fragmentation of at least one parent nucleic acid.
  • the library members are sequenced concomitantly.
  • the library members are amplified concomitantly before being analyzed by sequencing, microarray, or PCR assays.
  • a "nucleic acid molecule” can be a single nucleic acid molecule or a plurality of nucleic acid molecules. Also, a nucleic acid molecule can be of biological or synthetic origin. Examples of nucleic acid molecules include genomic DNA, cDNA, RNA, a DNA/RNA hybrid, amplified DNA, a pre-existing nucleic acid library, etc.
  • a nucleic acid may be obtained from a human sample, such as blood, serum, plasma, cerebrospinal fluid, cheek scrapings, biopsy, semen, urine, feces, saliva, sweat, etc.
  • a nucleic acid molecule may be subjected to various treatments, such as repair treatments and fragmentation treatments.
  • Fragmentation treatments include mechanical, sonic, and hydrodynamic shearing.
  • Repair treatments include nick repair via extension and/or ligation, polishing to create blunt ends, removal of damaged bases, such as deaminated, derivatized, abasic, or crosslinked nucleotides, etc.
  • a nucleic acid molecule of interest may also be subjected to chemical modification (e.g. , bisulfite conversion, methylation / demethylation), extension, amplification (e.g. , PCR, isothermal, etc.), etc.
  • the present invention provides a method for the simultaneous shearing and end-repair of nucleic acid molecules (e.g. , double-stranded DNA; dsDNA).
  • nucleic acid molecules e.g. , double-stranded DNA; dsDNA.
  • Such a method may reduce the time and strength of shearing (e.g. , hydrodynamic shear force) required to break a nucleic acid molecule to a desired size, thus enabling the use of devices that produce mild shear forces to generate small fragments of nucleic acids that can be readily amplified and/or sequenced.
  • the method may reduce the type and extent of damage done to the nucleic acid molecule during and consequent to shearing.
  • repairing DNA ends immediately after they are produced may reduce the need for repair by reducing additional chemical damage due to hydrolysis and free radicals thought to interfere with ligation or amplification. Eliminating the need to transfer the DNA from the tube used for shearing to the tube used for repair will reduce molecular loss and risk of contamination. Finally, concurrent shearing and repair may reduce the elapsed time and hands-on time to complete the process.
  • the method comprises binding nucleic acid repair enzymes to the termini of the nucleic acid molecules to reduce the hydrodynamic shear force and time necessary for breaking the nucleic acid strands, while simultaneously protecting the ends of the nucleic acids from indirect damage by contact with free radicals, oxygen, or other reactive molecules.
  • Reduction of the minimum hydrodynamic shear force is achieved due to the physical binding of the repair proteins to the nucleic acid termini at the time of shearing.
  • the bound terminal-binding proteins not only increase the hydrodynamic shearing force but also protect the pre-existing termini from indirect damage. Physical binding of the repair proteins to newly -formed termini provides for repair of new direct damage caused by fragmentation.
  • Ligase and adaptors may be added to the reactions to complete the creation of the adaptor-ligated library molecules to be sequenced without PCR amplification ("PCR-free prep") or to be amplified by PCR to create an amplified library.
  • PCR-free prep PCR amplification
  • reagents needed to perform "hot-start" PCR may be added to a reaction comprising end-repair proteins, ligase, and adaptors to complete the creation of an amplified library in the absence of exogenous manipulation (see U.S. Appln. Serial No. 14/250,538, which is incorporated herein by reference in its entirety).
  • the term "in the absence of exogenous manipulation” as used herein refers to there being modification of a DNA molecule without changing the solution in which the DNA molecule is being modified. In specific embodiments, it occurs in the absence of the hand of man or in the absence of a machine that changes solution conditions, which may also be referred to as buffer conditions. In further specific embodiments, changes in temperature occur during the modification.
  • the invention provides a multi-step procedure that can be performed in a single tube or in a micro-titer plate, for example, in a high-throughput format, said steps comprising fragmentation and repair of DNA ends, incorporation of known sequences at both ends of fragments, and at least one enzyme possessing strand-displacement or nick-translation activity.
  • the resulting libraries of molecules are then amplified by PCR using primers corresponding to the known sequences, resulting in several thousand-fold amplification of the entire genome or transcriptome.
  • the products of this amplification can be re-amplified additional times, resulting in amplification that exceeds, for example, several million fold.
