Patent Grant
10081827
5-hydroxymethylcytosine (5-hmC), 5-formylcytosine (5-fC), and 5-carboxycytosine (5-caC) were recently identified in mammalian brain and embryonic stem cells as products of the oxidation of 5-methylcytosine (5-mC) by cytosine oxygenases. The biological functions of 5-hmC, 5-fC, and 5-caC are not completely understood; however, several lines of evidence suggest that 5-hmC is involved in epigenetic regulation and DNA demethylation. Iterative oxidation of 5-hmC by cytosine oxygenase enzymes yields 5-fC and 5-caC which are hypothesized to be intermediates in the DNA demethylation process. Several challenges are associated with the identification these biologically modified nucleobases in genomic DNA samples due to their low abundance and temporal fluctuation. Mapping and quantifying 5-mC, 5-hmC, 5-fC, and 5-caC at the DNA level is, therefore, important for unraveling their role in the dynamics of gene expression and regulation.
The present invention provides a variety of reagents, kits and methods for selectively altering and identifying modified nucleotides in a nucleic acid such as DNA. The modified nucleotides that can be identified include, for example, 5-mC, 5-hmC, 5-fC and 5-caC. The methods, reagents and kits of the present invention permit not merely the determination that a modified cytosine residue is present, but also permit the discrimination among or between different types of modified nucleotides, such as 5-mC and 5-hmC, or between 5-hmC and 5-fC. In this way, the invention can be used to elucidate the precise state of a modified nucleic acid, which may be a genome or genome fragment, for example.
In some embodiments, the discrimination among or between different oxidation states of a nucleotide is facilitated by alternately glucosylating or glucosaminylating various modified nucleotides. The glucosylated or glucosaminylated forms can be distinguished based on their performance in various assays, such as by their differential sensitivity to certain restriction endonucleases. In this way, alternately reacting the nucleic acid with a UDP-GlcN (Uridine diphosphate glucosamine or UDP-Glucosamine) substrate and a UDP-Glc (Uridine diphosphate glucose or UDP-Glucose) substrate, places off and on switches for endonuclease cleavage of a nucleic acid containing modified nucleotides. By detecting the presence of cleavage-sensitive sites, the modified nucleotides can be located and identified.
In the present invention, the term glucosylation (and any form of glucosylation such as “glucosylating” or “glucosylated”) refers to the incorporation of a glucosyl moiety from UDP-Glc into the 5-hydroxy position of 5-hmC via the action of a glycosyltransferase to produce 5-gmC (5-glucosyloxymethylcytosine). Other names in common use for 5-gmC include glucosyl-5-hydroxymethyl-cytosine, glucosyl-5-hydroxymethylcytosine, glucosyl-oxy-5-methylcytosine, 5-glucosylhydroxymethylcytosine. In the present invention, the term glucosylation (and any form of glucosylation such as “glucosylating” or “glucosylated”) may also refer to the incorporation of an azido modified glucosyl moiety from UDP-Azido-Glc (for example, UDP-6-Azido-Glc or UDP-6-N3-Glc) into the 5-hydroxy position of 5-hmC via the action of a glycosyltransferase to produce a N3-5-gmC (for example, 6-azide-glucosyl-5-hydroxymethylcytosine or 6-N3gmC). In the present invention, the term glucosaminylation (and any form of glucosaminylation such as “glucosaminylating” or “glucosaminylated”) refers to the incorporation of a glucosaminyl moiety from UDP-GlcN into the 5-hydroxy position of 5-hmC via the action of a glycosyltransferase to produce 5-gNmC (5-glucosaminyloxymethylcytosine). Other names for 5-gNmC include glucosaminyl-5-hydroxymethyl-cytosine, glucosaminyl-5-hydroxymethylcytosine, 5-glucosylhydroxymethylcytosine, aminoglucosyl-5-hydroxymethyl-cytosine, aminoglucosyl-5-hydroxymethylcytosine, and 5-aminoglucosylhydroxymethylcytosine. If the glycosyltransferase is an inverting glycosyltransferase such as T4 β-glucosyltransferase (BGT or βGT or beta-GT) the product is formed with a beta glycosydic linkage (for example, 5-β-gmC, 5-β-6-N3gmC, 5-β-6-gNmC, and 5-β-2-gNmC). If the glycosyltransferase is a retaining glycosyltransferase such as T4 α-glucosyltransferase (AGT or aGT or alpha-GT) the product is formed with an alpha glycosydic linkage (for example, 5-α-gmC).
