WO2016145039A1 - Enhanced fragmentation and repair of nucleic acids - Google Patents

Abstract

Provided herein are method of simultaneous shearing and end-repair of nucleic acids. In some aspect, proteins that bind to the termini of a nucleic acid may be used in conjunction with hydrodynamic shearing to reduce the shear force required to fragment the nucleic acid.

Description

ENHANCED FRAGMENTATION AND REPAIR OF NUCLEIC ACIDS

This application claims benefit of priority to U.S. Provisional Application Serial No. 62/131,015 filed March 10, 2015, the entire contents of which are hereby incorporated by reference.

BACKGROUND OF THE INVENTION 1. Field of the Invention

[0001] 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

[0002] Cellular DNA is too long to sequence or amplify directly. 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. As the molecules are fragmented into smaller sizes, the rate of fragmentation decreases because as the length of the molecules is decreased so are the forces trying to "stretch" the molecules. Eventually after a long period of shearing with a constant power, the molecules are short enough that they no longer break into smaller molecules. The strong forces must be exerted for many minutes in order to produce fragments of the desired size (Bankier et al , 1987; Quail, 2010). [0003] To achieve high shearing forces, ultrasonic power is often focused into a small volume within a sample, and the bulk of the liquid is used to disperse heat. The non-uniform power density produces fragments of non-uniform length. The stronger and longer the hydrodynamic shearing is performed, the smaller the molecules become. 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. However, 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. Longer and stronger hydrodynamic shearing also leads to internal chemical damage to the nucleic acid molecules (e.g., deamination or oxidation of bases) or denaturation, which make accurate replication or sequencing of the nucleic acid problematic (Costello et al , 2013; Margolin et al , 2008; Ravanat et al, 2002; Finnegan et al, 1996; Lindahl, 1993; Bruskov et al , 2002; Kennedy et al , 1997; Fuciarelli et al, 1995; Milowska and Gabryelak, 2007; Burton and Ingold, 1981 ; Taghizadeh et al, 2008; Deininger, 1983). [0004] The field of molecular biology would benefit from methods to decrease direct and indirect damage caused by hydrodynamic shearing of nucleic acids and to increase the efficiency of repair of such direct and indirect damage.

SUMMARY OF THE INVENTION

[0005] Provided herein are methods to reduce the damage caused by fragmentation of nucleic acid molecules by decreasing the strength and duration of hydrodynamic shearing necessary to fragment nucleic acids while protecting the ends of the nucleic acids from indirect damage. Also provided herein are methods to increase the effectiveness of enzymes used to repair residual nucleic acid damage. The methods provided herein provide for more efficient shearing and repair as well as decreased hands-on and elapsed time to create DNA libraries for various applications, such as, for example, PCR, arrays, and sequencing.

[0006] In one embodiment, a method is provided 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. In some aspects, the shear force may be a hydrodynamic shear force, such as those generated by acoustic or mechanical means. In certain aspects, 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. In certain aspects, 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.

[0007] In some aspects, 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. For example, the protein may be a nucleic acid repair enzyme (e.g., T4 Polymerase, Klenow, and/or T4 polynucleotide kinase). In some aspects, 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. In various aspects, the second particle is an inert particle. An "inert particle" is a particle that lacks enzymatic activity, such as, for example, the protein albumin. In other certain aspects, 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. Thus, in certain aspects, the method may comprise incubating the nucleic acid fragments under conditions to allow for repair of the ends of the nucleic acid fragments.

[0009] In some aspects, 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. In some aspects, 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. In this aspect, 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. In certain aspects, a nucleic acid fragment produced by the method may be a ligation-competent DNA fragment. In some aspects, a ligation-competent DNA fragment may comprise a blunt end.

[0010] In some aspects, 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. In certain aspects, the method may be carried out in a single solution. In certain aspect, the process may occur in the absence of exogenous manipulation.