  • Exemplary applications for the invention include but are not limited a closed tube preparation and amplification of genomic libraries (e.g., from highly degraded serum, plasma, and/or urine (such as the supernatant fraction) DNA; formalin fixed, paraffin embedded tissues; fresh biopsy tissues; cell cultures, etc.).
  • genomic libraries e.g., from highly degraded serum, plasma, and/or urine (such as the supernatant fraction) DNA; formalin fixed, paraffin embedded tissues; fresh biopsy tissues; cell cultures, etc.
  • DNA amplification and re- amplification can be used as an in vitro "immortalization" process to maintain and generate necessary quantities of valuable but limited DNA samples for gene association studies, mutation and microsatellite instability detection in cancer diagnostics, research applications, etc.
  • the present invention may also provide for a one-step preparation and simultaneous immobilization of prepared DNA libraries on a solid support.
  • Some aspects of the present invention may comprise using at least one particle
  • a particle bound anywhere along the nucleic acid will increase the hydrodynamic frictional coefficient of the DNA. The larger and more numerous the particles, the greater the frictional coefficient. Increases in the frictional coefficient translate into greater forces on the nucleic acid in the presence of a shear force. If the particles(s) are specifically attached to the ends of the nucleic acid, the drag produced will most efficiently increase the tension in the nucleic acid strand and thus increase the probability that the nucleic acid will be sheared with a maximal force at the center of the DNA molecule (see FIG. 1C).
  • the particles(s) are specifically attached to the ends of the nucleic acid, the drag produced will most efficiently increase the tension in the nucleic acid strand and thus increase the probability that the nucleic acid will be sheared with a maximal force at the center of the DNA molecule (see FIG. 1C).
  • nucleic acid-binding particles e.g. , end repair proteins
  • random fragmentation refers to the fragmentation of a nucleic acid molecule in a non-ordered fashion, such as irrespective of the sequence identity or position of the nucleotide comprising and/or surrounding the break.
  • the shearing probability of a given length of a nucleic acid with particles at both ends will be substantially higher, perhaps 50% - 500% higher than the probability of the same length of naked nucleic acid (e.g. , dsDNA). This may allow for shearing in an end- repair mixture to be much faster than in buffer alone.
  • dsDNA naked nucleic acid
  • Hydrodynamic shearing of a nucleic acid can occur by any method known in the art, including passing the nucleic acid through a narrow capillary or orifice, referred to as "point-sink” shearing (Oefner et al , 1996; Thorstenson et al , 1998: Quail, 2010), acoustic shearing, or sonication. Hydrodynamic shearing produces DNA molecules with an appropriate and narrow size distribution. Such fragmentation usually results in double-strand breaks within a double-stranded DNA molecule.
  • double-stranded molecule refers to a molecule that is double stranded at least in part.
  • the shearing process may be computer controlled to select run parameters.
  • Acoustic shearing is the transmission of high-frequency acoustic energy waves to a nucleic acid (Larguinho et al , 2010).
  • the transducer is bowl shaped so that waves converge at the target of interest.
  • the commercially available focused-ultrasonicators in conjunction with miniTUBEs or microTUBEs (Covaris, Woburn, MA; U.S. Patent Nos. 8,459,121; 8,353,619; 8,263,005; 7,981,368; 7,757,561), can randomly fragment DNA with distributions centered between 2-5 kb and 0.1-1.5 kb, respectively. Sonication subjects nucleic acid to hydrodynamic shearing forces (Grokhovsky, 2006; Sambrook et al, 2006).
  • the commercially available Bioruptor (Diagenode; Denville, NJ; U.S. Patent Publn. No. 2012/0264228) use sonication to shear nucleic acids.
  • the methods of the present invention provide a means to increase the shearing force so that shorter exposure times and lower energy levels may be employed with acoustic or sonic nucleic acid shearing devices while still providing nucleic acid fragments of the desired length.
  • a point-sink shearing device that forces the nucleic acid solution through one or more orifices (e.g. , U.S. Patent Publn. No. 2012/0077283) without causing appreciable damage.
  • a nebulizer is a small device that uses compressed air to atomize liquids. DNA fragmentation by nebulization involves forcing a DNA solution through a small hole in the nebulizer which creates a fine mist that is then collected (Sambrook and Russell, 2006).
  • point-sink shearing uses a syringe pump to create hydrodynamic shear forces by pushing a nucleic acid through a small abrupt contraction.
  • the commercially available devices HydroShear and HydroShear Plus can randomly fragment DNA to within a two-fold size distribution with the average size of molecules ranging from 1.5 kb to 5 kb.
