Purification of sulfonated DNA

Abstract

This invention provides a method for purifying chemically modified, specifically sulfonated, DNA. [Solution] The method involves combining sulfonated DNA and a silica support in an acidic binding solution, wherein the acidic binding solution has a pH below the isoelectric point (pH(I)) of the sulfonated DNA, and the sulfonated DNA is bound to the silica support.

Description

Detailed Description of the Invention

[0001] 〔Technical Field〕 This application claims priority to U.S. Provisional Application No. 63 / 058,179, filed Jul. 29, 2020, which is incorporated herein by reference.

[0002] The present disclosure relates to the purification of sulfonated DNA. In addition, the present disclosure relates to methods and systems for bisulfite conversion of DNA, improved purification of sulfonated DNA having a support (e.g., a silica support), and use of acidic conditions for binding sulfonated DNA to a support.

[0003] 〔Background Art〕 DNA methylation is an epigenetic mechanism that occurs by adding a methyl group to DNA, specifically to the cytosine ring of DNA, thereby modifying the function of a gene. Detection and mapping of DNA methylation sites are essential for understanding epigenetic gene regulation and detecting abnormal DNA methylation involved in the development and progression of cancer and other disease states.

[0004] Mapping of methylation sites is currently achieved by the bisulfite method described by Frommmer et al. for the detection of 5-methylcytosine in DNA (which is incorporated herein by reference in its entirety for all purposes, Proc. Natl. Acad. Sci. USA 89:1827-31 (1992)), or a variation thereof. The bisulfite method for mapping 5-methylcytosine is based on the observation that cytosine, not 5-methylcytosine, reacts with bisulfite. Typically, this reaction involves a series of steps including reacting cytosine with bisulfite to form sulfonated cytosine, spontaneous deamination of the sulfonation reaction intermediate resulting in sulfonated uracil, and desulfonating uracil under alkaline conditions to form uracil. The difference in base pairing between uracil and 5-methylcytosine facilitates the detection of the uracil base pair with adenine and the 5-methylcytosine base with guanine. Therefore, methylated cytosine can be distinguished from unmethylated cytosine by various methods, such as bisulfite genome sequencing (Grigg G, & Clark S, Bioessays (1994) 16:431-36, Grigg G, DNA Seq. (1996) 6:189-98), or by methylation-specific PCR (MSP), as disclosed in, for example, U.S. Patent No. 5,786,146. Numerous variations in reaction conditions, reagent concentrations, etc., are known in the art for bisulfite conversion, and each variation can result in differences in DNA recovery rate and deamination level.

[0005] Generally, bisulfite treatment methods involve washing and buffer exchange steps to produce purified and converted DNA samples for base pair analysis. Various approaches facilitate steps such as spin column, ethanol purification, and solid support. However, methods using silica spin columns or ethanol purification often result in sample loss, which impairs the usefulness of the bisulfite method as a means of cytosine methylation. Although some improvements using solid support have been developed, these methods require large amounts of DNA as input and suffer from sample loss and reproducibility issues. In addition, conventional methods often take several days to complete and do not result in efficient conversion and recovery of converted DNA. Therefore, conventional methods only provide a qualitative measure of DNA methylation.

[0006] When attempting to optimize the bisulfite conversion of DNA, the focus has generally been on the bisulfite modification reaction itself, i.e., the step of sulfonating unmethylated cytosine in the DNA. Several methods to improve the recovery of the resulting sulfonated DNA have focused on increasing the chaotropic salt concentration, thereby disrupting the hydrogen bonds between water and DNA and promoting silica-DNA interactions. Other methods address the reduced recovery rate of cytosine-rich DNA by simplifying the binding buffer (for example, as found in U.S. Patent No. 9,315,853, whose entirety is incorporated herein by reference for all purposes) and removing additives such as alcohol and Tris.

[0007] [Summary of the Invention] The systems and methods disclosed herein increase the recovery of bisulfite-converted DNA, particularly for cytosine-rich targets, improve robustness to variations in the bisulfite treatment process, and increase the recovery of DNA across a wide range of sample types. The methods include neutralizing DNA charge to promote silica-DNA interactions, in addition to disrupting hydrogen bonds.

[0008] A method comprising binding sulfonated DNA to a silica support in an acidic solution is disclosed herein, wherein the acidic solution has a pH below the isoelectric point pH(I) of the sulfonated DNA. In some embodiments, the pH is less than 5. In some embodiments, the pH is between 2.5 and 4. The acidic solution may further contain a chaotropic salt or an agent. In some embodiments, the chaotropic salt is guanidine hydrochloride (GuHCl).

[0009] The method may further include incubating denatured non-sulfonated DNA with a sulfonating reagent to produce sulfonated DNA. As used herein, the term “sulfonating reagent” preferably refers to a reagent that selectively converts unmethylated cytosine nucleotides to uracil sulfonate nucleotides. In some embodiments, the sulfonating reagent is ammonium bisulfite.

[0010] The method may further include at least one or all of the following: washing a silica support bound to sulfonated DNA; desulfonating the sulfonated DNA to form desulfonated DNA; and eluting the desulfonated DNA from the silica support. In some embodiments, washing is performed with alcohol, for example, ethanol. In some embodiments, desulfonation includes incubation of sulfonated DNA under alkaline conditions, and in some embodiments, sulfonated DNA is directly added to a detection assay, for example, a polymerase chain reaction, and as a result, the sulfonated DNA is desulfonated under the conditions under which the detection assay is performed. In some embodiments, elution includes treatment with high heat.

[0011] This technology further encompasses the following embodiments, which are illustrated by the following embodiments.

[0012] 1. A method comprising combining sulfonated DNA and a silica support in an acidic binding solution, wherein the acidic binding solution has a pH below the isoelectric point (pH(I)) of the sulfonated DNA, and the sulfonated DNA is bound to the silica support.

[0013] 2. The method according to Embodiment 1, wherein the silica support comprises at least one of particles, beads, and fibers.

[0014] 3. The method according to Embodiment 1, wherein the silica support is magnetic.

[0015] 4. The method according to Embodiment 1, wherein the pH of the acidic bonding solution is less than 5.

[0016] 5. The method according to Embodiment 4, wherein the pH of the acidic bonding solution is 2.0 to 4.

[0017] 6. The method according to Embodiment 1, wherein the acidic bonding solution contains a chaotropic salt.

[0018] 7. The method according to Embodiment 6, wherein the chaotropic salt comprises at least one of guanidine hydrochloride (GuHCl) and guanidine isothiocyanate (GITC).

[0019] 8. The method according to Embodiment 1, further comprising incubating single-stranded unsulfonated DNA or partially sulfonated DNA with a sulfonating reagent to produce the sulfonated DNA.

[0020] 9. The method according to Embodiment 8, wherein the sulfonating reagent comprises at least one of ammonium bisulfite and sodium bisulfite.

[0021] 10.i) Washing the silica support bound to sulfonated DNA, ii) Desulfonating the sulfonated DNA to form desulfonated DNA, iii) further comprising at least one of eluting the desulfonated DNA from the silica support, the method according to embodiment 1.

[0022] 11. The method according to embodiment 8, wherein incubating the non-sulfonated DNA with a sulfonating reagent is carried out in a sulfonation reaction solution having a pH of not less than the pH(I) of the sulfonated DNA.

[0023] 12. The method according to embodiment 11, wherein at least a part of the sulfonation reaction solution is removed from the sulfonated DNA before combining the sulfonated DNA and the silica support in the acidic binding solution.

[0024] 13. The method according to embodiment 12, wherein substantially all of the sulfonation reaction solution is removed from the sulfonated DNA before combining the sulfonated DNA and the silica support in the acidic binding solution.

[0025] 14. The method according to embodiment 12, wherein at least a part of the sulfonation reaction solution is removed from the sulfonated DNA by size exclusion filtration.

[0026] 15. The method according to embodiment 11, wherein the acidic binding solution is formed by adding an acidic component to the sulfonated DNA in the sulfonation reaction solution.

[0027] 16. The method according to embodiment 15, wherein the acidic component is an acidic solution.

[0028] 17. The method according to embodiment 16, wherein the acidic component comprises at least one of acetic acid, glycine, malic acid, formic acid, or citrate buffer.

[0029] 18. The method according to embodiment 15, wherein the acidic component comprises a chaotropic salt.

[0030] 19. The method according to Embodiment 18, wherein the chaotropic salt comprises at least one of GuHCl GITC.

[0031] 20. A method for preparing desulfonated DNA, i) Incubating single-stranded unsulfonated DNA or partially sulfonated DNA with a sulfonation reagent to produce sulfonated DNA in a sulfonation reaction mixture, ii) The sulfonation reaction mixture a) Acidic components, b) Chaotropic salts, c) Combined with a silica support The formation of an acidic binding solution, wherein the acidic binding solution has a pH below the isoelectric point (pH(I)) of the sulfonated DNA, and the sulfonated DNA is bound to the silica support, iii) Separating the sulfonated DNA bound to the silica support from the acidic binding solution, iv) The method comprising treating the isolated sulfonated DNA under conditions in which the sulfonated DNA has been desulfonated.

[0032] 21. The method according to Embodiment 20, wherein the non-sulfonated DNA comprises one or more non-methylated cytosine nucleotides, and the desulfonated DNA comprises one or more deoxyuracil nucleotides.

[0033] 22. The method according to Embodiment 20, wherein processing the sulfonated DNA separated under conditions in which the sulfonated DNA is desulfonated includes combining the sulfonated DNA with a desulfonation solution.

[0034] 23. The method according to Embodiment 22, wherein the desulfonation solution is combined with sulfonated DNA bound to the silica support.

[0035] 24. A composition comprising sulfonated DNA and a silica support in an acidic binding solution containing a chaotropic salt, wherein the acidic binding solution has a pH less than or equal to the pH(I) of the sulfonated DNA, and the sulfonated DNA is bound to the silica support.

[0036] 25. The composition according to Embodiment 24, wherein the chaotropic salt comprises at least one of GuHCl and GITC.

[0037] 26. The composition according to Embodiment 24, wherein the acidic binding solution comprises at least one of acetic acid, glycine, malic acid, formic acid, or citrate buffer.

[0038] 27. The composition according to Embodiment 26, wherein the acidic binding solution comprises a citrate buffer.

[0039] 28. The composition according to Embodiment 24, wherein the acidic bonding solution contains a bisulfite.

[0040] 29. The composition according to Embodiment 28, wherein the bisulfite is ammonium bisulfite.

[0041] 30. The composition according to Embodiment 24, wherein the pH of the acidic bonding solution is less than 5.

[0042] 31. The composition according to Embodiment 30, wherein the pH of the acidic bonding solution is 2.0 to 4.

[0043] 32.i) A bisulfite reagent solution or components for preparing a bisulfite reagent solution, ii) Silica support and iii) A kit comprising an acidic component having a pH below pH(I) of sulfonated DNA.

