Patent Application
20170114333
The current invention relates to mutated transposases and methods to use them for fragmenting and tagging target DNA for use in next generation DNA sequencing.
Transposons, segments of DNA that can mobilize to other locations in a genome, are useful for insertion mutagenesis and for generation of priming sites for sequencing of DNA molecules. In vitro, transpositions using transposases and transposons can be used to generate mutagenized plasmid/fosmid libraries for large scale phenotypic screening. More recently, the ability of transposase and transposon end compositions to bring about fragmentation and 5′ tagging of DNA has been exploited in generating libraries of tagged DNA fragments for Next Generation sequencing platforms. Such applications for “cut and paste” DNA transposons Tn5 and Mu and the advantages of using them over methods involving mechanical fragmentation are disclosed in Published U.S. Patent Application 2011/0287435. For these uses, a transposon with minimal insertion bias is desired to allow complete coverage with minimal oversampling. Tn5 and Mu transposons show unfavorable insertional sequence bias. A modified Tn7 TnsABC-only system has low sequence bias but requires the expression and purification of several different subunits to form the active complex and is therefore cumbersome to exploit commercially. Moreover, the frequency of transposition is very low for most transposons and there is a requirement in the art for hyperactive transposases. The modified Hermes Transposase of the present invention is a substantial improvement for the above mentioned applications because of the combination of its higher activity and reduced insertional bias. Transposons have also been used in vivo in generating transgenic organisms as disclosed in Published U.S. Patent Application 2003/0150007. The modified form of Hermes Transposase can also be used for such in vivo applications. In vivo insertional mutagenesis methods using transposons in general e.g. Hermes is disclosed in Published U.S. Patent Application 2004/0092018. These patent applications are incorporated herein by reference to the extent permitted by applicable statute and regulation.
Described herein are Hermes transposases and reaction conditions that result in increased strand transfer in vitro, thereby increasing the efficiency of nucleic acid modification. Also described herein is the use of the wild type version of Hermes that may also be modified by mutation to prevent aggregation at particular KCL concentrations. The transposase mutants described herein are used for the development of DNA libraries and next generation sequencing. The mutant transposases disclosed in this invention are a modified form of the native Hermes Transposase, have a similar mechanism of action as the wild type, can easily be expressed in the bacterium, E. coli, and purified in large quantities. The transposases also have the additional advantage of not requiring a preformed transposase complex as in existing alternative transposons such as Tn5 and Mu.6. The transposases described herein, unlike alternatives that have to be incubated at 37° C., is fully active at room temperature at 23° C. up to 30° C. so that the reaction can be readily carried out on a laboratory benchtop.
The modified Hermes Transposases of the invention, as a result of the introduced mutations are octameric. These Hermes Transposases also have a higher transposition activity in vitro than do the wild type transposase. Compared to existing commercialized transposases, the modified Hermes Transposases have less insertional sequence bias when used for in vitro fragmentation of genomic DNA and 5′ end tagging followed by next generation sequencing.
Described herein is an improved hyperactive mutant transposase having a sequence selected from the group consisting of SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, and SEQ ID NO: 24.
The invention provides methods of fragmenting and tagging target DNA sequences comprising the steps of: providing ligand labeled Hermes LEs; reacting the labeled Hermes LEs with an improved mutant transposase and target DNA sequences whereby each target DNA sequence becomes fragmented and each DNA fragment is labeled at either end by one of the labeled Hermes LEs; purifying the labeled DNA fragments using an affinity system that binds the ligand.
In some cases, the affinity system employs beads that bind the ligand. For example, the beads are magnetic beads. The ligand is biotin or polyhistidine of at least six histidine residues and the affinity system is biotinstreptavidin or nickel or cobalt affinity material, respectively.
Optionally, the method further comprises using a DNA polymerase to fill in gaps. For example, the DNA polymerase is T4 polymerase.
In some cases, the method further comprises a step of enzymatically cutting the tagged DNA following the step of purifying to replace one of the labeled Hermes LEs on each fragment with a specific terminal sequence. For example, PCR, DNA ligase or DNA polymerase chain extension is used to add the specific terminal sequence.
In one aspect, the method further comprises the step of using a second transposon system to introduce a second tag into each DNA fragment. For example, the step of using a second transposon system follows the step of purifying. In some cases, the second transposon system is a piggy Bac transposon.
