FIELD

[0001] The present invention relates to compositions and methods for DNA sequencing and sequencing library preparation.

GOVERNMENT SUPPORT CLAUSE

[0002] This invention was made with Government support under contracts CA193694 and CA233254 awarded by the National Institutes of Health. The Government has certain rights in the invention.

BACKGROUND

[0003] DNA sequencing is a large and growing field of biotechnology with uses in both research and clinical practice. The throughput and cost of sequencing have decreased so much in recent years that the labor time of preparing sequencing libraries from DNA may be a more substantial concern than the sequencing itself. The preparation of a DNA library suitable for sequencing generally requires multiple enzymatic modifications of the DNA molecules performed sequentially, often requiring intervening “cleanup” steps to remove the reagents and/or byproducts of a previous reaction. For example, in order to be “sequencing ready”, genomic DNA or other template DNA molecules must undergo a series of modifications including fragmentation into smaller sizes suitable for amplification or cloning, optional “end repair”, A-tailing to incorporate or leave a single adenine overhang at the 3' ends of the DNA molecules, ligation of adapter oligonucleotides to the ends of the DNA molecules, degradation or removal of excess adapter molecules, and amplification of the DNA, typically using a polymerase chain reaction ("PCR") based method.

[0004] With respect to genomic DNA, the initial three steps of DNA fragmentation/end repair/A- tailing are required to produce DNA with ends suitable for adapter ligation and PCR, that is double-stranded DNA with 5 ’-phosphate and 3 ’-hydroxyl groups at the terminal ends. While current methods provide for (i) fragmentation/end repair/A-tailing and (ii) adapter ligation/degradation/PCR to be performed in a single container, the workflows involve multiple distinct steps often including a “cleanup” step to remove unligated adapter oligonucleotides following ligation and prior to amplification. Typically, the enzymes for ligation, adapter degradation, and PCR are kept separate due to the need for one or more intervening “cleanup” steps, and these reactions are performed at separate times.

[0005] There is a need to further streamline the process of preparing a DNA library to be suitable for sequencing. The present invention addresses this need.

BRIEF SUMMARY

[0006] The present invention provides methods and related compositions that advantageously simplify the preparation of DNA for sequencing by providing a single reaction mixture and a single set of reaction conditions for ligation of adapter oligonucleotides, degradation of unligated adapter molecules, and amplification of DNA-adapter molecules.

[0007] In one aspect, the invention provides a method for producing a sequencing ready DNA library, the method including forming a reaction mixture by contacting a plurality of DNA fragments with an enzyme mixture that includes a DNA ligase, a uracil-DNA glycosylase, a DNA polymerase, a set of double-stranded adapter oligonucleotides such as stem loop adapter oligonucleotides, a set of polymerase amplification primers complementary to at least a portion of the adapter oligonucleotides, deoxynucleoside triphosphates (dNTPs), adenosine triphosphate (ATP) or nicotinamide adenine dinucleotide (NAD), and a buffer suitable for activity of the polymerase, glycosylase, and ligase, where the DNA fragments of the plurality consist of double-stranded DNA molecules having terminal 5’phosphate and 3’hydroxyl groups, optionally with a single adenine overhang at each 3' end, subjecting the reaction mixture to conditions suitable for activity of the DNA ligase, thereby producing adapter-linked DNA fragments, subjecting the reaction mixture to conditions suitable for activity of the uracil- DNA glycosylase, subjecting the reaction mixture to conditions suitable for activity of the DNA polymerase, thereby amplifying the adapter-linked DNA fragments, thereby producing a sequencing ready DNA library.

[0008] The method may also include where the conditions suitable for activity of the DNA ligase include a temperature of from 4-50°C or from 4-37°C. In embodiments, the conditions suitable for activity of the DNA ligase include a temperature of about 4°C, about 16°C, about 25°C, or about 37°C.

[0009] The method may also include where the conditions suitable for activity of the uracil- DNA glycosylase include a temperature above 50°C, or from 65-98°C. The method may also include where the uracil-DNA glycosylase is active only at temperatures higher than a temperature at which the DNA ligase is active.

[0010] The method may also include where the conditions suitable for activity of the DNA polymerase include a series of extensions at a temperature of from 37-72°C and denaturations a temperature of from 95-98°C.

[0011] The method may also include where the double stranded or stem loop adapter oligonucleotide includes deoxyuridine residues. In some embodiments, the double stranded or stem loop adapter oligonucleotide includes a plurality of deoxyuridine residues located in the 5' stem region and/or in the loop region.