[0044] 33. The kit according to Embodiment 32, wherein the bisulfite reagent solution has a pH higher than the pH(I) of the sulfonated DNA.

[0045] 34. The kit according to Embodiment 32, wherein the acidic component is an acidic solution.

[0046] 35. The kit according to Embodiment 33, wherein the acidic component comprises at least one of acetic acid, glycine, malic acid, formic acid, or citrate buffer.

[0047] 36. The kit according to embodiment 32, further comprising chaotropic salts.

[0048] 37. The kit according to Embodiment 36, wherein the acidic component comprises a chaotropic salt.

[0049] 38. The kit according to Embodiment 36, wherein the chaotropic salt comprises at least one of GuHCl and GITC.

[0050] 39. The kit according to Embodiment 32, wherein the silica support comprises at least one of particles, beads, and fibers.

[0051] 40. The kit according to Embodiment 32, wherein the silica support is magnetic.

[0052] 41. The kit according to Embodiment 32, wherein the pH of the acidic component is less than 5.

[0053] 42. The kit according to Embodiment 41, wherein the pH of the acidic component is 2.0 to 4.

[0054] 43. A method comprising combining sulfonated DNA and a silica support in an acidic binding solution, wherein the acidic binding solution has a pH below the isoelectric point (pH(I)) of the sulfonated DNA, the sulfonated DNA is bound to the silica support, the silica support preferably comprises at least one of particles, beads, and fibers, and the silica support is preferably magnetic.

[0055] 44. The method according to Embodiment 43, wherein the pH of the acidic bonding solution is less than 5, preferably less than 4, and preferably about 2.0 to 4.

[0056] 45. The method according to Embodiment 43 or Embodiment 44, wherein the acidic bonded solution comprises a chaotropic salt, and the chaotropic salt preferably comprises at least one of guanidine hydrochloride (GuHCl) and guanidine isothiocyanate (GITC).

[0057] 46. ​​The method according to any one of embodiments 43 to 45, further comprising incubating single-stranded unsulfonated DNA or partially sulfonated DNA with a sulfonating reagent to produce the sulfonated DNA.

[0058] 47. The method according to Embodiment 46, wherein the sulfonating reagent comprises at least one of ammonium bisulfite and sodium bisulfite.

[0059] 48.i) Washing the silica support bound to sulfonated DNA, ii) Desulfonating the sulfonated DNA to form desulfonated DNA, The method according to any one of embodiments 43 to 47, further comprising at least one of the following: iii) eluting the desulfonated DNA from the silica support.

[0060] 49. The method according to Embodiment 48, wherein the incubation of non-sulfonated DNA with a sulfonation reagent is carried out in a sulfonation reaction solution having a pH equal to or greater than the pH(I) of the sulfonated DNA.

[0061] 50. The method according to Embodiment 49, wherein at least a portion of the sulfonation reaction solution is removed from the sulfonated DNA before combining the sulfonated DNA and the silica support in the acidic binding solution.

[0062] 51. The method according to Embodiment 50, wherein substantially all of the sulfonation reaction solution is removed from the sulfonated DNA before combining the sulfonated DNA and the silica support in the acidic binding solution.

[0063] 52. The method according to Embodiment 50 or 51, wherein at least a portion of the sulfonation reaction solution is removed from the sulfonated DNA by size exclusion filtration.

[0064] 53. The method according to any one of Embodiments 49 to 52, wherein the acidic binding solution is formed by adding an acidic component to the sulfonated DNA in the sulfonation reaction solution.

[0065] 54. The method according to Embodiment 53, wherein the acidic component is an acidic solution.

[0066] 55. The method according to Embodiment 53 or 54, wherein the acidic component comprises at least one of acidic acetic acid, glycine, malic acid, formic acid, or a citric acid solution.

[0067] 56. The method according to any one of Embodiments 53 to 55, wherein the acidic component comprises a chaotropic salt, and the chaotropic salt preferably comprises at least one of GuHCl GITC.

[0068] 57. A method for preparing desulfonated DNA, i) Incubating single-stranded unsulfonated DNA or partially sulfonated DNA with a sulfonation reagent to produce sulfonated DNA in a sulfonation reaction mixture, ii) The sulfonation reaction: a) Acidic components, b) Chaotropic salts, c) Silica support, in combination with The formation of an acidic binding solution, wherein the acidic binding solution has a pH below the isoelectric point (pH(I)) of the sulfonated DNA, and the sulfonated DNA is bound to the silica support, iii) Separating the sulfonated DNA bound to the silica support from the acidic binding solution, iv) Processing the sulfonated DNA isolated under conditions in which the sulfonated DNA is desulfonated, including, Preferably, the method wherein the non-sulfonated DNA comprises one or more non-methylated cytosine nucleotides, and the desulfonated DNA comprises one or more deoxyuracil nucleotides.

[0069] 58. The method according to Embodiment 57, wherein the sulfonated DNA isolated under conditions in which the sulfonated DNA is desulfonated is combined with a desulfonation solution.

[0070] 59. The method according to Embodiment 58, wherein the desulfonation solution is combined with sulfonated DNA bonded to the silica support.

[0071] 60. A composition comprising sulfonated DNA and a silica support in an acidic binding solution containing a chaotropic salt, wherein the acidic binding solution has a pH less than or equal to the pH(I) of the sulfonated DNA, and the sulfonated DNA is bound to the silica support. The composition wherein the chaotropic salt preferably comprises at least one of GuHCl and GITC, and the acidic binding solution preferably comprises at least one of acetic acid, glycine, malic acid, formic acid, or citrate buffer, preferably citrate buffer.

[0072] 61. The composition according to Embodiment 60, wherein the acidic bonding solution comprises a bisulfite, preferably ammonium bisulfite.

[0073] 62. The composition according to Embodiment 60 or Embodiment 61, wherein the pH of the acidic bonding solution is less than 5, preferably 2.0 to 4.

[0074] 63.i) A bisulfite reagent solution is preferably one having a pH higher than the pH(I) of sulfonated DNA, and the bisulfite reagent solution or components for preparing the bisulfite reagent solution, ii) Silica support and iii) A kit comprising an acidic component, preferably an acidic solution, having a pH less than or equal to the pH(I) of sulfonated DNA, wherein the acidic component preferably comprises at least one of acetic acid, glycine, malic acid, formic acid, or citrate buffer, preferably citrate buffer.

[0075] 64. The kit according to Embodiment 63, further comprising a chaotropic salt, preferably the acidic component comprising a chaotropic salt, and preferably the chaotropic salt comprising at least one of GuHCl and GITC.

[0076] 65. The kit according to embodiment 63 or 64, wherein the silica support comprises at least one of particles, beads, and fibers.

[0077] 66. The kit according to any one of embodiments 63 to 65, wherein the silica support is magnetic.

[0078] 67. The kit according to any one of embodiments 63 to 66, wherein the pH of the acidic component is less than 5, and preferably the pH of the acidic component is 2.0 to 4.

[0079] In any particular preferred embodiment of the embodiments described herein, single-stranded DNA is isolated from a sample, for example, a biological sample. For example, in some preferred embodiments, single-stranded DNA is isolated from a sample from a human subject, such as a subject having or suspected of having cancer, for example, from a plasma sample, a stool sample, or another sample from the subject. In other embodiments, single-stranded DNA is a synthetic compound. In preferred embodiments, single-stranded DNA is formed by exposing double-stranded DNA to denaturing conditions (e.g., alkaline conditions, high temperature, etc.) that cause base pairs of DNA strands to separate and form single strands. In some embodiments, desulfonated DNA is measured, for example, to measure one or more methylated DNA markers (MDMs) in the DNA. For example, any of the DNAs named herein (in the experimental sections or drawings, including MDM disclosed in Tables 1-7, Figures 3, 7-13, 15A, and 15B, 16, and 18) can be detected or measured from any type of sample (e.g., plasma, stool, tissue, etc.) alone, in combination, or in any partial combination such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, etc.)

[0080] Other aspects and embodiments of this disclosure will become apparent in light of the following detailed description and accompanying drawings.

[0081] [Brief explanation of the drawing] [Figure 1] This is an overview of one embodiment of a bisulfite treatment method, including the method disclosed herein.

[0082] [Figure 2] This is a schematic diagram of the proposed coupling mechanism.

[0083] [Figure 3] Provides graphs of the bound pH versus log strand recovered for six different marker DNAs.

[0084] [Figure 4] This graph shows the percentage of converted cytosine versus the maximum binding pH. The percentage of converted cytosine (uracil sulfonate nucleotides / total nucleotides) for each DNA strand is plotted against the maximum estimated binding pH required for maximum strand recovery. A linear fitting of these points is shown.

[0085] [Figure 5] Graph of the percentage of converted cytosine versus maximum binding pH for DNA strands using the same primer / probe combination. Three of the DNA strands (BMP3, β-actin ("BTACT"), and NDRG4 strands) were modified to create sets of DNA that retain the same primer and probe binding sequences for each variant of a particular DNA, but contain different numbers of unmethylated C in other regions of the DNA strand. To further illustrate the strength of this correlation, lines were fitted to each set of sequences.

[0086] [Figure 6] Graphs showing total DNA post-conversion and cleanup using unadjusted binding pH (A) or low binding pH (B).

[0087] [Figure 7] Graphs of recovered strands per stool sample volume (mL) for four different DNA sequences (LASS4, PPP2R5C, LRRC4, and ZDHHC1) at unadjusted binding pH (top panel) or low binding pH (bottom panel). ZDHHC1 DNA was tested twice.

[0088] [Figure 8A] A table is provided showing chain recovery from different lots of ammonium bisulfite from binding reactions carried out at low pH and unadjusted pH.

[0089] [Figure 8B] A table is provided showing chain recovery from different concentrations of ammonium bisulfite from binding reactions carried out at low pH and unadjusted pH.

[0090] [Figure 9] A table is provided showing the effects of contaminants present in binding reactions carried out at low pH and unadjusted pH.

[0091] [Figure 10] Provides a table showing logs of recovered chains using various silica beads with an unadjusted pH binding reaction.

[0092] [Figure 11] Provides a table showing the logs of chains recovered using various silica beads with the low pH binding reaction described in Example 1.

[0093] [Figure 12] A table is provided showing the chains recovered using a binding reaction adjusted to an acidic pH ("BNDpH") in the range of 4.85 to 2.13.

[0094] [Figure 13] A table is provided showing the strands recovered for DNA molecules of different sizes and concentrations using unadjusted or low pH binding reactions.

[0095] [Figure 14] A table comparing the reaction conditions provided by Protocol 1, Protocol 2, and Protocol 3 is shown.

[0096] [Figure 15A] A table comparing chain recovery using Protocol 1, Protocol 2, and Protocol 3 is shown.