Also provided is a method of fragmenting and tagging target DNA sequences comprising the steps of: providing tagged Hermes LEs bearing at least one specific sequence tag; and reacting the tagged Hermes LEs with an improved mutant transposase and target DNA sequences whereby each target DNA sequence becomes fragmented and each DNA fragment is labeled at either end by one of the tagged Hermes LEs.
Optionally, the method further comprises the step of employing a DNA polymerase to fill in gaps. For example, the DNA polymerase is T4 polymerase.
In some cases, the method further comprises the step of using a second transposon system to introduce a second tag into each DNA fragment. For example, the second transposon system is a piggy Bac transposon. In one aspect, the method further comprises the step of enzymatically cutting the tagged DNA following the step of purifying to replace one of the tagged Hermes LEs on each fragment with a specific terminal sequence. For example, PCR, DNA ligase or DNA polymerase chain extension is used to add the specific terminal sequence.
Improved transposases that are hyperactive in vitro and useful for DNA fragmentation include: E55K, A290S, G366E, G366W, I370V, F433S, E459G, I498T, S499L, and T500M each of which being numbered in accordance with HermesI-612 wildtype (SEQ ID NO: 21).
Polynucleotides, polypeptides, or other agents are purified and/or isolated. Specifically, as used herein, an “isolated” or “purified” nucleic acid molecule, polynucleotide, polypeptide, or protein, is substantially free of other cellular material, or culture medium when produced by recombinant techniques, or chemical precursors or other chemicals when chemically synthesized. Purified compounds are at least 60% by weight (dry weight) the compound of interest. Preferably, the preparation is at least 75%, more preferably at least 90%, and most preferably at least 99%, by weight the compound of interest. For example, a purified compound is one that is at least 90%, 91%, 92%, 93%, 94%, 95%, 98%, 99%, or 100% (w/w) of the desired compound by weight. Purity is measured by any appropriate standard method, for example, by column chromatography, thin layer chromatography, or high-performance liquid chromatography (HPLC) analysis. A purified or isolated polynucleotide (ribonucleic acid (RNA) or deoxyribonucleic acid (DNA)) is free of the genes or sequences that flank it in its naturally-occurring state. A purified or isolated polypeptide is free of the amino acids or sequences that flank it in its naturally-occurring state. Purified also defines a degree of sterility that is safe for administration to a human subject, e.g., lacking infectious or toxic agents.
Similarly, by “substantially pure” is meant a nucleotide or polypeptide that has been separated from the components that naturally accompany it. Typically, the nucleotides and polypeptides are substantially pure when they are at least 60%, 70%, 80%, 90%, 95%, or even 99%, by weight, free from the proteins and naturally-occurring organic molecules with they are naturally associated.
“Conservatively modified variations” of a particular polynucleotide sequence refers to those polynucleotides that encode identical or essentially identical amino acid sequences, or where the polynucleotide does not encode an amino acid sequence, to essentially identical sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given polypeptide. For instance, the codons CGU, CGC, CGA, CGG, AGA, and AGG all encode the amino acid arginine. Thus, at every position where an arginine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid variations are “silent substitutions” or “silent variations,” which are one species of “conservatively modified variations.” Every polynucleotide sequence described herein which encodes a polypeptide also describes every possible silent variation, except where otherwise noted. Thus, silent substitutions are an implied feature of every nucleic acid sequence which encodes an amino acid. One of skill will recognize that each codon in a nucleic acid (except AUG, which is ordinarily the only codon for methionine) can be modified to yield a functionally identical molecule by standard techniques.
Similarly, “conservative amino acid substitutions,” in one or a few amino acids in an amino acid sequence are substituted with different amino acids with highly similar properties are also readily identified as being highly similar to a particular amino acid sequence, or to a particular nucleic acid sequence which encodes an amino acid. Such conservatively substituted variations of any particular sequence are a feature of the present invention. Individual substitutions, deletions or additions which alter, add or delete a single amino acid or a small percentage of amino acids (typically less than 5%, more typically less than 1%) in an encoded sequence are “conservatively modified variations” where the alterations result in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. See, e.g., Creighton (1984) Proteins, W.H. Freeman and Company, incorporated herein by reference.