Specifically, for example, the invention permits the differentiation of 5-β-glucosyloxymethylcytosine (5-β-gmC) from 5-β-2-glucosaminyloxymethylcytosine (5-β-2-gNmC) in a nucleic acid, by reacting the nucleic acid with an endonuclease capable of cleaving a nucleic acid at a glucosylated nucleotide but not at an glucosaminylated nucleotide. One suitable endonuclease is AbaSI. Other useful endonucleases include AbaAI, AbaCI, AbaDI, AbaTI, AbaUI, AcaPI, and PxyI, or one of the ZZYZ proteins or its variants described in US Patent Application Publication No. 2012/0301881, the complete disclosure of which is hereby incorporated by reference. Accordingly, one of these endonucleases, or a polypeptide at least 70% (e.g. at least 75%, at least 80%, at least 82%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to one of those endonucleases or an active fragment thereof) can be used to selectively cleave a target nucleic acid. By controlling the conversion of glucosylation and glucosaminylation of various forms of naturally occurring modified nucleotides, the modified nucleotides can be located and identified in a manner distinguishing their original forms, such as methylcytosine, hydroxymethylcytosine, formylcytosine, and/or carboxycytosine.
Accordingly, in one aspect the invention provides methods for selectively altering modified nucleotides in a nucleic acid containing hydroxymethylated nucleotides and other modified nucleotides such as, 5-fC or 5-caC. The methods generally include reacting a first subset of the modified nucleotides (e.g. 5-hmC) in the nucleic acid with UDP-GlcN in the presence of a β-glycosyltransferase, such as T4-β-glucosyltransferase, to convert hydroxymethylated nucleotides in the nucleic acid to glucosaminylated nucleotides.
In some embodiments, the methods include the subsequent step of reacting the nucleic acid with a reducing agent, such as NaBH4, and a UDP derivative, such as UDP-Glc or UDP-azido-glucose, to convert a second subset of nucleotides in the nucleic acid to glucosylated or azido-glucosylated nucleotides. The reducing agent promotes the reduction of a nucleotide in a higher oxidation state, such as 5-fC or 5-caC, to a hydroxymethylated nucleotide, which is then glucosylated or azido-glucosylated in a reaction typically catalyzed by a glycosyltransferase such as an α-glycosyltransferase or a β-glycosyltransferase. The glycosyltransferase may or may not be the same glycosyltransferase used to catalyze the previous conversion of hydroxymethylated nucleotides to glucosaminylated nucleotides. The method can optionally differentiate pre-existing 5-hmC in a nucleic acid from newly formed as a result of the reduction of 5-fC or 5-caC, for example. When a UDP-azido-glucose such as UDP-6-azido-glucose is used, 5-hmC in the nucleic acid can be converted to a β-6-azide-glucosyl-5-hydroxymethylcytosine in the presence of a β-glycosyltransferase; Further derivatization of the azido moiety via azide-alkyne Huisgen cycloaddition using a copper(I) catalyst (“click chemistry”) or via copper-free variants (for example, using strained cyclooctyne derivatives) can then optionally be used to label the nucleotide, such as with a biotin label, permitting the subsequent use of avidin to selectively isolate the labeled nucleotide.
In other embodiments, following the conversion of hydroxymethylated nucleotides in the nucleic acid to glucosaminylated nucleotides, the nucleic acid is reacted with an oxidizing agent, a UDP-Glc derivative, and a glycosyltransferase to convert a second subset of modified nucleotides in the nucleic acid to glucosylated nucleotides. Suitable oxidizing agents include those capable of oxidizing 5-mC to 5-hmC, such as mYOX1, a TET enzyme, or an inorganic oxidizing agent such as KRuO4. The reaction with UDP-Glc is typically conducted in the presence of a glycosyltransferase such as an α-glycosyltransferase or a β-glycosyltransferase. These methods can, for example, differentiate pre-existing 5-hmC in a nucleic acid from 5-hmC generated from a 5-mC precursor.
In any of these methods, whether incorporating a reducing agent or an oxidizing agent, an endonuclease can be used to characterize the reaction products. Typically, the endonuclease is specific for the glucosylated (or azidoglucosylated) nucleotides, and not for glucosaminylated nucleotides, i.e., the endonuclease has a higher enzymatic activity for a glucosylated or azidoglucosylated nucleic acid that it has for the same nucleic acid with an glucosaminylated nucleotide at the same position(s). Endonucleases that may be used include, for example, AbaSI, AbaAI, AbaCI, AbaDI, AbaTI, AbaUI, AcaPI, and/or PxyI. An adapter molecule can then be ligated to the cleaved end of the endonuclease reaction product, facilitating the subsequent purification, amplification or sequencing of the nucleic acid.