[0097] [Figure 15B] A table comparing chain recovery using Protocol 1, Protocol 2, and Protocol 3 is shown.

[0098] [Figure 16] A table showing pairwise comparisons of strand retrieval for different methylated marker DNAs using Protocols 1, 2, and 3 is presented, as shown in Figures 15A-15B.

[0099] Figure 17 shows a graph comparing strand retrievals for different methylated marker DNAs, as shown in the ratio of retrieval using Protocol 2 versus retrieval using Protocol 1 or Protocol 3, as shown in Figure 16. "Protocol X" for each data point represents either Protocol 1 or Protocol 3 for that data point.

[0100] [Figure 18] A table is shown comparing the recovery of different DNAs when using an unadjusted pH during the binding step, or when reducing the pH during the binding of the shown sulfonated DNA to silica beads using glycine, citrate, malic acid, or formate buffer.

[0101] definition The section headings used in this section and throughout the disclosure herein are for organizational purposes only and are not intended to limit the scope of the information.

[0102] To facilitate understanding of the present invention, several terms and phrases are defined below. Additional definitions are provided throughout the detailed description.

[0103] Throughout this specification and the claims, the following terms have the meaning expressly relating to this specification unless otherwise clearly indicated by the context. The phrase “in one embodiment” may, but not necessarily, refer to the same embodiment. Furthermore, the phrase “in another embodiment” may, but not necessarily, refer to a different embodiment. Thus, various embodiments of the Art can be readily combined without departing from the scope or spirit of the Art, as described below.

[0104] As used herein, the terms “comprise,” “include,” “having,” “has,” “can,” “contain,” and their variations are intended to be open-ended transitional phrases, terms, or words that do not preclude the possibility of additional acts or structures. Furthermore, as used herein, the term “or” is an inclusive “or” operator and is equivalent to the term “and / or” unless otherwise explicitly indicated by the context. The term “based on” is not exclusive and allows for the basing on additional factors not listed, unless otherwise explicitly indicated by the context.

[0105] The singular forms “one,” “and,” and “it” include multiple references unless explicitly indicated otherwise in the context. This disclosure also contemplates other embodiments that “include,” “consist of,” and “essentially consist of,” the embodiments or elements presented herein, whether expressly described or not.

[0106] The transitional phrase “essentially consisting of” when used in the claims of this application limits the claims to a specific substance or step of the claimed invention that “does not substantially affect the basic and novel features” as stated in In re Herz, 537F.2d 549,551-52,190 USPQ 461,463 (CCPA 1976). For example, a “essentially consisting of” composition may contain unlisted contaminants, although they are present in the listed elements, to the extent that the contaminants do not alter the function of the listed composition compared to a pure composition, i.e., a composition “consisting of” the listed components.

[0107] For the purposes of enumerating numerical ranges in this specification, each numerical value that intersects them with the same degree of precision is explicitly intended. For example, for the range 6–9, the digits 7 and 8 are intended in addition to 6 and 9, and for the range 6.0–7.0, the digits 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are explicitly intended.

[0108] Unless otherwise defined herein, scientific and technical terms used in connection with this disclosure shall have meanings generally understood by those skilled in the art. For example, any nomenclature and techniques used in connection with molecular biology and protein, as well as nucleic acid chemistry and hybridization described herein, are well known and commonly used in the art. The meaning and scope of terms shall be clear. However, in the event of potential ambiguity, the definitions provided herein shall take precedence over dictionary or external definitions. Furthermore, unless otherwise required by context, singular terms shall include plural forms, and plural terms shall include singular forms.

[0109] As used herein, “DNA fragment,” “low molecular weight DNA,” or “short DNA” means DNA consisting of approximately 200 base pairs or nucleotides in length or less.

[0110] As used herein, “methylated” or “methylated” generally refers to the presence or absence of a methyl group at position 5 of the cytosine base (i.e., whether a particular cytosine is 5-methylcytosine), for example, in relation to the methylation status of cytosine at the CpG locus. Methylation can be determined directly, as demonstrated by routine methods for analyzing the methylation status of cytosine, for example, by determining the sensitivity (or lack thereof) of a particular C residue to conversion to uracil by treatment with bisulfite. For example, cytosine residues in a sample that are not converted to uracil when the sample is treated with bisulfite in a manner that would be expected to convert the residue if it were unmethylated (e.g., under conditions where most or all unmethylated cytosines in the sample are converted to uracil) can generally be considered “methylated.”

[0111] As used herein, the terms “methylcytosine,” “methyl C,” “methylated cytosine,” “methylated C,” and “MeC” are used interchangeably and encompass both 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC).

[0112] The term “bisulfite reagent” refers to a reagent comprising bisulfite, disulfite, hydrogen sulfite, or a combination thereof, which is useful for distinguishing methylated CpG dinucleotide sequences from unmethylated CpG dinucleotide sequences, as disclosed herein. Methods for such treatment are known in the art (e.g., PCT / EP2004 / 011715 and WO2013 / 116375, each of which is incorporated by reference in whole). In some embodiments, the bisulfite treatment is carried out in the presence of a denaturing solvent, such as n-alkylene glycol or diethylene glycol dimethyl ether (DME), or in the presence of dioxane or a dioxane derivative, though not limited to these embodiments. In some embodiments, the denaturing solvent is used at a concentration of 1% to 35% (v / v). In some embodiments, the bisulfite reaction is carried out in the presence of a scavenger such as a chroman derivative, e.g., 6-hydroxy-2,5,7,8-tetramethylchroman 2-carboxylic acid or trihydroxybenzoic acid, and their derivatives, e.g., gallic acid (see PCT / EP2004 / 011715, which is incorporated in whole by reference). In certain preferred embodiments, the bisulfite reaction involves treatment with ammonium hydrogensulfite, also known as ammonium bisulfite, as described, for example, in International Publication WO2013 / 116375.

[0113] As used herein, “sulfonated DNA” refers to DNA containing cytosine or uracil that has been sulfonated as a result of treatment with a sulfonating reagent, such as a bisulfite reagent. Sulfonated DNA may be partially sulfonated so that the DNA contains unmethylated cytosine(s) that have not been sulfonated to form uracil sulfonates, or the DNA may be completely sulfonated so that all unmethylated (or otherwise unprotected) cytosine(s) are sulfonated to form uracil sulfonates. Sulfonated DNA may also be synthesized using, for example, standard nucleic acid synthesis chemistry, to mimic DNA treated with a bisulfite reagent. For example, DNA strands may be synthesized from nucleotide monomers to form strands containing one or more uracil sulfonate nucleotides.

[0114] As used herein, “unsulfonated DNA” refers to DNA that has not been treated with a sulfonating reagent, such as a bisulfite reagent, under conditions in which cytosine is converted to uracilsulfonate nucleotides. Unsulfonated DNA may contain one or more unmethylated cytosine nucleotides. In preferred embodiments, unsulfonated DNA does not contain uracilsulfonate nucleotides.

[0115] Where used herein in relation to solutions or conditions for binding DNA to a solid support, the term "low pH" refers to a binding solution or conditions having a pH below the pH(I) of the solid support to which the DNA, e.g., sulfonated DNA, is bound. In some embodiments, low pH binding conditions are achieved by using a binding solution containing a binder, e.g., a chaotropic salt such as guanidine hydrochloride, combined with an acidic component, e.g., a buffer having an acidic pH. In some embodiments, the acidic component is pre-mixed with the binder before the binding mixture is combined with the sulfonated DNA, while in some embodiments, the acidic component is combined with the sulfonated DNA either before or after the binder is combined with the sulfonated DNA.

[0116] In this specification, when used in reference to solutions or conditions for binding sulfonated DNA to a solid support, the terms “unadjusted pH,” “standard pH,” and “high pH” are used interchangeably and refer to binding reaction solutions or conditions that do not contain acidic components to reduce the pH of the binding solution or conditions compared to a binding solution containing only sulfonated DNA and a binder, e.g., GuHCl. Generally, an unadjusted pH binding solution has a pH equal to or greater than the pH(I) of the solid support, particularly a silica support. In some embodiments, the unadjusted pH binding solution is a mixture containing a sample of DNA, sulfonated DNA, and some or all of the sulfonating reagent used to sulfonate the binder, e.g., GuHCl. In some embodiments, the pH of the unadjusted binding solution is equal to or near the pH of the sulfonating reagent alone.

[0117] As used herein, the term “sample” is used in its broadest sense. For example, a sample suspected to contain human genes or chromosomes or sequences related to human chromosomes may include cells, chromosomes isolated from cells (e.g., broad metaphase chromosomes), genomic DNA (in solution or bound to a solid support for Southern blotting, etc.), RNA (in solution or bound to a solid support for Northern blotting, etc.), cDNA (in solution or bound to a solid support), cell-free DNA (e.g., circulating cell-free DNA from plasma, fragmented DNA from bodily fluids such as plasma, urine, and feces), exosomes or other microvesicles, or DNA isolated from bodily fluids, etc. In some embodiments, a sample means including specimens or cultures (e.g., microbial cultures), and in other embodiments, a sample means including both biological and environmental samples (e.g., suspected to contain target sequences, genes, or templates). In some embodiments, a sample may include samples of synthetic origin. A sample may be unpurified, partially or completely purified, or otherwise processed. Samples "suspected of containing" nucleic acids may or may not contain the target nucleic acid molecule.

[0118] This technique is not limited by the type of biological sample used or analyzed. It is useful for a wide variety of biological samples, including, but not limited to, tissues (e.g., heart, liver, brain, lungs, stomach, intestines, spleen, kidneys, pancreas, and reproductive organs), glands, skin, and muscle), cells (e.g., blood cells (e.g., lymphocytes or red blood cells), muscle cells, tumor cells, and skin cells), gases, bodily fluids (e.g., blood or a portion thereof, serum, plasma, urine, semen, saliva, etc.), or solid samples (e.g., feces) obtained from humans (e.g., adults, infants, or embryos), or animals (e.g., cattle, poultry, mice, rats, dogs, pigs, cats, horses, etc.). In some embodiments, the biological sample may be solid food and / or feed products and / or components, such as dairy products, vegetables, meat and meat by-products, and waste. Biological specimens may be obtained from all families of domesticated animals, as well as from feral or wild animals, but are not limited to, ungulates, bears, fish, lagomorphs, rodents, and pygmy dinosaurs.

[0119] Biological specimens include biopsies and tissue sections (e.g., biopsies or sections of tumors, growths, rashes, infections, or paraffin-embedded sections), medical or hospital specimens (e.g., blood specimens, saliva, oral swabs, cerebrospinal fluid, pleural fluid, milk, colostrum, lymph, sputum, vomit, bile, semen, oocytes, cervical cells, amniotic fluid, urine, feces, hair, and sweat), laboratory specimens (e.g., intracellular fractions), forensic specimens (e.g., blood or tissue (e.g., sputtered or residual material), hair and skin cells containing nucleic acids), and archaeological specimens (e.g., fossil organisms, tissues, or cells).