By “isolated nucleic acid” is meant a nucleic acid that is free of the genes which flank it in the naturally-occurring genome of the organism from which the nucleic acid is derived. The term covers, for example: (a) a DNA which is part of a naturally occurring genomic DNA molecule, but is not flanked by both of the nucleic acid sequences that flank that part of the molecule in the genome of the organism in which it naturally occurs; (b) a nucleic acid incorporated into a vector or into the genomic DNA of a prokaryote or eukaryote in a manner, such that the resulting molecule is not identical to any naturally occurring vector or genomic DNA; (c) a separate molecule such as a cDNA, a genomic fragment, a fragment produced by polymerase chain reaction (PCR), or a restriction fragment; and (d) a recombinant nucleotide sequence that is part of a hybrid gene, i.e., a gene encoding a fusion protein. Isolated nucleic acid molecules according to the present invention further include molecules produced synthetically, as well as any nucleic acids that have been altered chemically and/or that have modified backbones. For example, the isolated nucleic acid is a purified cDNA or RNA polynucleotide. Isolated nucleic acid molecules also include messenger ribonucleic acid (mRNA) molecules.
The transitional term “comprising,” which is synonymous with “including,” “containing,” or “characterized by,” is inclusive or open-ended and does not exclude additional, unrecited elements or method steps. 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.
Other features and advantages of the invention will be apparent from the following description of the preferred embodiments thereof, and from the claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All published foreign patents and patent applications cited herein are incorporated herein by reference. Genbank and NCBI submissions indicated by accession number cited herein are incorporated herein by reference. All other published references, documents, manuscripts and scientific literature cited herein are incorporated herein by reference. In the case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.
The following description is provided to enable any person skilled in the art to make and use the invention and sets forth the best modes contemplated by the inventors of carrying out their invention. Various modifications, however, will remain readily apparent to those skilled in the art, since the general principles of the present invention have been defined herein specifically to provide improved embodiments of modified Hermes transposases.
Transposons are mobile genetic elements that are an important source of genetic variation and are useful tools for genome engineering, mutagenesis screens, and vectors for transgenesis including gene therapy.
For example, cell free systems for inter-molecular transposition for DNA sequencing, to create deletions or insertions into genes, and for studying protein domain functions have been developed for Tn7 (1), for Tn5 (2), and for Mu (3).
Hermes is a 2479 bp long hAT family DNA transposon element derived from the Maryland strain of the common housefly Musca domestica. Its use in creating transgenic insects was disclosed both in a research publication (4), and in U.S. Pat. No. 5,614,398, which is incorporated herein by reference to the extent permissible under applicable statute and regulation.
The Hermes transposase gene has since been cloned (SEQ ID NO:2) and encodes a 612 amino acid polypeptide chain (
This scheme is illustrated in
The full-length native Hermes transposase (Hermes; residues 1-612) was subcloned into pET-15b (Novagen) for expression in Escherichia coli as an N-terminal His-tag fusion protein and purified. The full-length Hermes transposase (residues 1-612) is soluble, but not readily amenable to crystallization for structural studies because it forms large aggregates in solution when expressed as an N-terminally histidine (His)-tagged fusion protein in E. coli. However, removal of the N-terminal 78 residues results in a version of Hermes that is readily crystallized. The structure of Hermes79-612 was solved using X-ray crystallography (6).
Size-exclusion chromatography and sedimentation equilibrium experiments revealed that Hermes forms multimers in solution and examination of the structure revealed an explanation for the multimerization of Hermes253-612 is provided by the presence of a second interface (interface 2) through which heterodimers can form heterotetramers. This interface arises by domain swapping of two helices between residues 497 and 516 that project away from each Hermes79-612 molecule.
The crystal structure of Hermes79-612 as well as a more recent unpublished structure solved by Alison Hickman and others that reveals the configuration of transposon ends within this structure, see
Therefore, several residues were mutated along the polypeptide chain and each mutant tested for its Transposition activity. Two mutants (
The polypeptide sequences and method of production of the “triple mutant” and the “delta497-516” mutants of Hermes Transposase for in vitro transposition and 5′ tagging of nucleic acids are disclosed herein. Methods of using the above hyperactive forms of the Hermes Transposase in generating genomic 5′ transposon tagged libraries for whole genome amplification and DNA sequencing are also disclosed. The wild type Hermes Transposase showed minimal insertional bias when a very large dataset of in vitro target sites were analyzed by using a standard method (8). Using this approach, in one example where half of a sequencing lane of an Illumina sequencing slide (Illumina, Inc., San Diego, Calif.) was used, 6.5× coverage of the yeast genome was obtained, i.e., on average, each base is contained in 6.5 reads, with only 7.02% of the genome not covered. It was confirmed that the triple mutant did not display any difference in insertional bias.