Accordingly, in one embodiment the invention provides a method for differentiating a 5-mC from 5-hmC in a genome or genome fragment. The genome or genome fragment may, for example, be mammalian in origin, such as a human genome or genome fragment. The method includes reacting the isolated genome or genome fragment containing 5-mC and 5-hmC with (i) UDP-GlcN in the presence of a glycosyltransferase catalyzing transfer of glucosamine to the 5-hmC; (ii) oxygenating any existing 5-mC residues to 5-hmC by the action of TET or mYOX1; (iii) reacting the newly created 5-hmC sites with UDP-Glc in the presence of a glycosyltransferase catalyzing transfer of glucose to the 5-hmC; (iv) cleaving the glucosylated template with a modification-dependent endonuclease that recognizes at least one of the modified nucleotides; and (v) differentiating the original 5-mC from the 5-hmC by an altered cleavage pattern.
In this embodiment, the oxygenation of any existing 5-mC residues to 5-hmC can be done in the presence of UDP-Glc and a glycosyltransferase to catalyze the transfer of glucose to 5-hmC as it is being formed from 5-mC. Alternatively, reaction conditions such as pH of the oxygenation reaction can be selected to optimize the yield of 5-hmC. See, for example, Ser. No. 13/827,087, “Compositions and Methods for Oxygenation of Nucleic Acids Containing 5-Methylpyrimidine,” filed on the same date as the present application and hereby incorporated by reference in its entirety.
In another embodiment, the invention provides a method for differentiating a 5-mC from one or more of its oxidation products in a genome or genome fragment containing 5-mC and 5-hmC. The method includes reacting the isolated genome or genome fragment with UDP-2-glucosamine in the presence of a β-glycosyltransferase (BGT) catalyzing the transfer of 2-glucosamine to the 5-hmC; reacting the isolated genome or genome fragment with mYOX1 or TET or a chemical oxidizing agent and optionally with a reducing agent; cleaving the template with a modification dependent endonuclease that is capable of selectively cleaving a 5-hmC and not a 5-glucosaminated hydroxymethylcytosine; and differentiating the 5-mC from one or more of its oxidation products by an altered cleavage pattern.
The invention also provides preparations useful for converting methylcytosine or an oxidized nucleotide, such as 5-fC or 5-caC, or to a glucosylated nucleotide. The preparations include a reducing agent, such as NaBH4, or an oxidizing agent, such as mYOX1, a TET enzyme, or an inorganic oxidizing agent such as KRuO4; a glycosyltransferase, such as an α-glycosyltransferase or a β-glycosyltransferase; a UDP-GlcN or a UDP derivative, such as UDP-Glc or UDP-azido-glucose.
The invention also provides preparations useful for modifying and selectively cleaving nucleic acids. The preparations include a glycosyltransferase and an endonuclease having an amino acid sequence at least 95% identical to an enzyme such as AbaSI, AbaAI, AbaCI, AbaDI, AbaTI, AbaUI, AcaPI, and/or PxyI. The preparations also include (a) UDP-Glc and an oxidizing agent, or (b) UDP-GlcN. Where UDP-GlcN is included, the glycosyltransferase may be a BGT to catalyze the transfer of glucose to hydroxymethylated pyrimidine residues. Where UDP-Glc and an oxidizing agent are included, the oxidizing agent, which may be a methylcytosine oxygenase such as mYOX1 or a TET enzyme or may be an inorganic oxidizing agent such as KRuO4, promotes the conversion of methylcytosine residues in a nucleic acid to hydroxymethylcytosine, which can be glucosylated by the combination of UDP-Glc and the glycosyltransferase, and can be recognized by the endonuclease.
The invention also provides kits useful for making these preparations and practicing these methods. For example, kits for modifying formylcytosine or carboxycytosine residues in a nucleic acid can include a reducing agent, such as sodium borohydride, permitting the reduction of formylcytosine or carboxycytosine residues to hydroxymethylcytosine; a glycosyltransferase (such as a β-glycosyltransferase); and a UDP derivative, such as UDP-Glc and/or UDP-azidoglucose (such as UDP-6-azidoglucose), permitting the transfer of a sugar or modified sugar to the hydroxymethylcytosine. These kits may also include an oxidizing agent (e.g., a methylcytosine oxygenase such as mYOX1 or a TET enzyme or an inorganic oxidizing agent such as KRuO4) to promote the conversion of methylcytosine residues in a nucleic acid to hydroxymethylcytosine; UDP-GlcN; a restriction endonuclease (e.g., having an amino acid sequence at least 95% identical to AbaSI, AbaAI, AbaCI, AbaDI, AbaTI, AbaUI, AcaPI, and/or PxyI); or any combination of the above.