[0120] Environmental samples include, but are not limited to, surface materials, soil, water (e.g., freshwater or seawater), algae, lichens, geological samples, air-containing materials containing nucleic acids, crystals, and industrial samples, as well as environmental materials such as food and dairy processing equipment, apparatus, instruments, disposable and non-disposable items.

[0121] Samples may be prepared by any desired or preferred method. In some embodiments, nucleic acids are analyzed directly from bodily fluids, feces, or other samples using methods and systems described in U.S. Patents 9,000,146 and 10,047,390, each of which is incorporated herein in whole by reference for any purpose.

[0122] However, the examples described above should not be construed as limiting the types of samples to which this technology can be applied (e.g., those suspected to include target sequences, genes, or templates (e.g., the presence or absence of such sequences can be determined using the compositions and methods of this technology)).

[0123] The term "target," when used in relation to nucleic acid detection or analysis methods, refers to a nucleic acid having a specific sequence of nucleotides to be detected or analyzed in a sample suspected of containing the target nucleic acid. In some embodiments, the target is a nucleic acid having a specific sequence for which a methylation state is desired. When used in relation to polymerase chain reactions, the "target" generally refers to a nucleic acid region bound by a primer used in the polymerase chain reaction. Thus, the "target" is required to be selected from other nucleic acid sequences that may be present in the sample. A "segment" is defined as a region of nucleic acid within the target sequence. The term "sample template" refers to a nucleic acid derived from a sample being analyzed for the presence of a target.

[0124] The term "template" refers to a nucleic acid chain with a flap structure, for example, an invasive cleavage structure, where upstream and downstream nucleic acids or nucleic acid regions hybridize to form an invasive cleavage structure. A template chain can function, for example, as a template for primer extension by polymerase in a PCR flap endonuclease assay, but the use of this term is not limited to polymerization assays or reactions.

[0125] As used herein, the term “nucleic acid molecule” refers to any nucleic acid-containing molecule, including, but not limited to, DNA or RNA. This term is not limited to, but includes four acetylcytosines, 8-hydroxy-N6-methyladenosine, aziridinylcytosine, pseudoisocytosine, 5-(carboxyhydroxyl-methyl)uracil, 5-fluorouracil, 5-bromouracil, 5-carboxymethylaminomethyl-2-thiouracil, 5-carboxymethylaminomethyluracil, dihydrouracil, inosine, N6-isopentenyladenine, 1-methyladenine, 1-methylpsuduracil, 1-methylguanine, 1-methylirinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguadenine, 3-methylcytosine, 5-methylisocytosine, 5-hydroxymethylcytosine, 5-carboxylcytosine, 5-formylcytosine, N6-methyladenine, 7-methylguanine, 5-methylami The sequence includes any known DNA and RNA base analogs, including nomethyluracil, 5-methoxy-amino-methyl-2-thiouracil, beta-D-mannoylqueosine, 5'-methoxycarbonylmethyluracil, 5-methoxyuracil, 2-methylthio-N-isopentenyladenine, uracil-5-oxyacetate methyl ester, uracil-5-oxyacetic acid, oxybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, N-uracil-oxyacetate methyl ester, uracil-5-oxyacetic acid, pseudouracil, queosine, 2-thiocytosine, and 2,6-diaminopurine.

[0126] A nucleic acid sequence or molecule may be either genomic or synthetic DNA or RNA, may be single-stranded or double-stranded, and may represent sense or antisense strands. Thus, a nucleic acid sequence may be dsDNA, ssDNA, mixed ssDNA, mixed dsDNA, dsDNA converted to ssDNA (e.g., via lysis, denaturation, helicase, etc.), A-, B-, or Z-DNA, triple-stranded DNA, RNA, ssRNA, dsRNA, mixed ssRNA and dsRNA, dsRNA converted to ssRNA (e.g., via lysis, denaturation, helicase, etc.), messenger RNA (mRNA), ribosomal RNA (rRNA), transfer RNA (tRNA), catalytic RNA, SnRNA, microRNA, or protein nucleic acid (PNA).

[0127] This technology is not limited by the type or source of nucleic acid used (e.g., sequence or molecule (e.g., target sequence and / or oligonucleotide)). For example, the nucleic acid sequence may be an amplified or constructed sequence (e.g., amplification or construction of nucleic acid sequences via synthesis (e.g., polymerization (primer extension (e.g., RNA-DNA hybrid primer technology)) and reverse transcription (e.g., transcription of RNA into DNA)) and / or amplification (e.g., polymerase chain reaction (PCR), rolling circle amplification (RCA), nucleic acid sequence-based amplification (NASBA), transcription-mediated amplification (TMA), ligase chain reaction (LCR), cycling probe technology, Q-beta replicase, strand displacement amplification (SDA), branched DNA signal amplification (bDNA), hybrid capture, and helicase-dependent amplification).

[0128] As used herein, the term “process control” refers to an exogenous molecule, such as an exogenous nucleic acid, added to a sample before the extraction of target DNA, which can be measured after extraction to assess the efficiency of the process and determine a success or failure mode. The nature of the process control nucleic acid used typically depends on the assay type and the material being measured. For example, if the assay used is for the detection and / or quantification of double-stranded DNA or mutations in it, the double-stranded DNA process control is typically spiked into the sample before extraction. Similarly, in the case of an assay monitoring mRNA or microRNA, the process control used is typically either an RNA transcript or synthetic RNA. Process controls typically assist in assessing the efficiency of the process and determining the success or failure of the process and process steps.

[0129] Where used herein with respect to non-target DNA, the term “exogenous” refers to non-target DNA that is isolated and purified from a source other than the source containing the target DNA or the sample. For example, as described in U.S. Patent No. 9,212,392, incorporated herein by reference, purified fish DNA is exogenous DNA with respect to a sample containing human target DNA. Exogenous DNA does not need to be of a different biological origin than the target DNA. For example, commercially available purified fish DNA is exogenous when added to a reaction configured to detect the target nucleic acid in a sample of a particular fish origin. In a preferred embodiment, the exogenous DNA is selected so as not to be detected by an assay configured to detect and / or quantify the target nucleic acid in a reaction to which the exogenous DNA is added.

[0130] As used herein, the term “fish DNA” refers to bulk (e.g., genomic) DNA isolated from fish, as described, for example, in U.S. Patent No. 9,212,392. Bulk purified fish DNA is commercially available and is supplied, for example, in the form of cod and / or herring sperm DNA (Roche Applied Science, Mannheim, Germany) or salmon DNA (USB / Affymetrix). “Fish DNA” is not, for example, a specific gene of fish origin in an isolated form, either synthesized separately or isolated from other DNA in the fish genome.

[0131] As used herein, the term “zebrafish DNA” refers to DNA isolated from Danio rerio or created in vitro (e.g., enzymatically, synthetically) and having a sequence of nucleotides found in DNA from Danio rerio. In preferred embodiments, zebrafish DNA is a methylated DNA added as a detectable control DNA, e.g., a process control for verifying DNA recovery through a sample processing step. In particular, zebrafish DNA containing at least a portion of the RASSF1 gene is used as a process control for human samples, as described in WO2018 / 017710A1, incorporated herein by reference, which describes the use of zebrafish DNA as a process control for human samples. As used herein, “ZFRASSF1” refers to a process control containing at least a portion of the zebrafish RASSF1 gene.

[0132] As used herein, the terms “recovered” and “recovered” refer, when used in reference to nucleic acid strands, to the amount or number of DNA strands measured in a sample after a process (e.g., a complete bisulfite conversion process) or after one or more process steps (e.g., matrix binding of nucleic acids, followed by elution of the bound strands). In some embodiments, the recovered strands are compared to a reference value, e.g., the amount or number of strands that would be added to or expected to be present in the sample before the process, or the amount measured to or expected to be present in the reference sample.

[0133] As used herein, the term “kit” refers to any delivery system for delivering a substance. In the context of nucleic acid purification systems and reaction assays, such a delivery system includes a system that enables the storage, transport, or delivery of reagents and devices (e.g., chaotropic salts, particles, buffers, denaturants, oligonucleotides, filters, etc., in appropriate containers) and / or supporting equipment (e.g., sample processing or sample storage containers, written instructions for performing the procedure, etc.) from one place to another. For example, a kit includes one or more enclosures (e.g., boxes) containing the relevant reaction reagents and / or supporting equipment. As used herein, the term “separated kit” refers to a delivery system comprising two or more separate containers, each containing a portion of the entire kit components. These containers may be delivered together or separately to the intended recipient. For example, the first container may contain materials and buffers for sample collection, and the second container may contain captured oligonucleotides and denaturants. The term “separated kit” is intended to include, but is not limited to, kits containing analyte-specific reagents (ASRs) regulated under Section 520(e) of the Federal Food, Drug, and Cosmetic Act. In practice, any delivery system comprising two or more separate containers, each containing a portion of the components of the entire kit, falls under the term "fragmented kit." In contrast, a "combined kit" refers to a delivery system that contains all the components of a reaction assay in a single container (e.g., a single box containing each of the desired components). The term "kit" encompasses both fragmented and combined kits.

[0134] As used herein, the term “system” refers to a collection of articles used for a particular purpose. In some embodiments, the articles include instructions for use as information provided, for example, on the articles, on paper, online (e.g., at a website or web address), or on a recordable medium (e.g., a diskette, CD, DVD, flash drive, etc.). In some embodiments, the instructions lead the user to an online location, for example, a website for viewing, listening to, and / or downloading instructions. In some embodiments, instructions or other information are provided, for example, as an application ("app") for a computer or a mobile device such as a smartphone.

[0135] As used herein, the term “information” refers to any collection of facts or data. With respect to information stored or processed using computer systems, including the Internet, the term refers to any data stored in any format (e.g., analog, digital, optical, etc.). As used herein, the term “information relating to a subject” refers to facts or data relating to a subject (e.g., a human, plant, or animal). The term “genomic information” refers to information relating to the genome, including but not limited to nucleic acid sequences, genes, allele frequencies, RNA expression levels, protein expression, and phenotypes correlated with genotype. “Allele frequency information” refers to facts or data relating to allele frequencies, including but not limited to allele identity, statistical correlations between the presence of alleles and characteristics of a subject (e.g., a human subject), the presence or absence of alleles in an individual or population, and the percentage probability of an allele being present in an individual having one or more specific characteristics.

[0136] [Modes for carrying out the invention] Preferred methods and materials are described below, but similar or equivalent methods and materials may be used in the implementation or testing of this disclosure. All publications, patent applications, patents, and other references referenced herein are incorporated in their entirety by reference. The materials, methods, and examples disclosed herein are illustrative and not intended to limit the scope of this disclosure.