  • the g-TUBE from Covaris or the Megaruptor from Diagenode can randomly fragment nucleic acids into 6-20 kb fragments.
  • the enhanced fragmentation achieved by binding repair enzymes or other particles to the termini of the nucleic acids will be used to create fragments much smaller in length (e.g., about 500 bp).
  • the amount of damage due to free radicals, hydronium, nicking, etc. may be reduced using these "point-sink” devices, increased the speed and efficiency of repair of the nucleic acid after fragmentation.
  • Very weak hydrodynamic shearing, such as vortexing the sample in a tube is known to break naked DNA to sizes about 10-30 kb. Even these weak forces may be used to cleave nucleic acid that is more susceptible to hydrodynamic forces.
  • the methods of the present invention provide a means to make a nucleic acid more fragile so that vortexing may be employed to break a large nucleic acid into small fragments.
  • proteins or types of proteins that specifically attach to the ends of a DNA molecule include Ku, DNA polymerases, polynucleotide kinases, DNA ligases, exonucleases, terminal transferases, alkaline phosphatases.
  • Ku is a protein that binds to DNA double-strand break ends and is required for the non-homologous end joining (NHEJ) pathway of DNA repair.
  • DNA polymerases include prokaryotic Pol I, Pol II, Pol III, Pol IV, and Pol V; eukaryotic Pol ⁇ , Pol a, telomerase; reverse transcriptase.
  • polynucleotide kinases include T4 polynucleotide kinase.
  • ligase refers to an enzyme that is capable of j oining the 3' hydroxyl terminus of one nucleic acid molecule to a 5' phosphate terminus of a second nucleic acid molecule to form a single molecule.
  • DNA ligases include E. coli DNA ligase, T4 DNA ligase, mammalian DNA ligases.
  • exonucleases include Exo I, Exo II, Exo III, Exo IV, Exo V, and Exo VIII.
  • Terminal transferases include terminal deoxynucleotidyl transferase (TdT).
  • Alkaline phosphatases include bacterial alkaline phosphatase, calf intestinal alkaline phosphatase, and shrimp alkaline phosphatase.
  • nucleic acid damage resulting from fragmentation are not well known, they can be divided into two categories: (1) damage caused directly from breakage of chemical bonds in the nucleic acids; and (2) damage caused indirectly through chemical interactions of the broken bonds with chemicals, such as water, oxygen, ions, and other solutes as well as unstable chemical species, such as free radicals that can be created by cavitation.
  • the extent of damage can be controlled by reducing the strength and duration of shearing and/or changing the composition of the liquid solution to eliminate stable or unstable reactive molecular species.
  • terminal enzymatic repair examples include, for example, using a kinase to add phosphate to the 5' ends and a DNA polymerase with 3' to 5' exonuclease activity and 5' to 3' polymerase activity to create a blunt end required for ligation to a blunt- end adaptor or to create a 3' single base overhang required for ligation to an adaptor with a 5' single base overhang.
  • the internal damage to nucleic acid molecules is often reversed with repair enzymes (e.g.
  • uracil deglycosylase [UDG] to remove deaminated cytosine bases from DNA coupled with API to replace the abasic sites with cytosine, or combinations of repair enzymes such as New England BioLab's PreCR Repair Mix.
  • Terminal and internal repair increases the cost and time of amplifying or sequencing nucleic acids as they increase the number of enzyme reactions that must be used to achieve accurate replication or sequencing of the nucleic acids. Lack of complete repair of internal or terminal damage reduces the yield of amplifiable or sequenceable nucleic acids or causes misincorporation of bases during replication, which introduces errors into the nucleic acid sequences and therefore leads to false sequence-based test results, errors in cloning of the nucleic acids, etc.
  • the generated fragment molecules may require conditioning or repair, herein defined as modification of the ends to facilitate further processing of the fragment.
  • a 3' end may require conditioning following fragmentation
  • a 5' end may require conditioning following fragmentation, or both.
  • the conditioning comprises modification of a 3' end lacking a 3' OH group.
  • said 3' end is conditioned through the exonuclease and/or extension activities of an enzyme such as T4 DNA polymerase or DNA polymerase I, including Klenow.
  • an enzyme such as T4 polynucleotide kinase is used.
  • an exonuclease enzyme such as exonuclease III, is used.
  • the method further comprises generation of at least one blunt end on said DNA fragments, such as is generated by T4 DNA polymerase, Klenow, or a combination thereof.