The Hermes transposase (Tnsp) ORF (612 amino acids) was amplified by polymerase chain reaction (PCR) from plasmid pBCHSHH1.9v and cloned between the NcoI and PvuII sites of plasmid pBAD/Myc-HisB (Invitrogen) to generate a Hermes-Myc-His fusion construct, pLQ4. E. coli strain Top10 (Invitrogen) transformed with the Hermes-Myc-His plasmid was grown overnight with shaking at 30° C. in LB medium containing 100 mg/ml carbenicillin. The following day the overnight culture was diluted 1:100 with fresh LB+carbenicillin, and cells were then grown to an absorbance at 600 nm of 0.6 at 30° C. The culture was then shifted to 16° C. and induced with 0.1% L-arabinose for 16 h. After induction, cells were washed by centrifugation at 4° C. with TSG (20 mM Tris-HCl, pH 7.9, 500 mM NaCl, 10% v/v glycerol), and frozen in liquid nitrogen; all subsequent steps were performed at 4° C. Frozen cells were resuspended in 10 ml TSG and lysed by sonication. The cleared lysate was loaded onto a pre-equilibrated Ni2+ Sepharose column (Amersham) and washed with ten column volumes of TSG, six column volumes of TSG+50 mM imidazole and six column volumes of TSG+100 mM imidazole. The Henries-Myc-His fusion protein was eluted with six column volumes of TSG+200 mM imidazole, dialyzed against TSG, and stored at −80° C.
Soluble Henries Transposase (both wild-type and mutants) was obtained by expression in E. coli BL21(DE3) cells which were grown at 310 K until OD600=0.6. Cells were then rapidly cooled on ice to 19° C. and protein expression was induced by addition of IPTG to a final concentration of 0.5 mM. Cells collected from an 8 liter culture were harvested 16-20 h post-induction. The pellet was resuspended in 300 mM NaCl, 12 mM phosphate pH 7.4, flash-frozen in liquid nitrogen and then stored at 193 K. Unless noted otherwise, all purification steps were performed at 4° C. After thawing, cells were lysed by sonication in the presence of 500 mM NaCl, 5 mM imidazole (Im), 25 mM Tris pH 7.5 and 2 mM β-mercaptoethanol (BME). Following centrifugation of the cell lysate at 100,000 g for 45 min, the supernatant was loaded onto a Hi-Trap metal-chelation column (Amersham Biosciences) previously equilibrated with NiSO4. The column was washed extensively with 20 mM Tris pH 7.5, 2 mM Im and 500 mM NaCl followed by the same buffer containing 22 mM Im. Hermes was eluted from the column using a gradient of 22-400 mM Im. After visualization on an SDS-PAGE gel, fractions containing Hermes 79-612 were combined and dialyzed against 20 mM Tris pH 7.5, 1 mM EDTA, 500 mM NaCl, 4 mM BME and 10% (w/v) glycerol. This was followed by dialysis against a single change of the same buffer containing 5 mM dithiothreitol (DTT) in place of BME (TSK buffer). To remove the polyhistidine tag, 10 units of thrombin (Sigma) were added per milligram of protein and incubated overnight. Thrombin was removed by passage over a 1 ml benzamidine Sepharose 4B (Pharmacia) column.
Method 3. Purification of Transposase without an Affinity Tag:
It is also possible to purify Hermes transposases in sufficient quantities by expressing a version of the protein that lacks an affinity purification tag. This was done by introducing a stop codon at the position where the sequence corresponding to the tag begins in the Hermes Transposase coding region of pLQ4 of method 1.