Kits for selectively modulating the susceptibility of modified nucleic acid residues to cleavage can include UDP-Glc; UDP-GlcN; a β-glycosyltransferase; and a reducing (e.g. sodium borohydride) or oxidizing agent (e.g., a methylcytosine oxygenase such as mYOX1 or a TET enzyme or an inorganic oxidizing agent such as KRuO4).
Kits useful for modifying and selectively cleaving nucleic acids can include, for example, a glycosyltransferase and an endonuclease having an amino acid sequence at least 95% identical to an enzyme such as AbaSI, AbaAI, AbaCI, AbaDI, AbaTI, AbaUI, AcaPI, and/or PxyI. These kits can also include (a) UDP-Glc and an oxidizing agent, and/or (b) UDP-GlcN.
Some embodiments of the kits or preparations of the present invention include an optional nucleic acid, such as a nucleic acid that is to be modified, that is undergoing modification, and/or a control nucleic acid. Accordingly, a nucleic acid, if present, may include 5-hmC, 5-gmC, 5-gNmC, 5-fC, 5-caC, 5-mC, or any combination of the above.
Endonucleases have been identified from bacteria and more will undoubtedly be discovered using routine BLAST searches based on the present disclosure that are capable of preferentially cleaving 5-β-gmC compared to C, 5-mC and 5-β-2-gNmC. In one example, the ZZYZ family members for example AbaSI (see for example WO 2011/091146), isoschizomers and mutants thereof preferentially cleave 5-β-gmC compared to C, 5-mC and 5-β-2-gNmC. For example, AbaSI has cleavage activity at 5-β-gmC that is 500 fold greater than 5-mC and 5-α-gmC. 5-β-gmC is the product of BGT mediated transfer of glucose from UDP-Glc to 5-hmC.
The specificity of AbaSI is demonstrated in the data shown in
Other endonucleases that differentiate between various forms of modified cytosine are also available, such as other ZZYZ family members described in WO 2011/091146 and enzymes described in Borgaro et al. (2013) “Characterization of the 5-hydroxymethylcytosine-specific DNA restriction endonucleases,” Nucleic Acids Research, doi: 10.1093/nar/gkt102, the entire disclosures of each of which are incorporated herein by reference. Several endonucleases that discriminate among various cytosine modifications are described in
It is expected that the use of endonucleases with preferential specificity for a specific modified nucleotide over other modified and unmodified nucleotides can be detected using the method described herein for 5-mC, 5-hmC, 5-fC, and 5-caC. In combination with the cofactor UDP-Glc, this system enables sequencing different epigenetic states of 5-mC and greatly enhances the ability to determine the epigenetic modification at a single base resolution level.
An embodiment of the method relies on modifying the non-target modified base (for example, 5-hmC) chemically and/or enzymatically in such a way that its reactivity to a endonuclease is completely or partially blocked, and then chemically or enzymatically reacting the target modified base to convert it into a newly formed 5-hmC (e.g., with a reducing or oxidant agent) which can be then be reacted with a glycosyltransferase to form 5-gmC which in turn can be cleaved by an endonuclease that recognizes 5-gmC preferentially in a positive identification.
By the appropriate choice of the substrate for modifying the non-target and target modified bases, this invention provides an on-off switch assay which is determined by enzyme specificity. For example, if in the first step UDP-2-GlcN is used to label pre-existing 5-hmC sites (resulting in an off-signal for AbaSI cleavage), and in the second step the sample is treated with a methylpyrimidine oxygenase (mYOX), BGT and UDP-Glc, an “on-signal” for AbaSI cleavage will be generated exclusively for 5-mC sites which underwent oxidation by mYOX. If UDP-Glc is used to label pre-existing 5-hmC sites (on-signal for AbaSI cleavage), and the sample is treated with an mYOX, BGT and UDP-2-GlcN, then an “off-signal” will be generated for all 5-mC sites which were converted into 5-hmC by mYOX-mediated oxidation.