  • blunt end refers to the end of a dsDNA molecule having 5' and 3' ends, wherein the 5' and 3' ends terminate at the same nucleotide position. Thus, a blunt end comprises no 5' or 3' overhang.
  • adaptors or linkers Supplementing DNA ends with additional short polynucleotide sequences, referred to as adaptors or linkers, is used in many areas of molecular biology.
  • the usefulness of adapted DNA molecules is illustrated by, but not limited to, several examples, such as ligation-mediated locus-specific PCR, ligation-mediated whole genome amplification, adaptor-mediated DNA cloning, DNA affinity tagging, DNA labeling, etc.
  • the attachment of a substantially known sequence to at least one 3' end of at least one DNA fragment comprises ligation of an adaptor molecule to at least one end of the DNA fragment.
  • the adaptor comprises at least one blunt end.
  • the adaptor comprises a single stranded region.
  • the adaptor comprises a stem-loop structure.
  • 7,803,550 shows the structure of a stem-loop adaptor with a non-replicable linker (which may be introduced chemically during oligonucleotide synthesis or introduced enzymatically during/after the attachment reaction) and shows detailed events occurring at a DNA end during the exemplary multi-enzyme attachment process (see also U.S. Patent Nos. 8,071,312; 8,399,199; and 8,728,737, each of which is incorporated herein by reference in its entirety).
  • the method further comprises generation of at least one blunt end of said DNA fragments, such as is generated by T4 DNA polymerase, Klenow, or a combination thereof.
  • hairpin and “stem-loop oligonucleotide” as used herein refer to a structure formed by an oligonucleotide comprised of 5' and 3' terminal regions that are inverted repeats and a non-self-complementary central region, wherein the self- complementary inverted repeats form a double-stranded stem and the non-self- complementary central region forms a single-stranded loop.
  • the adaptor in a specific embodiment, comprises a substantially known sequence.
  • substantially known refers to having sufficient sequence information in order to permit preparation of a DNA molecule, including its amplification. This will typically be about 100%, although in some embodiments some of the primer sequence is random.
  • substantially known refers to about 50% to about 100%, about 60% to about 100%, about 70% to about 100%, about 80% to about 100%, about 90% to about 100%, about 95% to about 100%, about 97% to about 100%, about 98% to about 100%, or about 99% to about 100%.
  • a blunt-end adaptor can be attached to the ends of double-stranded DNA fragments produced by the fragmentation methods of the present embodiments. Some methods require an additional step that involves a repair of the DNA ends by T4 DNA polymerase and/or Klenow fragment and the removal of 3' or 5' protrusions.
  • the structure of the blunt-end adaptor may be similar to an adaptor of U. S. Patent Nos. 6, 197,557 and 6,828,098, both incorporated by reference herein.
  • One important feature of such an adaptor is the blocking groups at both 3' ends that prevent adaptors from self-ligation. The phosphate group is present at one end of the adaptor to direct its ligation in only one orientation to DNA ends.
  • a single-stranded DNA adaptor with short 3' overhang containing 4 - 6 random bases and a phosphorylated recessed 5' end can be attached to the 3' ends of single stranded DNA molecules.
  • the adaptor may have blocking groups at both 3' ends that prevent adaptors from self-ligation.
  • the phosphate group is present at the recessed 5' end of the adaptor.
  • the 4 - 6 base 3' overhang of the adaptor may have a random base composition. In specific embodiments, it facilitates the annealing and ligation of the adaptor to single stranded DNA molecules.
  • Some methods require an additional step that involves a repair of the 3' ends of single stranded molecules by T4 DNA polymerase, Klenow fragment, and/or exonuclease I.
  • the structure of the single-stranded DNA adaptor may be similar to the adaptor design of U. S. Patent No. 6,828,098, incorporated by reference herein.
  • primer is meant to encompass any nucleic acid that is capable of priming the synthesis of a nascent nucleic acid in a template-dependent process, such as a single-stranded oligonucleotide or a single-stranded polynucleotide that is extended by covalent addition of nucleotide monomers during amplification.
  • primers are oligonucleotides from ten to twenty and/or thirty base pairs in length, but longer sequences can be employed.
  • Primers may be provided in double-stranded and/or single- stranded form, although the single-stranded form is preferred.
  • Oligonucleotide refers collectively and interchangeably to two terms of art, “oligonucleotide” and “polynucleotide.” Note that although oligonucleotide and polynucleotide are distinct terms of art, there is no exact dividing line between them and they are used interchangeably herein.