Protein was expressed in Top10 cells by growth at 37° C. until OD600 nm˜0.6, followed by cooling to 19° C. and then induction by addition of arabinose to a final concentration of 0.012%; cells were harvested after 16-18 hrs. Cells were lysed by sonication in Lysis Buffer (25 mM Tris pH 7.5, 0.5 M NaCl, 0.2 mM TCEP), centrifuged to remove cell debris, and the soluble material loaded onto Heparin Sepharose columns (GE Healthcare) previously equilibrated in 25 mM Tris pH 7.5, 0.1 M NaCl, 0.2 mM TCEP. After washing with the same buffer containing 0.5 M NaCl, protein was eluted using a linear gradient from 0.5 M to 1.0 M NaCl. For gel filtration, fractions containing Hermes were combined, concentrated, and loaded onto a preparative scale BioSep-SEC-S 3000 column (Phenomenex) equilibrated in 25 mM HEPES pH 7.3, 1.5 M NaCl, and 0.2 mM TCEP.
Pre-cleaved Hermes-L end for strand-transfer reactions to measure transposition activity was made by annealing the following oligonucleotides:
In some experiments, the oligonucleotide was radiolabeled at its 5′ end with y-P32-dATP (to demonstrate covalent attachment to target) (9 and 10) or, as in the example shown in
The dimeric forms of Hermes Transposase are efficient in strand transfer/covalent attachment to target DNA and fragment the target DNA as the reaction proceeds as shown in
The Strand transfer reaction is diagrammatically illustrated in
5′ Biotinylated oligo-Hermes LE Top strand, (SEQ ID No:11) 5′Biotin-ataagtagcaagtggcgcataagtatcaaaataagccaCTTGTTGTTGTTCTCTG and 5′phosphorylated oligo,-Hermes LE Bottom strand, (SEQ ID NO:12) 5′P-cCAGAGAACAACAACAAGtggcttattttgatacttatgcgccacttgctacttat (Synthesized by IDT) with the addition to 2.53 pM (2 μg in 100 μL) of proteinase K treated-phenol-chloroform purified Schizosaccharomyces pombe or Saccharomyces cerevisae genomic DNA in a buffer containing 25 mM MOPS pH 7.5, 100 mM NaCl, 10 mM MgCl2, 4% Glycerol, 2 mM DTT, 0.1 mg/mL BSA for 2-3 h at 30° C. The reaction was quenched by adding EDTA and SDS to a final concentration of 20 mM and 0.1% respectively and inactivating the enzyme at 65° C. for 20 min. Note that for SEQ ID NO:11 the uppercase nucleotides represent the 17 bp terminal inverted repeat while the lowercase nucleotides represent the biotin sequencing priming region. For SEQ ID NO:12 the uppercase nucleotides represent the 17 bp terminal inverted repeat while the lowercase nucleotides represent the sequencing priming region.
At this stage as shown in
The fragments can, at this stage, be subjected to an extension and strand displacement reaction using DNA polymerase. Arbitrary tags or specific Next gen sequencing platform specific tags (e.g. SEQ ID NOs:17-20) can be added onto the target DNA fragments by this method (see
Hermes L-end oligo (tag A-LE) with Illumina/arbitrary tag A sequencing priming region, 4 bp barcode and a 30 bp Hermes Transposon end is prepared by annealing:
For SEQ ID NO:17 the Illumina/arbitrary tag A is shown in uppercase while the sequencing priming region is shown in lower case with the 4 bp barcode in uppercase followed by a 30 bp Hermes Transposon end with the minimal 17 bp end shown in lower and uppercase. For SEQ ID NO:18 the 30 bp Hermes Transposon end with the minimal 17 bp end is shown in uppercase and lowercase with the 4 bp barcode in uppercase followed by the sequencing priming region in lowercase and the-Illumina/arbitrary tag A in uppercase.
A Henries L-end oligo (tagB-LE) with Illumina/arbitrary tag A sequencing priming region, 4 bp barcode and 30 bp Henries Transposon end is prepared by annealing
For SEQ ID NO:19 the Illumina/arbitrary tag B is shown in uppercase, the sequencing priming region is shown in lower case followed by a 4 bp barcode in uppercase and a 30 bp Hermes Transposon end with the minimal 17 bp end shown in lowercase and uppercase. For SEQ ID NO:20 the 30 bp Henries Transposon end with the minimal 17 bp end is shown in lowercase and uppercase followed by a 4 bp barcode in uppercase and a sequencing priming region and Illumina/arbitrary tag B in uppercase.
Arbitrary tags or specific Next gen sequencing platform specific tags can also be added onto the target DNA fragments by a modified method that does not need “suppression PCR” but provides a second distinct priming site using any “4-bp cutter”-restriction enzyme and a linker ligation mediated PCR approach.