[0137] The techniques provided herein relate to methods for improving the post-modification recovery of DNA treated with sulfonating reagents. In particular, the techniques provide a method for binding sulfonated DNA to a charged surface, such as a silica surface or a support, such as the surface of an assay well or slide, fibers, particles, beads, etc. While the techniques are considered in relation to a specific material and form of support, such as silica in the form of beads, it will be understood that the techniques are applicable to other support materials and supports of any form or configuration, including fibers, particles, tubes, etc.

[0138] This method provides conditions that promote highly stable binding of sulfonated DNA to beads. This facilitates the efficient recovery of bisulfite-treated DNA despite the highly basic reaction conditions of desulfonation, which, as those skilled in the art would expect, would disrupt the interaction between the DNA and the beads. Through the innovative combination of steps provided herein, this technique provides a method for preparing bisulfite-converted DNA with improved recovery rates of the input DNA.

[0139] This technique is demonstrated in experimental examples related to the experimental results described below. These examples illustrate the development and testing of reagents used for analyzing the methylation status of nucleic acids. In particular, this technique relates to a binding solution having an acidic pH such that the pH of the binding buffer is below the isoelectric point pH(I) of both silica and uracil sulfonate-rich DNA, thereby promoting the binding of sulfonated DNA to silica.

[0140] Common sulfonating reagents include sodium bisulfite and ammonium bisulfite. While ammonium bisulfite is used in the following example, the principle of this technique is readily applicable to any DNA treated in a way that generates a charge throughout the molecule, for example, a very negative charge. For example, DNA treated with other sulfonating reagents (e.g., sodium bisulfite) can be used with this technique.

[0141] The pH of the sulfonation reaction is primarily determined by ammonium bisulfite, which typically has a pH above the pH(I) of silica (as schematically shown in Figure 2) and above the pH(I) of sulfonated DNA, without the addition of acid. However, if the pH of the binding reaction is above the pH(I) of silica (or other supporting material) and / or DNA, electrostatic repulsion may prevent complete binding of DNA to silica. While this technique is not limited to a specific mechanism of action, it has been observed that, for example, performing the binding reaction at an acidic pH by adding acid to a sulfonated DNA-silica bead mixture can neutralize the silica, protonate the uracilsulfonic acid groups of DNA, thereby reducing electrostatic repulsion and promoting silica-sulfonated DNA interaction.

[0142] Suitable DNA for use with this technology is not limited to DNA isolated by a specific method; suitable DNA can be prepared by several methods, including the use of commercially available kits, columns, etc. Exemplary methods for isolating DNA from a sample, for example, from a subject, are described below.

[0143] Fecal DNA isolation In some embodiments, DNA may be isolated from a stool sample. Exemplary embodiments for isolating DNA from stool, suitable for use with the art of the present invention, are found in U.S. Patent No. 9,000,146, incorporated herein by reference. Briefly, a stool sample is homogenized with a buffer, the solid is removed, for example, by centrifugation, and the specific target DNA is captured from the resulting clarified supernatant using particles or beads containing oligonucleotides complementary to the target DNA(s). The target DNA bound to the captured oligonucleotide is separated from the solution, for example, by collecting magnetic beads using a magnetic field, and the captured DNA is optionally washed with a buffer before eluting from the captured oligonucleotide, for example, using denaturing conditions.

[0144] Cell lines and cell culture DNA For cell lines, cell-free genomic DNA can be isolated from cell-conditioned medium using, for example, the "Maxwell® RSC ccfDNA Plasma Kit (Promega Corp., Madison, WI)". Following the kit protocol, 1 mL of cell-conditioned medium (CCM) is used instead of plasma, and the sample is processed according to the kit's procedure. The elution volume is 100 μL, of which 70 μL is typically used for bisulfite conversion.

[0145] Blood or plasma DNA An exemplary procedure for isolating DNA from a 4 mL plasma sample, for example, from a human blood sample, is as follows: Add 300 μL of proteinase K (20 mg / mL) to 4 mL of plasma sample and mix.

[0146] • Add 1 μg / 1 μL of fish DNA to a plasma-proteinase K mixture.

[0147] Add 2 mL of plasma lysis buffer to the plasma.

[0148] Plasma lysis buffer is as follows:

[0149] -4.3M guanidine thiocyanate -10% IGEPAL CA-630 (octylphenoxypoly(ethyleneoxy)ethanol, branched) (5.3g of IGEPAL CA-630 combined with 45mL of 4.8M guanidine thiocyanate) Incubate the mixture at 55°C for 1 hour while shaking at 500 rpm.

[0150] • Add and mix: 03 mL of plasma lysis buffer o200μL magnetic silica bonded beads (16μg / μL beads) Add 0.2 mL of 100% isopropanol. (Optionally, each addition may be mixed afterwards, and / or, optionally, the lysis buffer and isopropanol may be pre-mixed before adding to the mixture.) Incubate at 30°C for 30 minutes while shaking at 500 rpm.

[0151] Place the tube on the magnet and allow the beads to be collected. Aspirate the supernatant and discard it.

[0152] Add 750 μL of GuHCl-EtOH to the container containing the binding beads and mix.

[0153] The GuHCl-EtOH wash buffer is as follows: -3M GuHCl (Guanidine Hydrochloride) -57% EtOH (ethyl alcohol) Shake at 400 rpm for 1 minute.

[0154] Transfer the sample to a deep well plate or a 2 mL microcentrifuge tube.

[0155] Place the tube on the magnet and allow it to collect beads for 10 minutes. Aspirate and discard the supernatant.

[0156] Add 1000 μL of washing buffer (10 mM Tris HCl, 80% EtOH) to the beads and incubate at 30°C for 3 minutes while shaking.

[0157] Place the tube on the magnet and allow the beads to be collected. Aspirate the supernatant and discard it.

[0158] Add 500 μL of washing buffer to the beads and incubate at 30°C for 3 minutes while shaking.

[0159] Place the tube on the magnet and allow the beads to be collected. Aspirate the supernatant and discard it.

[0160] Add 250 μL of washing buffer and incubate at 30°C for 3 minutes while shaking.

[0161] Place the tube on the magnet and allow the beads to be collected. Aspirate and discard the remaining buffer solution.

[0162] Add 250 μL of washing buffer and incubate at 30°C for 3 minutes while shaking.

[0163] Place the tube on the magnet and allow the beads to be collected. Aspirate and discard the remaining buffer solution.

[0164] Dry the beads at 70°C for 15 minutes while shaking.

[0165] Add 125 μL of elution buffer (10 mM Tris HCl, pH 8.0, 0.1 mM EDTA) to the beads and incubate at 65°C for 25 minutes while shaking.

[0166] Place the tube on the magnet and let it collect beads for 10 minutes.

[0167] • Aspirate the supernatant containing the DNA and transfer it to a new container or tube.

[0168] DNA sulfonation and purification In a typical bisulfite treatment method, during the bisulfite treatment step, unmethylated cytosine bases undergo deaminolysis and sulfonation to become negatively charged uracil sulfonates (Figure 1). To remove the conversion reagent, the sulfonated DNA sample can be isolated by alcohol precipitation or, for example, gel filtration. Sulfonated DNA may also be bound to a support such as silica, e.g., a silica-coated surface of a reaction vessel, silica particles or fibers, paramagnetic silica beads, etc., by adding a chaotropic salt, e.g., a guanidine salt such as guanidine hydrochloride (GuHCl) or guanidine isothiocyanate (GITC), at a concentration of, for example, about 6-7 M. Other chaotropic binding agents to promote the binding of denatured DNA to a solid matrix include iodide, perchlorates, and trichloroacetates.

[0169] A typical method for bisulfite treatment of DNA (without the low pH binding step of this technology) is as follows: I. Sulfonation of DNA using ammonium bisulfite 1. Combine 64 μL of DNA, 7 μL of 1N NaOH, and 9 μL of carrier solution containing 0.2 mg / mL of BSA and 0.25 mg / mL of fish DNA in each tube.

[0170] Incubate at 2.42°C for 20 minutes.

[0171] Add 120 μL of 3.45% ammonium bisulfite and incubate at 66°C for 75 minutes.

[0172] Incubate at 4.4°C for 10 minutes. II. Desulfonation using magnetic beads material • Magnetic beads (Promega MagneSil Paramagnetic Particles, Promega catalog number AS1050, 16 μg / μL).

[0173] • Binding buffer: 6.5-7M guanidine hydrochloride.

[0174] • Post-conversion washing buffer: 80% ethanol with 10 mM Tris HCl (pH 8.0).

[0175] • Desulfonation buffer: 70% isopropyl alcohol and 0.1N NaOH were selected as the desulfonation buffer.

[0176] The samples are mixed using any suitable device or technique, and are basically mixed or incubated at the temperatures and mixing rates described below. For example, a Thermomixer (Eppendorf) can be used for mixing or incubation of the samples. An example of desulfonation is as follows: 1. Mix the bead stock thoroughly in a vortex bottle for 1 minute. Transfer 2.50 μL of beads into a 2.0 mL tube (for example, from USA Scientific). 3. Add 750 μL of binding buffer to the beads. 4. Add 150 μL of sulfonated DNA to the mixture from Step I. 5. Mix (for example, at 30°C for 30 minutes at 1000 RPM). 6. Place the tube on the magnetic stand and leave it for 5 minutes. With the tube still on the stand, remove and discard the supernatant. Add 7.1,000 μL of washing buffer. Mix (e.g., 3 minutes at 30°C, 1000 RPM). 8. Place the tube on the magnetic stand and leave it for 5 minutes. With the tube still on the stand, remove and discard the supernatant. Add 9.250 μL of washing buffer. Mix (e.g., 3 minutes at 30°C, 1000 RPM). 10. Place the tube in the magnetic rack, remove the supernatant after 1 minute, and discard it. Add 11,200 μL of desulfonation buffer. Mix (e.g., 5 minutes at 30°C, 1000 RPM). 12. Place the tube in the magnetic rack, remove the supernatant after 1 minute, and discard it. Add 13.250 μL of washing buffer. Mix (e.g., 3 minutes at 30°C, 1000 RPM). 14. Place the tube in the magnetic rack, remove the supernatant after 1 minute, and discard it. 15. Add 250 μL of washing buffer to the tube. Mix (e.g., 3 minutes at 30°C, 1000 RPM). 16. Place the tube in the magnetic rack, remove the supernatant after 1 minute, and discard it. 17. Incubate all tubes with the caps open at 30°C for 15 minutes. 18. Remove the tubes from the magnetic rack and add 70 μL of elution buffer directly to the beads. 19. Incubate the beads in elution buffer (e.g., 40°C for 45 minutes at 1000 RPM). 20. Place the tube in a magnetic rack for about 1 minute, then remove the supernatant and store it.