The 5-mC may be chemically or enzymatically converted to 5-hmC by reacting the 5-mC with mYOX1 (see for example, “nMCO1” described in U.S. Provisional Application No. 61/723,427), TET enzymes or chemical oxidizing agents. Similarly, oxidation of 5-mC to 5-hmC to 5-fc to 5-CaC can be achieved by chemical or enzymatic oxidation using mYOX1 or TET. Specific chemical oxidation of 5-hmC to 5-fC in synthetic nucleotide oligomer single strand (ssDNA) containing 5-hmC can be achieved with potassium perruthenate, KRuO4 (Booth, et al. Science, 336:934-937 (2012)). KRuO4 can oxidize 5-hmC in double-stranded DNA (dsDNA), with an initial denaturing step before the addition of the oxidant, resulting in quantitative conversion of 5-hmC to 5-fC. Other oxidants known in art, such as Osmium (VIII)-based oxidants, Cerium (IV)-based oxidants, and Chromium (VI)-based oxidants may be used for the oxidation of 5-hmC to 5-fC.
A variety of sugars and modified sugars can be used to alter the propensity of a 5-hmC to trigger an endonuclease-mediated cleavage event. Some of these sugars and modified sugars are depicted in
The reduction of 5-fC and its conversion to 5-β-gmC was demonstrated in the experiment depicted in
In one embodiment the method comprises one or more of the following steps;
Reducing agents and conditions can be used herein that can convert the carboxylic acids into alcohols, e.g., NaBH4, CoCl2, i-Pr2NH, EtOH/H2O (Jagdale, et al. Synthesis, 660-664 (2009)); EDC, HOBt, NaBH4, THF/H2O (Morales-Serna, et al. Synthesis, 1375-1382 (2011)); and cyanuric chloride, NaBH4, NMM/H2O (Falorni, et al. Tetrahedron Lett., 4395-4396 (1999)) as well as other reducing agents known to a person of ordinary skilled in the art.
Indeed, many water-soluble metal or metalloid hydrides are able to reduce aldehydes and/or carboxylic acids to alcohols. Examples of such reducing agents are sodium borohydride and related compounds where from 1 to 3 of the hydrogens are replaced by other moieties, such as cyano and alkoxy containing up to about 5 carbon atoms. Examples of substituted borohydrides, all of which are sodium, potassium, or lithium salts, include cyanoborohydride, dicyanoborohydride, methoxyborohydride, dimethoxyborohydride, trimethoxyborohydride, ethoxyborohydride, diethoxyborohydride, triethoxyborohydride, propoxyborohydride, dipropoxyborohydride, tripropoxyborohydride, butoxyborohydride, dibutoxyborohydride, tributoxyborohydride, and so forth. Examples of other water-soluble metal hydrides include lithium borohydride, potassium borohydride, zinc borohydride, aluminum borohydride, zirconium borohydride, beryllium borohydride, and sodium bis(2-methoxyethoxy)aluminium hydride. Sodium borohydride can also be used in combination with a metal halide, such as cobalt(II), nickel(II), copper(II), zinc(II), cadmium (II), calcium (II), magnesium(II), aluminum(III), titanium (IV), hafnium(IV), or rhodium(III), each of which can be provided as a chloride, bromide, iodide, or fluoride salt. Alternatively, sodium borohydride can be used in combination with iodine, bromine, boron trifluoride diethyl etherate, trifluoroacetic acid, catechol-trifluoroacetic acid, sulfuric acid, or diglyme. Particular reducing strategies include the combination of potassium borohydride with lithium chloride, zinc chloride, magnesium chloride, or hafnium chloride; or the combination of lithium borohydride and chlorotrimethylsilane. Other reducing strategies include the use of borane, borane dimethyl sulfide complex, borane tetrahydrofuran complex, borane-ammonia complex, borane morpholine complex, borane dimethylamine complex, borane trimethylamine complex, borane N,N-diisopropylethylamine complex, borane pyridine complex, 2-picoline borane complex, borane 4-methylmorpholine complex, borane tert-butylamine complex, borane triphenylphosphine complex, borane N,N-diethylaniline complex, borane di(tert-butyl)phosphine complex, borane diphenylphosphine complex, borane ethylenediamine complex, or lithium ammonia borane. Alternative reducing strategies include the reduction of carboxylic acids via the formation of hydroxybenzotriazole esters, carboxy methyleniminium chlorides, carbonates, O-acylisoureas, acyl fluorides, cyanurates, mixed anhydrides, arylboronic anhydrides, acyl imidazolide, acyl azides, or N-acyl benzotriazoles, followed by reaction with sodium borohydride to give the corresponding alcohols.