[0012] The method may also include where there is no intervening post-ligation manipulation prior to amplifying the adapter-linked DNA fragments.

[0013] The method may also further include, in the enzyme mixture, a solid support having attached thereto a plurality of oligonucleotides including a 3' end complementary to a region of the adapter oligonucleotides.

[0014] The method may also further include, before the step of forming a reaction mixture, forming a plurality of DNA fragments from the double stranded template DNA. In embodiments, the double stranded template DNA is selected from genomic DNA, cDNA, cell- free DNA, amplicons, e.g., PCR amplicons, and DNA selected by an enrichment method such as immunoprecipitation.

[0015] The method may also include where the forming a plurality of DNA fragments from template DNA includes subjecting template DNA to digestion by one or more endonucleases, including restriction endonucleases. Other technical features may be readily apparent to one skilled in the art from the following figures, descriptions, and claims.

[0016] In another aspect, the invention provides a kit of parts including a first container that includes an enzyme mixture, the enzyme mixture including a DNA ligase, a uracil-DNA glycosylase, and a DNA polymerase; a second container including a reactant mixture, the reactant mixture includes deoxynucleoside triphosphates, adenosine triphosphate (ATP) or nicotinamide adenine dinucleotide (NAD), and a source of divalent cation such as magnesium; and an optional third container including double stranded or stem loop adapter oligonucleotide each having a plurality of deoxyuridine residues, optionally located in the 5' stem region and/or in the loop region, and optionally a set of polymerase amplification primers complementary to at least a portion of the adapter oligonucleotides. BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

[0017] Non-limiting embodiments of the present disclosure are described by way of example with reference to the accompanying drawings, which are schematic and not intended to be drawn to scale. The accompanying drawings are provided for purposes of illustration only, and the dimensions, positions, order, and relative sizes reflected in the figures in the drawings may vary. In the figures, identical or nearly identical or equivalent elements are typically represented by the same reference characters, with redundant description omitted. For purposes of clarity and simplicity, not every element is labeled in every figure, nor is every element of each embodiment shown where illustration is not necessary to allow those of ordinary skill in the art to understand the disclosure. The detailed description will be better understood in conjunction with the accompanying drawings as follows.

[0018] FIG. 1 is a flow chart illustrating the steps in a standard protocol for making a sequencing ready library from genomic DNA.

[0019] FIG. 2 is a flow chart illustrating the steps in a protocol for making a sequencing ready library from genomic DNA with reduced steps compared to the standard protocol.

[0020] FIG. 3 is a flow chart illustrating the steps in a protocol for making a sequencing ready library from genomic DNA in accordance with one embodiment.

[0021] FIG. 4 is a flow chart illustrating the steps in a protocol for making a sequencing ready library from genomic DNA in accordance with a second embodiment.

[0022] FIG. 5 shows comparative results of amplification of a DNA library using a uracil- DNA glycosylase (UDG) from either Escherichia coli (“low temp”, triangles) or Archaeoglobus fulgidus (“high temp”, circles) under a “two step” or “separate steps” protocol (left panel) and a “single step” or "combined steps" protocol (right panel).

DETAILED DESCRIPTION

[0023] The present invention provides new methods and related compositions for adapter ligation and amplification of DNA molecules to produce a sequencing ready DNA library. [0024] In particular, the disclosure provides methods and related kits for producing a sequencing ready DNA library by performing ligation and amplification in a single combined step, rather than in two separate steps. The methods described here advantageously provide for streamlining library production by performing adapter ligation and library amplification in a single reaction mixture, without the need for one or more intervening steps to remove unreacted materials, enzymes, buffers, etc., referred to herein as “cleanup” steps.

[0025] In embodiments, the methods comprise contacting a plurality of double stranded template DNA fragments with a single mixture of three enzymes in a buffer suitable for activity of each of the three enzymes, the three enzymes including a DNA ligase, a uracil-DNA glycosylase, and a DNA polymerase.

[0026] In embodiments, the methods comprises providing, in a single container, a plurality of double stranded template DNA fragments and contacting the plurality of DNA fragments with a single mixture of three enzymes in a buffer suitable for activity of the three enzymes, the three enzymes including a DNA ligase, a uracil-DNA glycosylase, and a DNA polymerase.