[0011] In some aspects, a method of fragmentation may be performed in the presence of an oligonucleotide adaptor, such as, for example, a stem-loop adaptor. In certain aspects, the oligonucleotide adaptor may lack a phosphate on its 5' end. In certain aspects, the adaptor may comprise a known sequence. In various aspects, the method may comprise attaching one strand of the oligonucleotide adaptor to the nucleic acid molecule to produce an oligonucleotide-attached nucleic acid fragment. In certain aspects, attaching may be further defined as ligating. In some aspects, ligating may comprise providing T4 DNA ligase to the adaptor and the nucleic acid fragment. In some aspects, attaching may produce a nick in the oligonucleotide-attached nucleic acid fragment. In certain aspects, the method may be carried out in a single solution. In certain aspect, the process may occur in the absence of exogenous manipulation. [0012] In some aspects, a nucleic acid molecule may be a double-stranded DNA, such as, for example, human genomic DNA.

[0013] In some aspects, 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.

[0015] 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. In further embodiments 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. In further embodiments the double-stranded nucleic acid molecules are comprised of complementary or partially complementary single strands. In further embodiments the first and second double-stranded molecules are products of the shearing reaction. In other embodiments the 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. In further embodiments the first double-stranded molecule is a product of the shearing reaction and the second double-stranded molecules is a stem-loop oligonucleotide. In further aspects, 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.

[0016] In an additional embodiment, there is a 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.

[0017] Additional embodiments of the invention include a library of DNA molecules prepared by the methods of the invention.

[0018] As used herein, "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.

[0019] As used herein the specification, "a" or "an" may mean one or more. As used herein in the claim(s), when used in conjunction with the word "comprising," the words "a" or "an" may mean one or more than one.

[0020] The use of the term "or" in the claims is used to mean "and/or" unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and "and/or." As used herein "another" may mean at least a second or more. [0021] Throughout this application, the term "about" is used to indicate that a value includes the inherent variation of error for the device, the method being employed to determine the value, or the variation that exists among the study subjects.

[0022] Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS [0023] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

[0024] 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). If both nucleic acid ends are bound to protein (or other particles), the greatest tension is produced; if only one end is bound to a protein, a lower tension is produced; and if neither end is bound to a protein, the tension is lowest. [0025] 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. DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

I. Aspects of the Present Invention

[0026] 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. Although 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. In a specific embodiment, the library members are sequenced concomitantly. In another specific embodiment, the library members are amplified concomitantly before being analyzed by sequencing, microarray, or PCR assays.

[0027] 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.

[0028] The present invention provides a method for the simultaneous shearing and end-repair of 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. In addition, the method may reduce the type and extent of damage done to the nucleic acid molecule during and consequent to shearing. Furthermore, 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.

[0029] In one aspect, 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.

[0030] 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. In some aspects, 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.

[0031] In specific embodiments, 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. [0032] 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.). 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.

II. Fragmentation of Nucleic Acids [0033] Some aspects of the present invention may comprise using at least one particle

(e.g. , protein) that binds to the terminus of a nucleic acid during random fragmentation, thereby accelerating the shearing process (see FIGS. 1A-C). 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 creation of a break in the nucleic acid will be quickly followed by the binding of particles(s) to the newly-created ends and thus allow one or more secondary shearing event(s) to take place (see FIG. 2). The use of specific nucleic acid- binding particles (e.g. , end repair proteins) to protect the ends of the nucleic acid during fragmentation will serve to control the size of the library molecules produced. The term "random fragmentation" as used herein 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.

[0034] 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. A. Methods of Fragmentation

[0035] 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. The term "double-stranded molecule" as used herein refers to a molecule that is double stranded at least in part. The shearing process may be computer controlled to select run parameters.

[0036] 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). For example, 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.

[0037] The damaging effects of strong hydrodynamic shearing can be largely overcome by using 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. For example, 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). As another example, point-sink shearing uses a syringe pump to create hydrodynamic shear forces by pushing a nucleic acid through a small abrupt contraction. For example, the commercially available devices HydroShear and HydroShear Plus (Digilab, Marlborough, MA) 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. In addition, the g-TUBE from Covaris or the Megaruptor from Diagenode can randomly fragment nucleic acids into 6-20 kb fragments.