[0177] Next, the converted DNA is used in a detection assay, such as a pre-amplification and / or flap endonuclease assay, as described below.

[0178] Magnetic silica beads The techniques provided herein relate to bisulfite treatment and DNA isolation for the quantitative measurement of DNA methylation. In some embodiments, magnetic beads are used for the treatment and isolation of DNA, for example, beads comprising a magnetic core and a silica coating. The silica coating binds to the DNA, and the magnetic core provides an efficient method for concentrating and isolating the beads (and bound DNA) using a magnet. In some embodiments, the silica-coated magnetic beads are MagneSil Paramagnetic Particles (Promega, Madison, WI; catalog numbers AS1220 or AS640A, Promega.com).

[0179] This technology is not limited to any particular type of magnetic beads. Embodiments of the technology described herein use any magnetic beads (e.g., paramagnetic beads) that have an affinity for nucleic acids. In some embodiments, the magnetic beads have a magnetite (e.g., Fe3O4) core and a coating containing silicon dioxide (SiO2). The bead structure (e.g., size, porosity, shape) and composition of the solution to which nucleic acids are bound to the beads can be varied to selectively bind different types (e.g., DNA or RNA in single-stranded, double-stranded, or other forms or conformations; nucleic acids of natural origin; chemically synthesized; enzymatically synthesized (e.g., by PCR)) and nucleic acid sizes (e.g., small oligomers, primers, genomes, plasmids, fragments, etc., consisting of 200 or fewer bases). These characteristics of the beads affect the binding and elution of nucleic acids to the beads. Related technologies are described, for example, in U.S. Patents No. 6,194,562, 6,270,970, 6,284,470, 6,368,800, and 6,376,194, which are incorporated herein by reference, respectively. Also envisioned are magnetic beads coated with, for example, organosilanes (as described in U.S. Patent No. 4,554,088), carboxylated polyacrylates (as described in U.S. Patent No. 5,648,124), cellulose (as described in U.S. Patent Application No. 10 / 955,974), hydroxysilanes (as described in U.S. Patent Application No. 11 / 459,541), and hydrophobic aliphatic ligands (as described in U.S. Patent Application No. 12 / 221,750).

[0180] This technology is not limited to magnetic beads of a specific size. Therefore, embodiments of this technology utilize magnetic beads of several different sizes. Smaller beads offer more surface area (in units of weight) for adsorption, but smaller beads limit the amount of magnetic material that can be incorporated into the bead core compared to larger beads. In some embodiments, the particles are distributed across a size range having a defined mean or median size suitable for the technology in which the beads are used. In some embodiments, the particles have a relatively narrow, unimodal particle size distribution.

[0181] In some embodiments, the beads used in this technique have pores accessible from the outside of the particle. Such pores have a controlled size range large enough to accept nucleic acids, such as DNA fragments, into the interior of the particle and bind to the inner surface of the pore. The pores are designed to provide a large surface area on which nucleic acids can bind. Furthermore, in one embodiment, this technique is not limited to a specific nucleic acid (e.g., DNA) binding and / or isolation method. Thus, in some embodiments, aspects of the technique related to the bisulfite reaction are combined with other suitable DNA isolation methods (e.g., precipitation, column chromatography (e.g., spin column)).

[0182] The beads (and binding material) are removed from the mixture using a magnetic field. In some embodiments, other forms of external force in addition to the magnetic field are used to isolate the biological target material by this technique. Suitable additional forms of external force include, but are not limited to, gravity filtration, vacuum filtration, and centrifugation.

[0183] Embodiments of this technology remove a composite from a medium by applying an external magnetic field. Such a magnetic field can be appropriately generated within the medium using one of several different known means. For example, a magnet can be placed on the outer surface of a container of a solution containing beads, causing the particles to move through the solution and accumulate on the inner surface of the container adjacent to the magnet. The magnet can then be kept in a position on the outer surface of the container so that the particles are held in the container by the magnetic field generated by the magnet while the solution is decanted and discarded from the container. Next, a second solution can be added to the container, and the magnet can be removed so that the particles move into the second solution. Alternatively, a magnetizable probe can be inserted into the solution, and by magnetizing the probe, the particles can accumulate on the end of the probe immersed in the solution. The probe can then be removed from the solution, still magnetized and immersed in the second solution, with the magnetic field no longer allowing the particles to enter the second solution. Commercial sources exist for magnets designed for use in both the magnetic removal and transfer techniques generally described above. For example, Promega Corporation, Magnetight See the MagneSphere Technology Magnetic Separation Stand or Poly A Tract Series 9600™ Multi-Magnet, available from Separation Stand (Novagen, Madison, Wis.) or Dynal Magnetic Particle Concentrator (Dynal, Oslo, Norway). Several embodiments involve the use of a magnetic device as described in U.S. Patent Application No. 13 / 089116, which is incorporated herein by reference in whole for all purposes. Furthermore, several embodiments intend to use a “jet channel” or pipette end magnetic separation (as described, for example, U.S. Patents No. 5,647,994 and 5,702,950). Several embodiments intend to use an immersion probe approach (as described, for example, U.S. Patent Nos. 6,447,729 and 6,448,092), as exemplified by the KingFisher system commercially available from Thermo Scientific.

[0184] Quantification of DNA strands In the examples described herein, strand recovery was measured using a flap endonuclease assay. For example, exemplary methods for extracting sample nucleic acids from blood, and for quantifying bisulfite-converted and unconverted DNA strands, are described, for example, in U.S. Patent No. 10,648,025, which is incorporated herein by reference for all purposes.

[0185] QuARTS assay An exemplary method for quantifying DNA strands generated according to this technology is the QuARTS flap assay technique. The QuARTS technique combines a polymerase-based targeted DNA amplification process with an invasive cleavage-based signal amplification process. This technique is described, for example, in U.S. Patent Applications Nos. 8,361,720, 8,715,937, 8,916,344, 9,212,392, and 15 / 841,006, each of which is incorporated herein by reference. The fluorescent signal generated by the QuARTS reaction is monitored in a manner similar to real-time PCR, enabling quantification of the amount of target nucleic acid in the sample.

[0186] An exemplary QuARTS reaction typically involves approximately 400–600 nmol / L (e.g., 500 nmol / L) of each primer and detection probe, approximately 100 nmol / L of invasive oligonucleotides, and approximately 600–700 nmol / L of each FRET cassette (e.g., FAM commercially available from Hologic, Inc., HEX commercially available from BioSearch Technologies, and BioSearch Technologies). The reaction includes Quasar 670 (commercially available from Technologies), 6.675 ng / μL of FEN-1 endonuclease (e.g., Cleavase® 2.0, Hologic, Inc.), 1 unit of Taq DNA polymerase (e.g., GoTaq® DNA polymerase, Promegacorp., Madison, WI) in 30 μL reaction volume, 10 mmol / L of 3-(n-morphorphino)propanesulfonic acid (MOPS), 7.5 mmol / L of MgCl2, and 250 μmol / L of dTPN. Exemplary QuARTS cycling conditions are shown in the table below. In some applications, the quantification cycle (C q The analysis of ) provides a measure of the number of the first target DNA strands in the sample (e.g., copy number).

[0187] [Table 1]

[0188] Multiple target pre-amplification of large amounts of bisulfite-converted DNA Large quantities of bisulfite-treated DNA can be used in a single, high-volume multiple amplification reaction to pre-amplify most or all of the bisulfite-treated DNA from the input sample. For example, DNA is extracted from cell lines (e.g., DFCI032 cell line (adenocarcinoma), H1755 cell line (neuroendocrine)) using, for example, the Maxwell Promega blood kit #AS1400 as described above. The DNA is then bisulfite-converted, for example, as described above.

[0189] Pre-amplification is performed using, for example, 7.5 mM MgCl2, 10 mM MOPS, 0.3 mM Tris-HCl, pH 8.0, 0.8 mM KCl, 0.1 μg / μL BSA, 0.0001% Tween-20, 0.0001% IGEPARCA-630, 250 μM each of dNTPs, oligonucleotide primers (e.g., 12 primers vs. 24 primers, equimolar amounts for 12 targets (e.g., including, but not limited to, the range of 200-500 nM for each primer), or individual primer concentrations adjusted to balance the amplification efficiency of different target regions), and 0.025 units / μL HotStart. The reaction is carried out in a reaction mixture containing GoTaq concentration and 20–50 volume% bisulfite-treated target DNA (e.g., 10 μL of target DNA in 50 μL of reaction mixture, or 50 μL of target DNA in 125 μL of reaction mixture). The thermal cycling time and temperature are selected to be appropriate for the volume of reactants and amplification vessel. For example, the reaction may be cycled as follows:

[0190] [Table 2]

[0191] After thermal cycling, the aliquot (e.g., 10 μL) of the pre-amplification DNA, with or without fish DNA, is diluted to 500 μL in 10 mM Tris and 0.1 mM EDTA. The diluted aliquot of pre-amplification DNA (e.g., 10 μL) is used in the QuARTS PCR-flap assay, for example, as described above. See also U.S. Patent Application No. 62 / 249,097 filed October 30, 2015, U.S. Patent Application No. 15 / 335,096 filed October 26, 2016, PCT / US16 / 58875 filed October 26, 2016, and U.S. Patent No. 10,648,025, each incorporated herein in whole by reference for all purposes.

[0192] This technique is not limited to the use of flap endonuclease assays, and in different embodiments, any method for analyzing bisulfite-converted DNA may be used. For example, in some embodiments, the analysis may include direct sequencing, pyrosequencing, methylation-sensitive single-strand structural analysis (MS-SSCA), high-resolution lysis analysis, methylation-sensitive single-nucleotide primer extension (MS-SnuPE), base-specific cleavage / mass spectrometry (e.g., by MALDI-TOF), methylation-specific PCR (MSP), microarray analysis, restriction digest analysis, INVADER assay, combined bisulfite restriction analysis, or methylated DNA immunoprecipitation (MeDIP). These and other methods are incorporated by reference in whole for any purpose, e.g., Fraga MF & Esteller M (2002), “DNA methylation: a profile of methods and applications”, Bio Techniques 33(3):632,634,636-49, El-MarriO(2003), “Methods:DNA methylation”, Advances in Experimental Medicine and Biology 544:197-204, Laird PW(2003), “The power and the promise of DNA methylation markers”, Nat.Rev.Cancer 3(4):253-66, Callinan PA & Feinberg AP (2006), “The emerging science of epigenomics”, Hum Mol Genet 15(90001):R95-101.

[0193] experiment Example 1 Effect of binding pH on the recovery of bisulfite-converted DNA In the examples described below, single-stranded DNA templates were sulfonated with ammonium bisulfite (ABS) as described in the exemplary sulfonation step above. After sulfonation of the DNA, the sulfonation reaction solution was removed by size exclusion filtration, and the sulfonated DNA was combined with potassium acetate (KOAc) at low (19 mM K+), medium (228 mM K+), and high (474 ​​mM K+) concentrations. As a control, a portion of the sulfonated DNA template was returned to a bisulfite solution instead of potassium acetate.