[0027] In accordance with the methods described here, the DNA fragments of the plurality consist of double-stranded DNA molecules having terminal 5’phosphate and 3 ’hydroxyl groups, optionally with a single adenine overhang at each 3' end. Suitable DNA fragments may be produced, for example, by random chemical fragmentation, by sonication, or by digestion with endonucleases, including restriction endonucleases. Terminal 5’phosphate and 3 ’hydroxyl groups which may be generated simultaneously with fragmentation, for example by restriction endonuclease digestion, or may be generated in a separate step, for example using end repair. “End repair” may involve for example using a DNA polymerase to fill in or digest overhangs and using a polynucleotide kinase to phosphorylate 5’ ends and dephosphorylate 3’ ends. The termini of the DNA fragments may be either blunt ends, with or without A-tailing, or overhangs, including single 3' adenine overhangs, or other “sticky ends” including short overhangs from one to several bases in length, for example from 1 -15 bases, or from 1-10 bases, or from 1-5 bases in length.

[0028] In accordance with an embodiment of the methods described here, a reaction mixture is formed when the plurality of DNA fragments is contacted with the single enzyme mixture comprising a DNA ligase, a uracil-DNA glycosylase, and a DNA polymerase. The reaction mixture also contains appropriate primers and adapter oligonucleotides containing deoxyuridine, for example, stem loop adapter oligonucleotides containing deoxyuridine located, for example, in the 5' stem region and/or in the loop region of the adapter, and polymerase amplification primers complementary to at least a portion of the adapter oligonucleotides. The reaction mixture also contains appropriate reagents for performing an adapter ligation reaction and a DNA amplification reaction, for example deoxynucleoside triphosphates (dNTPs), polyethylene glycol, a source of magnesium such as magnesium chloride, and either adenosine triphosphate (ATP) or nicotinamide adenine dinucleotide (NAD).

[0029] In accordance with the methods described here, the reaction mixture is subjected to conditions suitable for activity of the DNA ligase, thereby producing adapter-linked DNA molecules; followed by conditions suitable for activity of the uracil-DNA glycosylase, thereby excising uracil and generating abasic sites in the adapters which function as replication stops; and conditions suitable for activity of the DNA polymerase, thereby producing and amplifying the adapter-linked DNA molecules in a single reaction mixture without an intervening postligation cleanup step prior to amplification. In embodiments, the activities of the uracil-DNA glycosylase and the DNA polymerase are concurrent or sequential.

[0030] In accordance with the methods described here, adapter oligonucleotides are incorporated at each terminal end of the fragmented DNA molecules to provide a target site for the amplification primers and, optionally, for sequencing primers.

[0031] In embodiments, the adapter oligonucleotides are stem-loop oligonucleotides. The stem-loop structure, also referred to as a hairpin, is formed by the intramolecular base pairing between two complementary regions of the oligonucleotide, which may also be referred to as inverted repeat or palindromic regions, i.e., a region that is the same as its reverse complement. The stem-loop adapters contain one or more deoxyuridine residues which are excised by the uracil-DNA glycosylase after adapter ligation and before amplification. This base excision produces abasic sites along the phosphate backbone of the DNA strand. An abasic site, which may also be referred to as an apurinic/apyrimidinic site, is a location in DNA that lacks a purine or a pyrimidine base. In embodiments, these abasic sites are cleaved by a second enzyme with apurinic/apyrimidinic lyase activity such as endonuclease VIII or by spontaneous hydrolysis at high temperature, e.g., a denaturation temperature such as a temperature above 90 or 95°C. Following cleavage, the former loop becomes two non-complementary strands to which two distinct PCR primers can be annealed for library amplification. In another embodiment, the one or more deoxyuridine residues are utilized to introduce at least one replication stop into the adapter molecule during polymerase chain reaction amplification of the library via conversion of deoxyuridine residues into abasic sites through the activity of the uracil-DNA glycosylase. The reaction proceeds through ligation of the stem-loop adapter oligonucleotides to the ends of the DNA fragments. Adapter degradation and fill-in are performed by the uracil-DNA glycosylase and DNA polymerase enzymes, respectively. Thus, the ligase enzyme attaches a 3' end of a stem-loop oligonucleotide to a 5' phosphate of a DNA fragment producing an adaptor-linked DNA fragment comprising a nick having a 3' hydroxyl group. During amplification of the resulting nicked adapter-DNA fragments, the polymerase displaces the 5' portion of the adapter from the nicked site and extends the 3' end of the DNA fragment until it reaches a non-replicable abasic gap, e.g., within the adapter loop region. The denaturation step of the PCR cycle creates breaks at the abasic sites and eliminates the 5' portion of the stem-loop oligonucleotide. Alternatively, in isothermal amplification, a hot denaturation step may be introduced for this purpose. This provides a mechanism for elimination of the inverted repeat of the hairpin structure from the ends of the ligated DNA- adapter molecules and prevents the formation of amplifiable adapter dimers.