[0038] However, such flow devices do not have sufficient force to break nucleic acids into the small fragments (e.g., <1000 bp) necessary for most sequencing instruments. Liquid flow through orifices is slower than ultrasonic shearing, in part because multiple passes through the orifice are usually required in order to achieve uniform molecular lengths of even thousands of bases. The advantage of liquid flow through orifices is that local heating and cavitation are avoided and therefore damage to the nucleic acid reduced. The methods of the present invention provide a means to make a nucleic acid more sensitive to hydrodynamic forces so that the more gentle methods of mechanical flow may be employed to break a large nucleic acid into small 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). Preferably, 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. [0039] 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.

B. Examples of Nucleic Acid Binding Proteins

[0040] Examples of 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.

[0041] 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. Examples of DNA polymerases include prokaryotic Pol I, Pol II, Pol III, Pol IV, and Pol V; eukaryotic Pol β, Pol a, telomerase; reverse transcriptase. Examples of polynucleotide kinases include T4 polynucleotide kinase. The term "ligase" as used herein 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. Examples of DNA ligases include E. coli DNA ligase, T4 DNA ligase, mammalian DNA ligases. Examples of 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.

III. Nucleic Acid End-Repair and Enzymatic Processing

[0042] While the mechanisms of 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. In addition, reducing the time that elapses between fragmentation of the nucleic acid and repair of the nucleic acid could have enormous benefit on reducing damage to the nucleic acid, for example by repairing the primary damage caused by the shearing process before the unstable reactive molecular species can create additional damage. [0043] Because shearing causes multiple types of damage to nucleic acids, current methods to make ligation-based libraries that involve initial shearing require terminal enzymatic repair. Examples of terminal enzymatic repair 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.

[0044] A skilled artisan will recognize that following fragmentation of DNA, the generated fragment molecules may require conditioning or repair, herein defined as modification of the ends to facilitate further processing of the fragment. For example, a 3' end may require conditioning following fragmentation, a 5' end may require conditioning following fragmentation, or both. In a specific aspect, the conditioning comprises modification of a 3' end lacking a 3' OH group. In an additional specific aspect 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. In a further aspect, an enzyme such as T4 polynucleotide kinase is used. In yet a further aspect, an exonuclease enzyme, such as exonuclease III, is used.

[0045] In a further specific embodiment, 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. The term "blunt end" as used herein 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. IV. Further Processing of Fragmented Nucleic Acids A. Ligation of Adaptors

[0046] 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.

[0047] Thus, in another specific embodiment, 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. In a specific embodiment, the adaptor comprises at least one blunt end. In another specific embodiment, the adaptor comprises a single stranded region. In another specific embodiment, the adaptor comprises a stem-loop structure. U.S. Patent No. 7,803,550 (incorporated herein by reference in its entirety) 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). In a further specific embodiment, 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.

[0048] The terms "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.

[0049] The adaptor, in a specific embodiment, comprises a substantially known sequence. A skilled artisan recognizes that "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. Thus, in specific embodiments, 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%.

[0050] 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.

[0051] 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.

B. Amplification

[0052] The term "primer," as used herein, 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. Typically, 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. [0053] "Oligonucleotide," as used herein, 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."

[0054] 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. Once hybridized, 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.

[0055] 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.

[0056] 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. In specific embodiments, 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. In a particular embodiment, the variable region is considered degenerate. [0057] A number of template-dependent processes are available to amplify the nucleic acids present in a given template sample. One of the best known amplification methods is the polymerase chain reaction (referred to as PCR™) which is described in detail in U.S. Patent Nos. 4,683,195, 4,683,202, and 4,800,159 and in Innis et al , 1990, each of which is incorporated herein by reference in their entirety. Briefly, 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. In a series (typically 30-35) of temperature cycles, 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. Thus, the template region between the two primers is amplified exponentially, rather than linearly.

[0058] "Amplification," as used herein, 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.

[0059] "Nucleotide," as used herein, 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.

[0060] 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. For example, 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. One may say that one incorporates dUTP into DNA even though there is no dUTP moiety in the resultant DNA. Similarly, one may say that one incorporates deoxyuridine into DNA even though that is only a part of the substrate molecule. [0061] 'Incoiporating," as used herein, means becoming part of a nucleic acid polymer.