[0194] Subsequently, each sample was incubated with chaotrope and silica beads to promote binding. Table 1 below illustrates that the bisulfite solution inhibited the binding reaction due to the high salt concentration of each template. The binding pH was 4.8–4.9.

[0195] [Table 3]

[0196] Six DNA templates (markers) with varying cytosine content (see Table 2 below) were treated with bisulfite and then bound to silica using a binding solution with a pH ranging from 5.0 to 3.4.

[0197] [Table 4]

[0198] The results are shown in Figure 3. The pH required for maximum logarithmic strand recovery varied depending on the marker and appeared to correlate with the percentage of cytosine sequences converted (i.e., the percentage of all nucleotides in the chain that were unmethylated cytosine nucleotides before treatment and uracil sulfonate nucleotides in the binding step). DNA with a high unmethylated cytosine content and therefore a high content of bases converted to uracil sulfonate in the binding step required a lower pH for maximum strand recovery.

[0199] A total of 17 different DNA sequences were tested in the same manner as described above. The percentage of converted cytosine for each DNA was plotted against the highest estimated binding pH required for maximum strand recovery. Linear fitting of these points (Figure 4) was performed with a R of 0.76. 2 This suggests a strong relationship between the percentage of converted cytosine and the binding pH.

[0200] To further illustrate the strength of the correlation between converted cytosine percentage and binding pH, three of the DNA strands in Figure 3 (BMP3, β-actin (bisulfite-treated actin, or "BTACT"), and NDRG4) were modified to generate sets of DNA that retained the same primer and probe binding sequences for each test sequence but had different numbers of unmethylated C in other regions of the sequence. For the sequences in each set, a linear relationship was fitted to the measurements of the captured strand (Figure 5). The slopes and intercepts of all four fittings (Figures 4 and 5) were similar.

[0201] Comparison of DNA sample types Strand recovery from binding reactions performed at low pH (citrate buffer) and unadjusted pH (standard conditions) was compared for multiple sample types: single-stranded 126-mer synthetic DNA ("126"), DNA isolated from fecal samples spiked with purified cell line DNA ("ECLD"), and DNA isolated from fecal samples previously characterized as giving a high signal to methylated DNA ("HMS"). Six replications for each combination of sample type and binding conditions are shown in Table 3 below, and the strand recovery results are presented. All DNA from the fecal samples was isolated using the capture process described above, and the synthetic 126-mer DNA strands were combined with exogenous fish DNA as a carrier.

[0202] [Table 5]

[0203] [Table 6]

[0204] The results in Table 3 show that the use of low pH binding reactions produced similar or enhanced chain recovery for each of these sample types.

[0205] Surrogate blood samples containing low (1×) or high (10×) nucleosome matrices were prepared. Nucleosome DNA was prepared from HCT116 cells using the Active Motif Nucleosome Kit (Active Motif, catalog no. 53504). To mimic methylated DNA marker (MDM)-positive plasma samples, nucleosome DNA was spiked into pooled plasma collected from healthy donors using LBgard® blood tubes (Exact Sciences, Inc.) or into SERACON-negative diluents. The use of nucleosome matrix preparations ensures that MDM-positive DNA has a size distribution similar to that expected in samples from cancer-positive patients.

[0206] The samples were processed to extract DNA using the plasma extraction procedure described above, or using the QIASYMPHONY DSP Circulating DNA Kit (Qiagen, Inc.). The extracted DNA samples were then bisulfite-treated and bound in the standard (unadjusted pH) binding reaction solution (7M GuHCl binding buffer added to the ABS reaction) and the low pH binding reaction solution (7M GuHCl, pH 2.2 binding buffer with 133 mM citrate buffer).

[0207] Comparison of 14 DNA samples showed increased strand recovery for 15 / 34 conditions compared to t-tests (Tables 4, 5, 6, and 7 below). Overall, lower binding pH increased strand recovery for various sample types.

[0208] [Table 7]

[0209] [Table 8]

[0210] [Table 9]

[0211] [Table 10]

[0212] DNA isolated from the clarified supernatant of a fecal sample. Cell line DNA-spiked stool samples with sample input volumes ranging from 4 to 14 mLs (standard) were processed to capture cell line DNA, followed by bisulfite treatment and isolation using standard pH (Figure 6A) and low-binding pH (Figure 6B) solutions. A comparison of Figures 6A and 6B shows improved linearity of total DNA and strand recovery at the low-binding pH. Samples used for unadjusted (standard) and low-binding pH have different amounts of spiked DNA template, and therefore the shown total strand recovery should not be directly compared across binding conditions.

[0213] The amount of DNA recovered from each volume (mL) of stool sample using unadjusted or low pH-bound conditions is shown in the eight panels of Figure 7.

[0214] The effect of different batches of ammonium bisulfite Four ammonium bisulfite (56%) lots were tested under unadjusted and low-binding pH conditions as controls. The ABS lots varied in age, with the oldest being 9 years old, having the lowest pH, and the ammonium bisulfite potentially degrading by as much as 2 percent per year. Chain recovery, without pH adjustment, showed the highest recovery rate with the oldest material and varied across lots (Figure 8A). Recovery alignment improved at lower binding pH.

[0215] Effect of ammonium bisulfite concentration After titrating with ammonium bisulfite at concentrations of 57–43%, synthetic DNA templates bound with low binding pH (citrate buffer) were tested, or without pH adjustment. Using 56.6% as the baseline, the percentage difference in binding for each indicated marker DNA at lower ABS concentrations was calculated. The results are shown in the table in Figure 8B. Strand recovery alignment across ABS concentrations improved at lower binding pH levels.

[0216] It was found that contaminants in an unknown binding solution (7M guanidine-HCl) introduced during manufacturing suppressed the recovery of the selection marker. When comparing recovery of low-binding pH and unadjusted chains with and without contaminants, the low-binding pH condition reduced the decrease in recovery compared to the unadjusted binding condition (Figure 9).

[0217] Recovery using beads from different manufacturers Silica beads from multiple vendors were tested for use in bisulfite conversion at unadjusted pH or at a low binding pH, and the results are shown in Figures 10 and 11, respectively. Some beads did not show chain recovery at unadjusted binding pH (Figure 10), but showed improved recovery as the binding pH decreased (Figure 11). The bead concentrations were not adjusted to be equal to the initial screening in Figure 10, but were aligned for the tests shown in Figure 11.

[0218] DNA recovery across a wide pH range Acidic conditions can lead to loss of strand recovery due to acid hydrolysis of the DNA template. When the binding pH was examined in the range of 4.9 to 2.1, strand recovery was most consistent at approximately pH 3.9 to 2.5 (see Figure 12).

[0219] Influence of DNA size and concentration DNA size and concentration can vary across and within sample types, such as tissue, blood, and stool. In this experiment, calf thymic DNA was titrated at 300, 2700, and 5400 ng / run at sizes of 200 bp, 3.6 reaction, and 20 kbp. DNA samples were spiked with a 126 bp synthetic target molecule and treated with bisulfite using unadjusted and low-binding pH to capture sulfonated DNA. The results are shown in Figure 13.

[0220] As DNA size decreased and concentration increased, strand recovery significantly decreased with unadjusted bound pH chemistry. Strand recovery with low bound pH chemistry was significantly improved (up to 374%) compared to unadjusted bound pH. In addition, the coefficient of variation (CV) improved with low bound pH chemistry.

[0221] Example 2 Comparison of three coupling protocols The following examples compare three different procedures for binding sulfonated DNA to silica.

[0222] Protocol 1 is a standard protocol that combines the sulfonation reaction with a conjugated solution of 7M guanidine hydrochloride (GuHCl), but without the addition of an acid to lower the pH.

[0223] In Protocol 2, the sulfonation reaction mixture is combined with a conjugation solution of 7M GuHCl, 133mM citrate, and pH 2.2.

[0224] In Protocol 3, the sulfonation reaction mixture is combined with a 7M GuHCl binding solution, but no acid is added to lower the pH.

[0225] In each of the procedures described, the bead-bound sulfonated DNA is desulfonated on a solid support, for example, as described in U.S. Patent No. 9,315,853, which is incorporated herein by reference in its entirety.

[0226] The conversion protocol is described below for the performance of the Hamilton STARlet automated pipetting system, but can be performed manually using equivalent equipment.

[0227] Protocol 1 This procedure describes the processing of DNA captured from a sample, such as a stool sample, using sequence-specific capture beads containing capture oligonucleotides complementary to the target DNA. Degeneration: 1. To wash the captured bead pellet, add 20 μL of 90 ng / μL BSA, 10 mM Tris (pH 8.0), 1 mM EDTA, and 160 μL of 100 mM NaOH.

[0228] 2. Incubate at 2.43°C, mixing at 1500 RPM for 45 seconds.

[0229] 3. Incubate at 43°C for another 19 minutes and 15 seconds without mixing (total 20 minutes).

[0230] 4. Connect on the magnet for 5 minutes.

[0231] 5. Transfer 80 μL of the denatured sample to a new deep-well plate. Sulfonation: 1. Add 120 μL of 57% (by weight) ammonium bisulfite (ABS) to the denatured sample.

[0232] 2. Mix at 1200 RPM for 3 minutes on a heater shaker at 2.65.5°C.

[0233] 3. Incubate for another 72 minutes without mixing (total 75 minutes). Sulfonated DNA binding: 1. Add 750 μL of 7M GuHCl bonded solution to the sample.

[0234] 2. Mix the bead stock through a pipette for 5 cycles, and add 50 μL of 16 mg / mL paramagnetic silica beads to each sample.

[0235] 3. Incubate at 30.5°C with a 1200 RPM mix for 30 minutes.

[0236] 4. Place the beads on a magnet for 5 minutes to bind, then aspirate and discard the supernatant. Alcohol cleaning 1 and 2: 1. Add 1 mL of 80% ethanol (EtOH) and a 10 mM Tris (pH 8.0) conversion washing solution to the bead pellet.

[0237] 2. Mix at 1200 RPM for 3 minutes.

[0238] 3. Place the beads on a magnet for 5 minutes to bind, then aspirate and discard the supernatant.

[0239] 4. Add 250 μL of 80% EtOH and 10 mM Tris (pH 8.0) conversion washing solution to the bead pellet.

[0240] 5. Mix at 1200 RPM for 3 minutes.

[0241] 6. Place the beads on a magnet for 2 minutes to bind, then aspirate and discard the supernatant. Desulfonation: 1. Add 200 μL of 70% isopropyl alcohol (IPA) and 105 mM NaOH to the bead pellet.