[0032] In embodiments, the adapter-linked DNA fragments are attached to a solid support. For example, following formation of adapter-linked DNA fragments comprising a nick having a 3' hydroxyl group, polymerization of the adapter-linked DNA fragment to the abasic gap generates a 5' overhang which hybridizes to a complementary oligonucleotide covalently attached to the solid support, thereby forming adapter-linked DNA fragments attached to the solid support.

[0033] Examples of stem-loop adapter oligonucleotides that may be used include, for example, those disclosed in US 7,803,550.

[0034] In accordance with the methods described here, the uracil-DNA glycosylase is selected to be active only at temperatures higher than those required for the DNA ligase activity, for example higher than 16°C, or higher than 25°C, or higher than 37°C, or higher than 50°C. An exemplary uracil-DNA glycosylase for use in the claimed methods is produced by the hyperthermophile Archaeoglobus fulgidus and is commercially available, for example from New England Biolabs.

[0035] Any suitable DNA polymerase may be used to perform DNA amplification in accordance with the methods described here. For example, the polymerase may be a thermostable DNA polymerase, or one suitable for isothermal amplification, including loop- mediated isothermal amplification (LAMP). Exemplary thermostable DNA polymerases suitable for use in the methods described here include Taq polymerase, Tfl polymerase, Tth polymerase, Pfu polymerase, and Pfx polymerase, and modified versions of the foregoing. Other exemplary polymerases include Bst polymerase and phi29 polymerase, and modified versions thereof. A suitable DNA polymerase for performance of DNA amplification in accordance with the methods described here is not required to have exonuclease activity, since nick extension proceeds by strand displacement.

[0036] Any suitable DNA ligase may be used to perform ligation of the adapter oligonucleotides to the DNA fragments in accordance with the methods described here. An exemplary DNA ligase for use in the methods described here is a T4 DNA ligase. Other exemplary DNA ligases include T3 DNA ligase, T7 DNA ligase, E. Coli DNA ligase, etc. [0037] The methods and compositions described here can be incorporated into a workflow to provide a DNA library suitable for sequencing from genomic DNA. In embodiments, the methods and compositions described here may also be incorporated into other methods, for example a method of DNA methylation profiling using a restriction endonuclease that specifically cuts at methylated DNA; and a three-step RAD-seq protocol, in which restriction digest products are sequenced for narrowly targeted genome coverage.

[0038] An exemplary workflow incorporating the compositions and methods described here is illustrated in Foley et al. 2023, which describes a method of DNA methylation profiling incorporating aspects of the methods described here for library production. The library protocol utilizes sequencing adapters with 5' overhangs of 4 random bases (4N) for efficient sticky-end ligation to the corresponding overhangs of unknown bases resulting from digestion by a restriction endonuclease whose motif includes methylated cytosine. The stem sequence’s uracils are excised before PCR by uracil-DNA glycosylase (UDG), leaving abasic sites that further deter replication and may be fully destroyed by hydrolysis at PCR denaturation temperature, thereby helping to prevent formation of adapter dimers. The library protocol specifies the use of UDG from the hyperthermophile Archae globus fulgidus in a combined ligation/loop-breaking/PCR master mix. As discussed above, the traditional Escherichia coli UDG interferes with the low-temperature ligation and would require an additional step between ligation and PCR, whereas the hyperthermophilic UDG is inert at ligation temperatures.