[0062] A reverse transcriptase PCR™ (RT-PCR™) 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-PCR™ are described in U.S. Patent No. 5,882,864. [0063] Nucleic acids useful as templates for amplification are generated by methods described herein. In a specific embodiment, 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).

[0064] The amplification product may be detected or quantified. In certain applications, the detection may be performed by visual means. Alternatively, 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).

C. Library Preparation

[0065] In another object of the present invention, there is 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. In a specific embodiment, the method further comprises concomitantly sequencing the plurality of primer-linked fragments. V. Uses of Fragmented DNA

A. Ligation-Mediated Amplification of Unknown Regions Flanking a Known DNA Sequence

[0066] Libraries generated by DNA fragmentation and addition of an adaptor (e.g., a universal adaptor) to one or both DNA ends may be used to amplify (by PCR) and sequence DNA regions adjacent to a previously established DNA sequence (see, for example, U.S. Patent No. 6,777,187 and references therein, all of which are incorporated by reference herein in their entirety). 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.

B. Ligation-Mediated Whole Genome Amplification

[0067] Libraries generated by DNA fragmentation and subsequent attachment of an adaptor (e.g., a universal adaptor) to both DNA ends were used to amplify whole genomic DNA (whole genome amplification, or WGA) (see, for example, U.S. Patent Publn. No. 2004/0209299 and U.S. Patent No. 7,718,403 and references therein, all of which are incorporated by reference herein in their entirety). 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.

C. Adaptor-Mediated DNA Cloning

[0068] Adaptors (or linkers) 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.

VI. Examples

[0069] The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

Example 1 - Evaluation of Simultaneous Nucleic Acid Shearing and Repair

[0070] To demonstrate effective simultaneous nucleic acid shearing and repair, DNA and commercial repair enzymes will be combined in a single tube during shearing. To measure the magnitude of the improvement caused by the binding of repair proteins to the termini of dsDNA, the following experiment will be performed.

[0071] Six samples of high molecular weight genomic DNA from cell line NA12878 will be sonicated using the Covaris M220 instrument using the protocol recommended by the manufacturer to shear naked DNA to an average size of 600 bp. Two ionic conditions and three concentrations of the repair enzymes in the Rubicon ThruPLEX DNA-seq Kit (cat. # R400407) will be tested according to the matrix provided in Table 1.

Table 1. Test conditions.

[0072] After sonication, one aliquot of 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. In addition, 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.

* * *

[0073] All of the methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.

REFERENCES

The following references, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference.

U.S. Patent No. 4,683,195

U.S. Patent No. 4,683,202

U.S. Patent No. 4,800,159

U.S. Patent No. 5,882,864

U.S. Patent No. 6,197,557

U.S. Patent No. 6,777,187

U.S. Patent No. 6,828,098

U.S. Patent No. 7,718,403

U.S. Patent No. 7,757,561

U.S. Patent No. 7,803,550

U.S. Patent No. 7,981,368

U.S. Patent No. 8,071,312

U.S. Patent No. 8,263,005

U.S. Patent No. 8,353,619

U.S. Patent No. 8,399,199

U.S. Patent No. 8,459,121

U.S. Patent No. 8,728,737

U.S. Appln. Serial No. 14/250,538

U.S. Patent Publn. No. 2004/0209299

U.S. Patent Publn. No. 2012/0264228

U.S. Patent Publn. No. 2012/0077283

PCT Publn. No. WO 90/07641

Bankier et al., Random cloning and sequencing by the M13/dideoxynucleotide chain termination method. Methods in Enzymology, 155:51-93, 1987.

Bruskov et al., Heat-induced formation of reactive oxygen species and 8-oxoguanine, a biomarker of damage to DNA. Nucleic Acids Res., 30: 1354-1363, 2002. Burton and Ingold, Autoxidation of biological molecules. 1. Antioxidant activity of vitamin E and related chain-breaking phenolic antioxidants in vitro. J. Am. Chem. Soc , 103:6472-6477, 1981.

Costello et al , Discovery and characterization of artifactual mutations in deep coverage targeted capture sequencing data due to oxidative DNA damage during sample preparation, Nuc. Acids Res. , 41 :e67, 2013.