In this method as shown in
The bound DNA is digested at 37° C. overnight. The beads are washed and Msel-specific linkers (obtained by annealing Linker/adapter Top strand (SEQ ID NO:13) and Linker/adapter bottom strand (SEQ ID NO:14) are ligated to the Msel-digested ends of the Henries L-end attached DNA. The beads are washed to remove non-ligated linkers. The DNA bound to the beads are used as a template for the PCR amplification of the Henries L-end insertion site junctions using the 5′ transposon end specific primer, that has i) 5′ Illumina tag sequence fused to ii) an Illumina proprietary sequence (sequencing primer), 4-by barcode and the Hermes L-end complementary sequence (SEQ ID NO:15) and the 3′ linker/adapter specific primer, that has the 3′ Illumina tag (SEQ ID NO:16). The PCR mix is separated from the Dynal beads, concentrated, the amplicons size-selected on an agarose gel and purified by gel extraction. Massively parallel sequencing is then carried out on the illumina Hi-Seq HTS platform.
For SEQ ID NO:15 the Illumina tag A and the 4 bp barcode are in uppercase while the sequencing priming region and inverted repeat are in lowercase. For SEQ ID NO:15 the Illumina tag B is in uppercase while the linker adapter PCR priming region is in lower case.
In another variation of the above embodiment (shown in
Yet another variation (shown in
Also described herein are Hermes transposases and reaction conditions (25 mM HEPES pH 7.5, 5 mM MgCl2, 5 mM NaCl, 100-300 mM KCl, 10 mM DTT, 0.1 mg/ml BSA, 250 nanogram pUC19, 100 ng L end Hermes, 60 ng Hermes) that result in increased strand transfer in vitro, thereby increasing the efficiency of nucleic acid modification. Also described herein is the use of the wild type version of Hermes that may also be modified by mutation (e.g., C519S) to prevent aggregation at particular KCL concentrations (e.g., 100-300 mM KCL).
The materials and methods for
The sequence of Hermes wild type from PLQ4-Hickman (Q2E because of cloning) with added TGA STOP codon after C-terminal I612 (Hermes1-612 wildtype; SEQ ID NO: 21) is as follows:
The sequence of Hermes C519S from pLQ4-Hickman Hermes Q2E; C519S, with STOP added after C-terminal I612 (Hermes1-612 C519S; SEQ ID NO: 22) is as follows:
The sequence of plasmid pLQ4-Hickman Hermes Q2E/STOP after Hermes I612 (Hermes ORF in UC) (pLQ4 Hermes1-612 wildtype “613” STOP; SEQ ID NO: 23) is as follows:
The sequence of plasmid pLQ4-Hickman Q2E; C519S; STOP after Hermes I612 (Hermes ORF in UC) (pLQ4 Hermes1-612 C519S “613” STOP; SEQ ID NO: 24) is as follows:
The following claims are thus to be understood to include what is specifically illustrated and described above, what is conceptually equivalent, what can be obviously substituted and also what essentially incorporates the essential idea of the invention. Those skilled in the art will appreciate that various adaptations and modifications of the just-described preferred embodiment can be configured without departing from the scope of the invention. The illustrated embodiment has been set forth only for the purposes of example and that should not be taken as limiting the invention. Therefore, it is to be understood that, within the scope of the appended claims, the invention may be practiced other than as specifically described herein.
The following references are provided to aid in understanding the invention and are incorporated herein by reference to the extent permitted by applicable statute and regulation.
While the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims Other aspects, advantages, and modifications are within the scope of the following claims.
The patent and scientific literature referred to herein establishes the knowledge that is available to those with skill in the art. All United States patents and published or unpublished United States patent applications cited herein are incorporated by reference. All published foreign patents and patent applications cited herein are hereby incorporated by reference. Genbank and NCBI submissions indicated by accession number cited herein are hereby incorporated by reference. All other published references, documents, manuscripts and scientific literature cited herein are hereby incorporated by reference.
While this invention has been particularly shown and described with references to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims.
This application claims the benefit of priority under 35 U.S.C. §119(e) to U.S. Provisional Application No. 61/978,498, filed on Apr. 11, 2014, which is incorporated herein by reference in its entirety.
This invention was made with U.S. government support under grant number NIH GM076425. The U.S. government has certain rights in the invention.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2015/025261 | 4/10/2015 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 61978498 | Apr 2014 | US |