In one embodiment, 5-hmC sequencing can use BGT and UDP-Glc to generate AbaSI-active 5-β-gmC sites, and T4-α-glucosyltransferase (AGT) and UDP-Glc to generate AbaSI-inactive 5-α-gmC sites as a negative control. In another embodiment, 5-hmC sequencing can use BGT and UDP-Glc to generate the AbaSI-active 5-β-gmC sites, and BGT and UDP-2-GlcN to generate AbaSI-inactive 5-β-2-gNmC as a negative control for 5-hmC.
In one embodiment, newly generated 5-hmC sites can be differentiated from pre-existing 5-hmC sites by sequentially transferring distinct sugar moieties from UDP-2-GlcN or native UDP-Glc using BGT.
In a further embodiment of the invention, UDP-Glc modified by any of keto, thiol, chloro, fluoro, bromo, iodo, nitro, boron, and other substituents may be transferred onto 5-hmC using BGT and may block AbaSI activity. Keto, thiol, chloro, fluoro, bromo, iodo, nitro, boron, and other substituents modifying glucose containing 5-hmC residues in a nucleic acid may facilitate cytosine modification mapping and inhibit AbaSI cleavage.
All references cited herein are incorporated by reference.
The locations of 5-hmC in a nucleic acid sample can be determined using differential cleavage.
A BGT can transfer glucose (“Glc”) from UDP-Glc to 5-hmC to form the glucoylated residue 5-β-2-gmC. Glucosylation enhances the sensitivity of the nucleic acid to a glucosylation-sensitive restriction enzyme such as AbaSI (see
An exemplary process for mapping 5-mC residues in a nucleic acid is depicted in
As shown in
Bioinformatic analysis of the sequencing reads is facilitated by the presence of adapter A which marks the enzyme cleavage sites. After mapping the read back to the reference genome, the modified cytosine can be mapped at fixed distance away from the cleavage sites.
Exemplary processes for identifying 5-fC or 5-caC residues in a nucleic acid are depicted in
As shown in
As described in Example 2, the DNA is digested with a 5-β-gmC-dependent restriction enzyme, such as AbaSI, cleaving at a fixed distance from 5-β-gmC and leaving a sticky end (2-base 3′-overhang). Since AbaSI does not recognize C or 5-β-2-gNmC, the only sticky ends created are those resulting from 5-β-gmC residues, which in turn are exclusively associated to 5-fC and/or 5-caC sites. A biotinylated adaptor A is then ligated onto the cleaved ends. The ligated DNA is then subjected to random fragmentation to an average size of about 200 bp. Avidin beads are used to pull out the fragments with the ligated adaptor A. After polishing the ends, adaptor P is then ligated onto the DNA fragments on the beads. The adaptor-specific PCR and the adapter ligated DNA enters the library preparation pipeline for specific sequencing platform where the end-sequencing is done from the adaptor A.
Bioinformatic analysis of the sequencing reads is facilitated by the presence of adapter A which marks the enzyme cleavage sites. After mapping the read back to the reference genome, the modified cytosine can be mapped at fixed distance away from the cleavage sites.
Another exemplary process for mapping the locations of 5-fC and/or 5-caC is shown in
The sticky ends are ligated to an adapter A. The resulting DNA is fragmented and ligated to a second adapter P1. The azido moiety can derivatized with a biotin label via azide-alkyne Huisgen cycloaddition using a copper(I) catalyst (“click chemistry”) or via copper-free variants (for example, using strained cyclooctyne derivatives) and avidin beads can then be used to selectively purify the fragments containing the azidoglucosylated residues.
An exemplary process for mapping the locations of 5-caC in a nucleic acid sample is provided in
As shown in
This application is a continuation-in-part of U.S. application Ser. No. 13/826,395 filed Mar. 14, 2013.
This invention was made with government support under GM096723 awarded by the National Institutes of Health. The government has certain rights in this invention.
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| Number | Date | Country | |
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| 20160138079 A1 | May 2016 | US |
| Number | Date | Country | |
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| 61724041 | Nov 2012 | US | |
| 61723427 | Nov 2012 | US | |
| 61722968 | Nov 2012 | US | |
| 61611295 | Mar 2012 | US |
| Number | Date | Country | |
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| Parent | 13826395 | Mar 2013 | US |
| Child | 14993590 | US |