[0039] FIG. 1 depicts nine sequential steps forming an exemplary standard protocol in the prior art for making a sequencing ready library starting from genomic DNA. In the first step, DNA fragmentation 102, genomic DNA is broken into a range of smaller fragments by any one of several methods. For example, genomic DNA may be broken into fragments having a range of size distributions, for example less than 3 kilobasepairs (kb), less than 2 kb, or less than 1 kb, for example in a range of from 100-250 bp, from 150-350 bp, from 200-450 bp, from 300- 700 bp, or from 500 bp to 1 kb. Suitable DNA fragments may be produced, for example, by random chemical fragmentation, by sonication, or by digestion with endonucleases using standard protocols such as those described in Molecular Cloning: A l aboratory Manual (Fourth Edition) 2012 Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. In an optional second step, End repair 104, the ends of the DNA fragments are modified to generate terminal 5’phosphate and 3’hydroxyl groups. This step is optional where endonucleases are used to fragment the DNA. At this stage, the termini of the DNA fragments may be either blunt ends or single or multiple base overhangs, depending on the type of endonuclease used in the first or optional second step. Following fragmentation and end repair, the ends of the fragmented DNA are further modified to include a single adenine overhang at each 3' end to facilitate ligation of adapter oligonucleotides using a process referred to as A-tailing 106 or dA-tailing. Standard protocols for end repair and A-tailing may also be found, for example, in Molecular Cloning: A Laboratory Manual (Fourth Edition) 2012 Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Prior to the next step of adapter ligation 110 a cleanup 108 step is performed to remove reagents and enzymes from the fragmentation, end repair, and A-tailing steps. A futher cleanup 112 step is performed after adapter ligation 110 in order to remove unligated adapter oligonucleotides prior to amplification 114. Amplification may by a polymerase chain reaction (PCR) or by an isothermal amplification method, including loop-mediated isothermal amplification (LAMP). A final cleanup 116 follows amplification. [0040] FIG. 2 depicts the six sequential steps forming an exemplary prior art protocol in which DNA fragmentation, end repair, and A-tailing are performed 202 in a single container in a single incubation with one reaction mixture containing the necessary enzymes and reagents. The next step of adapter ligation 204 is performed in a second incubation in the same container after addition of a mixture containing ligase, adapter oligonucleotides, and other required reagents, such as salts. Following adapter ligation, unligated adapter oligonucleotides are degraded 206 in a third incubation followed by a cleanup 208 step to remove degraded oligos, enzymes and other materials from the earlier steps, leaving the DNA fragments ligated to adapter oligonucleotides. The DNA fragments are then subjected to amplification 210 by a polymerase chain reaction, followed by a final cleanup 212 step. An exemplary prior art protocol of this type is described in the New England Biolabs NEBNEXT Ultra II FS kit.

[0041] FIG. 3 depicts one exemplary protocol for preparing a sequencing ready DNA library utilizing the methods and compositions described here. In this example, the input fragmented DNA having terminal 5 ’phosphate and 3 ’hydroxyl groups and a single adenine overhang at each 3' terminal end is prepared using a prior art protocol for performing DNA fragmentation, end repair, and A-tailing in a single incubation 302. Suitable enzymes and protocols for performing this step 302 are commercially available, for example from New England Biolabs. In the second step 304, a reaction mixture containing a DNA ligase, a uracil-DNA glycosylase, and a DNA polymerase in a buffer suitable for activity of all three enzymes is added to the container of fragmented DNA, without any intervening cleanup step. The reaction mixture also contains appropriate amplification primers and adapter oligonucleotides, for example, stem loop adapter oligonucleotides containing deoxyuridine and amplification primers complementary to at least a portion of the adapter oligonucleotides. The reaction mixture also contains appropriate reagents for performing the ligation and amplification reactions, for example deoxynucleoside triphosphates (dNTPs), adenosine triphosphate (ATP), and a source of magnesium. DNA amplification is followed by a final cleanup 306. DNA amplification may by a polymerase chain reaction (PCR) or by an isothermal amplification method, including loop-mediated isothermal amplification (LAMP).

[0042] FIG. 4 depicts another exemplary protocol for preparing a sequencing ready DNA library utilizing the methods and compositions described here. In this example, the input fragmented DNA having terminal 5 ’phosphate and 3 ’hydroxyl groups and a single adenine overhang at each 3' terminal end is prepared using a Restriction Enzyme Digestion 402. In the second step 404 a reaction mixture containing a DNA ligase, a uracil-DNA glycosylase, and a DNA polymerase in a buffer suitable for activity of all three enzymes is added to the container of fragmented DNA, without any intervening cleanup step. The reaction mixture also contains appropriate amplification primers and adapter oligonucleotides, for example, stem loop adapter oligonucleotides containing deoxyuridine and amplification primers complementary to at least a portion of the adapter oligonucleotides. The reaction mixture also contains appropriate reagents for performing the ligation and amplification reactions, for example deoxynucleoside triphosphates (dNTPs), adenosine triphosphate (ATP), and a source of magnesium. DNA amplification is followed by a final cleanup 406. DNA amplification may by a polymerase chain reaction (PCR) or by an isothermal amplification method, including loop-mediated isothermal amplification (LAMP).