  • the term “adaptor” may also be used interchangeably with the terms “oligonucleotide” and “polynucleotide.”
  • Pairs of primers designed to selectively hybridize to nucleic acids are contacted with the template nucleic acid under conditions that permit selective hybridization. Depending upon the desired application, high stringency hybridization conditions may be selected that will only allow hybridization to sequences that are completely complementary to the primers. In other embodiments, hybridization may occur under reduced stringency to allow for amplification of nucleic acids containing one or more mismatches with the primer sequences.
  • the template-primer complex is contacted with one or more enzymes that facilitate template-dependent nucleic acid synthesis. Multiple rounds of amplification, also referred to as "cycles,” are conducted until a sufficient amount of amplification product is produced.
  • the method may further comprise the step of designing the primers such that they purposefully are substantially non-self-complementary and substantially noncomplementary to other primers in the plurality.
  • the method may also further comprise the step of amplifying a plurality of the molecules comprising a known nucleic acid sequence to produce amplified molecules. Such amplification may comprise polymerase chain reaction, such as that utilizing a primer complementary to the known nucleic acid sequence.
  • the primers may comprise a constant region and a variable region, both of which include nucleic acid sequence that is substantially non-self-complementary and substantially non-complementary to other primers in the plurality.
  • the constant region is preferably known and may be a targeted sequence for a primer in amplification methods.
  • the variable region may or may not be known, but in preferred embodiments is known.
  • the variable region may be randomly selected or may be purposefully selected commensurate with the frequency of its representation in a source DNA, such as genomic DNA.
  • the nucleotides of the variable region will prime at target sites in a source DNA, such as a genomic DNA, containing the corresponding Watson-Crick base partners.
  • the variable region is considered degenerate.
  • PCRTM polymerase chain reaction
  • two synthetic oligonucleotide primers which are complementary to two regions of the template DNA (one for each strand) to be amplified, are added to the template DNA (that need not be pure), in the presence of excess deoxynucleotides (dNTP's) and a thermostable polymerase, such as, for example, Taq (Thermus aquaticus) DNA polymerase.
  • dNTP's deoxynucleotides
  • a thermostable polymerase such as, for example, Taq (Thermus aquaticus) DNA polymerase.
  • the target DNA is repeatedly denatured (around 90°C), annealed to the primers (typically at 50-60°C) and a daughter strand extended from the primers (72°C). As the daughter strands are created they act as templates in subsequent cycles.
  • the template region between the two primers is amplified exponentially, rather than linearly.
  • Amplification refers to any in vitro process for increasing the number of copies of a nucleotide sequence or sequences. Nucleic acid amplification results in the incorporation of nucleotides into DNA or RNA. As used herein, one amplification reaction may consist of many rounds of DNA replication. For example, one PCR reaction may consist of 30-100 "cycles" of denaturation and replication.
  • Nucleotide is a term of art that refers to a base-sugar- phosphate combination. Nucleotides are the monomeric units of nucleic acid polymers, i.e. , of DNA and RNA. The term includes ribonucleotide triphosphates, such as rATP, rCTP, rGTP, or rUTP, and deoxyribonucleotide triphosphates, such as dATP, dCTP, dUTP, dGTP, or dTTP.
  • ribonucleotide triphosphates such as rATP, rCTP, rGTP, or rUTP
  • deoxyribonucleotide triphosphates such as dATP, dCTP, dUTP, dGTP, or dTTP.
  • a "nucleoside” is a base-sugar combination, i.e. , a nucleotide lacking a phosphate. It is recognized in the art that there is a certain inter-changeability in usage of the terms nucleoside and nucleotide.
  • the nucleotide deoxyuridine triphosphate, dUTP is a deoxyribonucleoside triphosphate. After incorporation into DNA, it serves as a DNA monomer, formally being deoxyuridylate, i.e. , dUMP or deoxyuridine monophosphate.
  • dUTP a DNA monomer
  • dUMP deoxyuridine monophosphate.
  • one may say that one incorporates deoxyuridine into DNA even though that is only a part of the substrate molecule.
  • 'Incoiporating means becoming part of a nucleic acid
  • a reverse transcriptase PCRTM (RT-PCRTM) amplification procedure may be performed to quantify an mRNA by amplification of its cDNA.