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Claims

WHAT IS CLAIMED IS;

1. A method of 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.

2. The method of claim 1, wherein the shear force is a hydrodynamic shear force.

3. The method of claim 2, wherein the hydrodynamic shear force is generated by acoustic or mechanical means.

4. The method of claim 1, wherein the nucleic acid molecule is a purified nucleic acid molecule.

5. The method of claim 1, wherein the nucleic acid molecule is essentially free of chromatin proteins.

6. The method of claim 1, wherein the particle-bound nucleic acid molecule does not comprise particles covalently bound to the nucleic acid molecule.

7. The method of claim 1, wherein the nucleic acid fragments have an average size of about 200 basepairs (bp), about 300 bp, about 400 bp, about 500 bp, about 1000 bp, or about 2000 bp.

8. The method of claim 1, wherein the nucleic acid molecule has 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.

9. The method of claim 1, wherein the particle that selectively binds to a terminus of the nucleic acid molecule is a protein.

10. The method of claim 9, wherein the protein is a nucleic acid repair enzyme.

11. The method of claim 10, wherein the nucleic acid repair enzyme is bound to a second particle.

12. The method of claim 11, wherein the second particle is a protein, a high-molecular weight polymer, or a nanoparticle.

13. The method of claim 11, wherein the second particle is bound to the nucleic acid repair enzyme covalently.

14. The method of claim 11, wherein the second particle is bound to the nucleic acid repair enzyme non-covalently.

15. The method of claim 11, wherein the second particle is an inert molecule.

16. The method of claim 10, wherein the nucleic acid end-repair enzyme is T4 Polymerase, Klenow, and/or T4 polynucleotide kinase.

17. The method of claim 16, wherein the nucleic acid fragment is a ligation-competent DNA fragment.

18. The method of claim 17, wherein the ligation-competent DNA fragment comprises a blunt end.

19. The method of claim 9, wherein the protein is bound to a bead.

20. The method of claim 1, wherein the particle that selectively binds to a terminus of the nucleic acid molecule is a bead.

21. The method of claim 20, wherein the method is performed in the presence of at least one nucleic acid repair enzyme.

22. The method of claim 21, further comprising incubating the nucleic acid fragments under conditions to allow for repair of the ends of the nucleic acid fragments.

23. The method of claim 1, wherein the method is 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.

24. The method of claim 23, wherein the method is carried out in a single solution.

25. The method of claim 23, wherein the process occurs in the absence of exogenous manipulation.

26. The method of claim 16, wherein the method is performed in the presence of an oligonucleotide adaptor.

27. The method of claim 26, wherein the oligonucleotide adaptor is a stem-loop adaptor.

28. The method of claim 26, wherein the oligonucleotide adaptor lacks a phosphate on its 5' end.

29. The method of claim 26, wherein the adaptor comprises a known sequence.

30. The method of claim 26, wherein the adaptor comprises a random barcode sequence.

31. The method of claim 26, further comprising attaching one strand of the oligonucleotide adaptor to the nucleic acid fragment to produce an oligonucleotide-attached nucleic acid fragment.

32. The method of claim 31, wherein attaching is further defined as ligating.

33. The method of claim 32, wherein ligating comprises providing T4 DNA ligase to the adaptor and the nucleic acid fragment.

34. The method of claim 31, wherein attaching produces a nick in the oligonucleotide- attached nucleic acid fragment.

35. The method of claim 31, wherein the method is carried out in a single solution.

36. The method of claim 31, wherein the process occurs in the absence of exogenous manipulation.

37. The method of claim 1, wherein the nucleic acid molecule is double-stranded DNA.

38. The method of claim 37, wherein the double-stranded DNA is human genomic DNA.

39. The method of claim 1, further comprising preparing a library of nucleic acid fragments.

40. The method of claim 39, further comprising determining at least a partial sequence of a plurality of the library of nucleic acid fragments.

41. The method of claim 1 , further comprising amplifying at least one of the nucleic acid fragments.

42. The method of claim 41 , further comprising determining at least a partial sequence of at least one or more of the nucleic acid fragments.