[0043] FIG. 5 shows the results of a comparative example of an embodiment of the present invention and a standard “two step” protocol. Human genomic DNA from the Jurkat cell line was digested with MspJI restriction endonuclease (New England Biolabs) according to the manufacturer’s instructions. To the digestion product a second reaction mix was added, containing the ligation reagents T4 DNA ligase, PEG 4000, and ATP (Thermo Fisher); PCR buffer, hot-start polymerase, and dNTPs (Roche); PicoGreen and ROX dyes (Thermo Fisher); a pair of stem-loop adapters with a 5’ 4 nucleotide overhang and uracil replacing thymine in the 5’ portion of the stem and a pair of PCR primers matching the adapter sequences (Integrated DNA Technologies); and uracil-DNA glycosylase (UDG) from either Escherichia coli (“low temp”) or Archaeoglobus fulgidus (“high temp”) (New England Biolabs).

[0044] The samples were incubated 5 min at 16 C then transferred to a qPCR thermal cycler and incubated 3 min at 72 C, 2 min at 85 C, and 2 min at 98 C before a standard PCR program according to the manufacturer’s instructions, with fluorescence readings taken each cycle. Two protocols were performed. In the first protocol, only the ligation reagents and adapters were added before the 16 C incubation, followed by the remaining reagents before the qPCR program. In the second protocol, all of the reagents were added at the same time, before the 16 C incubation. The results of the first protocol are depicted in the left panel of FIG. 5 (separate steps), which shows similar performance for both UDG enzymes when the ligation and amplification steps were performed separately. However, in the second protocol, depicted in the right panel of FIG. 5 (combined steps) there was a significant loss of yield when the E. coli UDG was present during ligation while the A. fulgidus UDG achieved similar yields in the combined steps protocol compared to the separate steps protocol. This shows that the commonly used low-temperature UDG from E. coli is unsuitable for a combined steps protocol and further demonstrates that the combined steps protocol is made possible by substituting the high-temperature UDG from A. fulgidus, which has no loss of yield when the protocol is simplified from two separate steps of ligation and amplification to a single combined step in which both ligation and amplification are performed, either simultaneously or sequentially in the same container.

[0045] While various embodiments and aspects of the present invention are shown and described herein, it will be obvious to those skilled in the art that such embodiments and aspects are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. [0046] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. All documents, or portions of documents, cited in the application including, without limitation, patents, patent applications, articles, books, manuals, and treatises are hereby expressly incorporated by reference in their entirety for any purpose.

[0047] The abbreviations used herein have their conventional meaning within the chemical and biological arts. The chemical structures and formulae set forth herein are constructed according to the standard rules of chemical valency known in the chemical arts.

[0048] While the invention herein disclosed has been described by means of specific embodiments and applications thereof, numerous modifications and variations could be made thereto by those skilled in the art without departing from the scope set forth in the claims.

[0049] It will be appreciated that the present invention is set forth in various levels of detail in this application. In certain instances, details that are not necessary for one of ordinary skill in the art to understand the invention, or that render other details difficult to perceive may have been omitted. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting beyond the scope of the appended claims. Unless defined otherwise, technical terms used herein are to be understood as commonly understood by one of ordinary skill in the art to which the disclosure belongs.

[0050] It should be understood that, as described herein, an “embodiment” (such as illustrated in the accompanying Figures) may refer to an illustrative representation of a process or article or component in which a disclosed concept or feature may be provided or embodied, or to the representation of a manner in which just the concept or feature may be provided or embodied. However such illustrated embodiments are to be understood as examples (unless otherwise stated), and other manners of embodying the described concepts or features, such as may be understood by one of ordinary skill in the art upon learning the concepts or features from the present disclosure, are within the scope of the disclosure. Thus, it is intended that the present subject matter covers such modifications and variations as come within the scope of the appended claims and their equivalents.