  • Methods of reverse transcribing RNA into cDNA are well known and described in Sambrook et al, 1989. Alternative methods for reverse transcription utilize thermostable DNA polymerases. These methods are described in WO 90/07641. Polymerase chain reaction methodologies are well known in the art. Representative methods of RT-PCRTM are described in U.S. Patent No. 5,882,864.
  • Nucleic acids useful as templates for amplification are generated by methods described herein.
  • the DNA molecule from which the methods generate the nucleic acids for amplification may be isolated from cells, tissues or other samples according to standard methodologies (Sambrook et al , 1989).
  • the amplification product may be detected or quantified.
  • the detection may be performed by visual means.
  • the detection may involve indirect identification of the product via chemiluminescence, radioactive scintigraphy of an incorporated radiolabel or fluorescent label, or via a system using electrical and/or thermal impulse signals (Affymax technology).
  • a method of preparing a library of DNA molecules comprising obtaining a plurality of DNA molecules; randomly fragmenting at least one of the DNA molecules to produce DNA fragments; attaching a primer having a substantially known sequence to at least one end of a plurality of the DNA fragments to produce primer-linked fragments; and amplifying a plurality of the primer- linked fragments.
  • the method further comprises concomitantly sequencing the plurality of primer-linked fragments.
  • Libraries generated by DNA fragmentation and addition of an adaptor e.g., a universal adaptor
  • an adaptor e.g., a universal adaptor
  • the adaptor can be ligated to the 5' end, the 3' end, or both strands of DNA.
  • the adaptor can have a 3' or 5' overhang. It can also have a blunt end, especially in the cases when DNA ends are polished or conditioned after DNA fragmentation.
  • the terms “polished” and “conditioned” as used herein refers to the repair of dsDNA fragment termini that may be enzymatically repaired, wherein the repair constitutes the fill in of recessed 3' ends or the exonuclease activity trimming back of 5' ends to form a "blunt end” compatible with adaptor ligation.
  • Ligation-mediated PCR amplification is achieved by using a locus-specific primer (or several nested primers) and a primer complementary to the adaptor sequence.
  • an adaptor e.g., a universal adaptor
  • WGA whole genomic DNA
  • the adaptor can be ligated to both strands of DNA or only to the 3' end followed by extension.
  • the adaptor can have a 3' or 5' overhang, depending on the structure of the DNA end generated by fragmentation and repair. It can also have a blunt end, such as in the cases where DNA ends are repaired and polished or conditioned after fragmentation.
  • Whole genome PCR amplification is achieved by using one or two universal primers complementary to the adaptor sequence(s), in specific embodiments.
  • Adaptors are frequently used for DNA cloning (see, for example, Sambrook et al, 1989). Ligation of double stranded adaptors to DNA fragments produced by fragmentation, followed by restriction digestion within the adaptors allows production of DNA fragments with 3' or 5' protruding ends that can be efficiently introduced into a vector sequence and cloned.
  • each sample will be run on an Agilent Bioanalyzer to determine the molecular weight profile of the fragmented genomic DNA. The average and median molecular weights will be determined to confirm the ability of repair enzyme binding to increase the effectiveness of the sonication.
  • each of the samples will be made into ThruPLEX DNA-seq libraries and sequenced in order to determine whether the binding of the repair proteins increases the fraction of genomic DNA molecules that are successfully repaired and sequenced.

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Abstract

La présente invention concerne un procédé simultané de cisaillement et de réparation terminale d'acides nucléiques. Selon certains aspects, des protéines qui se lient aux terminaisons d'un acide nucléique peuvent être utilisées conjointement avec le cisaillement hydrodynamique afin de réduire la force de cisaillement requise pour fragmenter l'acide nucléique.
PCT/US2016/021503 2015-03-10 2016-03-09 Fragmentation et réparation améliorées d'acides nucléiques WO2016145039A1 (fr)

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WO2023004066A1 (fr) * 2021-07-23 2023-01-26 F. Hoffmann-La Roche Ag Procédés et dispositifs d'extraction d'acide nucléique par épitacophorèse

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20170283788A1 (en) * 2016-03-30 2017-10-05 Covaris, Inc. EXTRACTION OF cfDNA FROM BIOLOGICAL SAMPLES
US10781439B2 (en) * 2016-03-30 2020-09-22 Covaris, Inc. Extraction of cfDNA from biological samples
WO2023004066A1 (fr) * 2021-07-23 2023-01-26 F. Hoffmann-La Roche Ag Procédés et dispositifs d'extraction d'acide nucléique par épitacophorèse

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