[0051] The presently disclosed embodiments are to be considered in all respects as illustrative and not restrictive, the scope of the claimed subject matter being indicated by the appended claims, and not limited to the foregoing description or particular embodiments or arrangements described or illustrated herein. [0052] In the foregoing description and the following claims, the following will be appreciated. The phrases “at least one”, “one or more”, and “and/or”, as used herein, are open- ended expressions that are both conjunctive and disjunctive in operation. The terms “a”, “an”, “the”, “first”, “second”, etc., do not preclude a plurality. For example, the term “a” or “an” entity, as used herein, refers to one or more of that entity. As such, the terms “a” (or “an”), “one or more” and “at least one” can be used interchangeably herein.

[0053] The term “about” when used before a numerical designation, e.g., temperature, time, amount, concentration, and such other, including a range, indicates approximations which may vary by ( + ) or ( - ) 10%, 5%, 1%, or any subrange or subvalue there between. Preferably, the term “about” means that the value may vary by +/- 10%.

[0054] The term “comprises/comprising” does not exclude the presence of other elements, components, features, regions, integers, steps, operations, etc. Additionally, although individual features may be included in different claims, these may possibly advantageously be combined, and the inclusion in different claims does not imply that a combination of features is not feasible and/or advantageous. By contrast, the transitional phrase “consisting of’ excludes any element, step, or ingredient not specified in the claim. The transitional phrase “consisting essentially of’ limits the scope of a claim to the specified materials or steps “and those that do not materially affect the basic and novel characteristic(s)” of the claimed invention.

[0055] The term “complement,” refers to a nucleotide (e.g., RNA or DNA) or a sequence of nucleotides capable of base pairing with a complementary nucleotide or sequence of nucleotides. Complementarity is determined by the ability of an associated nitrogenous base of a nucleotide, also referred to as a “nucleobase” or simply a “base”, to hydrogen bond with the nitrogenous base of a different nucleotide, e.g., a nucleotide on a different nucleic acid. This interaction may also be referred to as “base pairing”. The base adenine binds to thymine or uracil and the base guanine binds to cytosine. Adenine may therefore be referred to as the complement of thymine or uracil and guanine may be referred to as the complement of cytosine, and vice versa. Thus, a complement may include a sequence of nucleotides that base pair with corresponding complementary nucleotides of a second nucleic acid sequence.

[0056] The term “contacting” is used in accordance with its plain ordinary meaning and refers to the process of allowing at least two distinct species such as chemical compounds, biomolecules, and enzymes, to become sufficiently proximal to react, interact or physically touch. [0057] The term “endonuclease” refers to an enzyme which possesses endonucleolytic catalytic activity for polynucleotide cleavage. For example, an endonuclease can cleave a phosphodiester bond of an oligonucleotide or polynucleotide. An endonuclease cleaves at a phosphodiester bond within or adjacent to its recognition site sequence, which spans at least 4 base pairs in length. Types of endonucleases include, but are not limited to restriction enzymes, AP endonuclease, T7 endonuclease, T4 endonuclease, Bal 31 endonuclease, Endonuclease I, etc.

[0058] The term “nucleic acid” refers to a polymer of nucleotides, e.g., deoxyribonucleotides or ribonucleotides, and may be used herein as shorthand for deoxyribonucleic acid (DNA) or ribonucleic acid (RNA).

[0059] The term “nucleoside” refers, in the usual and customary sense, to a glycosylamine including a nitrogenous base, also referred to as a “nucleobase”, and a five-carbon sugar, z.e., ribose or deoxyribose. Non limiting examples of nucleosides include cytidine, uridine, adenosine, guanosine, thymidine and inosine.

[0060] The term “nucleotide” refers, in the usual and customary sense, to the monomeric units of nucleic acids, each unit consisting of a nucleoside and a phosphate.

[0061] The term “base” as used herein with reference to sequences of nucleic acids refers to the nucleobase moiety of the nucleoside, e.g., cytosine, adenine, guanine, thymine, and uracil.

[0062] The terms “oligonucleotide,” “nucleic acid sequence,” and “polynucleotide” are used interchangeably and are intended to include a polymeric form of nucleotides covalently linked together that may have various lengths, either deoxyribonucleotides or ribonucleotides, or analogs, derivatives or modifications thereof. An oligonucleotide is typically composed of a sequence of nucleotides comprising nucleobases selected from adenine (A), cytosine (C), guanine (G), thymine (T), and uracil (U). Thus, the term “polynucleotide sequence” may refer to the alphabetical representation of a polynucleotide molecule; alternatively, the term may be applied to the polynucleotide molecule itself.