[0242] 2. Incubate at 30.5°C with a mixing rate of 1200 RPM for 7 minutes.

[0243] 3. Place the beads on a magnet for 2 minutes to bind, then aspirate and discard the supernatant. Alcohol cleaning 3 and 4: 1. Add 250 μL of 80% EtOH and 10 mM Tris (pH 8.0) conversion washing solution to the bead pellet.

[0244] 2. Mix at 1200 RPM for 3 minutes.

[0245] 3. Place the beads on a magnet for 2 minutes to bind, then aspirate and discard the supernatant.

[0246] 4. Add 250 μL of 80% EtOH and 10 mM Tris (pH 8.0) conversion washing solution to the bead pellet.

[0247] 5. Mix at 1200 RPM for 3 minutes.

[0248] 6. Place the beads on a magnet for 2 minutes to bind, then aspirate and discard the supernatant. Dried beads and eluted DNA: 1. Incubate the bead pellets at 71.5°C for 15 minutes while mixing at 1200 RPM, then allow to dry.

[0249] Add 70 μL of 2.10 mM Tris (pH 8.0) and 0.1 mM EDTA to the dried beads.

[0250] Mix at 66.5 °C at 1200 RPM and incubate for 25 minutes.

[0251] Place on the magnet for 8 minutes to cool and bind the beads.

[0252] The converted DNA is ready for the assay.

[0253] Protocol 2 This procedure describes the processing of DNA captured from a sample, e.g., a fecal sample, using sequence-specific capture beads containing capture oligonucleotides complementary to the target DNA. Denaturation: To wash the capture bead pellet, add 50 μL of 36 ng / μL BSA, 50 μL of 10 mM Tris (pH 8.0), 1 mM EDTA, and 160 mM NaOH.

[0254] Mix at 32.5 °C at 1500 RPM and incubate for 3 minutes.

[0255] Bind on the magnet for 5 minutes.

[0256] Transfer 80 μL of the denatured sample to a new deep well plate. Sulfonation: Add 120 μL of 52 wt / wt% ABS to the denatured sample.

[0257] Place on a heater shaker at 62.5 °C and mix at 1200 RPM for 3 minutes.

[0258] Incubate for an additional 85 minutes without shaking (total 88 minutes). Sulfonated DNA Binding: Add 750 μL of 7 M GuHCl, 133 mM citrate, pH 2.2 binding solution to the sample.

[0259] 2. Mix the bead stock through a pipette for 5 cycles, and add 50 μL of 16 mg / mL paramagnetic silica beads to each sample.

[0260] 3. Incubate at 32.5°C for 15 minutes while mixing at 1200 RPM.

[0261] 4. Place the beads on a magnet for 5 minutes to bind, then aspirate and discard the supernatant. Alcohol cleaning 1 and 2: 1. Add 1 mL of 75% IPA and 20 mM Tris (pH 7.8) conversion washing solution to the bead pellet.

[0262] 2. Mix at 1200 RPM for 3 minutes.

[0263] 3. Place the beads on a magnet for 5 minutes to bind, then aspirate and discard the supernatant.

[0264] 4. Add 250 μL of 75% IPA and 20 mM Tris (pH 7.8) conversion washing solution to the bead pellet.

[0265] 5. Mix at 1200 RPM for 3 minutes.

[0266] 6. Place the beads on a magnet for 2 minutes to bind, then aspirate and discard the supernatant. Desulfonation: 1. Add 200 μL of 70% IPA and 30 mM NaOH to the bead pellet.

[0267] 2. Incubate at 2.32.5°C with a mixing rate of 1200 RPM for 3 minutes.

[0268] 3. Place the beads on a magnet for 2 minutes to bind, then aspirate and discard the supernatant. Alcohol cleaning 3 and 4: 1. Add 250 μL of 75% IPA and 20 mM Tris (pH 7.8) conversion washing solution to the bead pellet.

[0269] Mix at 2.1200 RPM for 3 minutes.

[0270] 3. Place on the magnet for 2 minutes to bind the beads, aspirate and discard the supernatant.

[0271] 4. Add 250 μL of 75% IPA and 20 mM Tris (pH 7.8) conversion wash solution to the bead pellet.

[0272] 5. Mix at 1200 RPM for 3 minutes.

[0273] 6. Place on the magnet for 2 minutes to bind the beads, aspirate and discard the supernatant. Dry Beads and Eluted DNA: 1. Incubate the bead pellet at 71.5 °C for 15 minutes with mixing at 1200 RPM to dry.

[0274] 2. Add 70 μL of 3 mM Tris (pH 7.8) and 0.1 mM EDTA to the dry beads.

[0275] 3. Mix at 1200 RPM at 66.5 °C and incubate for 25 minutes.

[0276] 4. Place on the magnet for 25 minutes to cool and bind the beads.

[0277] 5. The converted DNA is ready for the assay.

[0278] Protocol 3 This procedure describes the processing of a sample, e.g., DNA extracted from plasma, using chaotrope-mediated binding to a silica support. Elute the DNA from the support prior to the following denaturation steps. Denaturation: 1. Add 11 μL of 90 ng / μ LBSA, 10 mM Tris (pH 8.0), 1 mM EDTA, and 11 μL of 1.0 M NaOH to 75 μL of the extracted DNA sample.

[0279] 2. Incubate at room temperature for 1 minute while mixing at 1200 RPM. Sulfonation: 1. Add 120 μL of 52 wt / wt% ABS to the denatured sample.

[0280] 2. Place on a heater shaker and mix at 65.5°C for 3 minutes at 1200 RPM.

[0281] 3. Incubate for another 72 minutes without mixing (total 75 minutes). Sulfonated DNA binding: Add 750 μL of 1.7 M GuHCl bonded solution to the sample.

[0282] 2. Mix the bead stock through a pipette for 5 cycles, and add 50 μL of 16 mg / mL paramagnetic silica beads to each sample.

[0283] 3. Mix at 30.5°C at 1200 RPM and incubate for 30 minutes.

[0284] 4. Place the beads on a magnet for 5 minutes to bind, then draw the supernatant into the waste. Alcohol cleaning 1 and 2: 1. Add 1 mL of 80% EtOH and 10 mM Tris (pH 8.0) conversion washing solution to the bead pellet.

[0285] 2. Mix at 1200 RPM for 3 minutes.

[0286] 3. Place the beads on a magnet for 5 minutes to bind, then draw the supernatant into the waste.

[0287] 4. Add 250 μL of 80% EtOH and 10 mM Tris (pH 8.0) conversion washing solution to the bead pellet.

[0288] 5. Mix at 1200 RPM for 3 minutes.

[0289] 6. Place the beads on a magnet for 2 minutes to bind, then aspirate and discard the supernatant. Desulfonation: 1. Add 200 μL of 75% IPA and 87.5 mM NaOH to the bead pellet.

[0290] 2. Incubate at 30.5°C with a mixing rate of 1200 RPM for 7 minutes.

[0291] 3. Place the beads on a magnet for 2 minutes to bind, then aspirate and discard the supernatant. Alcohol cleaning 3 and 4: 1. Add 250 μL of 80% EtOH and 10 mM Tris (pH 8.0) conversion washing solution to the bead pellet.

[0292] 2. Mix at 1200 RPM for 3 minutes.

[0293] 3. Place the beads on a magnet for 2 minutes to bind, then aspirate and discard the supernatant.

[0294] 4. Add 250 μL of 80% EtOH and 10 mM Tris (pH 8.0) conversion wash solution to the bead pellet.

[0295] 5. Mix at 1200 RPM for 3 minutes.

[0296] 6. Place the beads on a magnet for 2 minutes to bind, then aspirate and discard the supernatant. Dried beads and eluted DNA: 1. Incubate the bead pellets at 71.5°C for 15 minutes while mixing at 1200 RPM, then allow to dry.

[0297] Add 80 μL of 2.4 mM Tris (pH 8.0) and 0.1 mM EDTA to the dry beads.

[0298] 3. Mix at 1200 RPM at 66.5°C and incubate for 25 minutes.

[0299] 4. Place the beads on the magnet for 20 minutes to cool and bond. 5. The converted DNA is ready for assay. The above conditions were summarized and compared in the table in Figure 14.

[0300] To compare the treatment protocols described above, genomic DNA was extracted from OE33 cells (esophageal cancer) using the Maxwell system (PromegaCorp., Fichburg, WI) and the RSC Blood DNA Kit (PromegaCorp., catalog number ASB1400), both according to the manufacturer's instructions. The prepared DNA was used in all three protocols and added directly to the denaturation step of each protocol without the intervening DNA capture step. Strand recovery for each of protocols 1, 2, and 3 was tested for DNA concentrations of 26.7, 17.8, 11.87, 7.91, 5.27, and 3.52 ng / μL. Four copies were tested for each condition. The sequences tested with the prepared DNA were the methylation marker genes ZNF568, BMP3, B3GALT6, NDRG4, VAV3, and ZNF682. Detailed data are shown in Figures 15A and 15B.

[0301] Figure 16 shows a table with paired comparisons of strand retrieval from Protocol 2 (using low pH binding) compared to Protocols 1 and 3 (both without pH adjustment for binding) for different methylated marker DNAs, based on the data shown in Figures 15A-15B.

[0302] Figure 17 shows a graph illustrating the percentage change using Protocol 2 versus Protocol 1 or Protocol 3 (collectively grouped as "Protocol X") at the indicated DNA concentrations. The dashed line represents 100%, i.e., equal recovery rates using Protocol 2 and Protocol X. With the exception of the VAV3 marker DNA at lower DNA concentrations tested, where Protocol 1 yielded higher strand recovery, the method of Protocol 2 (using low pH-bound conditions) showed higher strand recovery rates compared to both of the other two protocols (both using standard, unadjusted pH-bound conditions).

[0303] Buffer comparison Protocol 2 was used as described above, along with a modified sulfonated DNA binding step method that uses glycine, citrate, malate, or formate buffer in a GuHCl-bound solution.

[0304] For the non-buffered control reaction, GuHCl was used without adding buffer during the binding step. The results are shown in Figure 18.

[0305] All documents and similar materials cited herein, including but not limited to patents, patent applications, articles, books, papers, manufacturers' manuals, product casings, and internet web pages, are expressly incorporated by reference in their entirety for any purpose. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to whom the various embodiments described herein belong. Where, when the definitions of these terms appear to differ from those provided in the teachings of the invention when used by reference, the definitions provided in the teachings of the invention shall prevail.

[0306] Various modifications and variations of the compositions, methods, and uses of the scientific and technological invention described herein will be obvious to those skilled in the art without departing from the scope and spirit of the scientific and technological invention as described herein. Although the scientific and technological invention has been described in relation to specific exemplary embodiments, it should be understood that the invention as described in the claims should not be unduly limited to such specific embodiments. In fact, various modifications of the described form for carrying out the invention which will be obvious to those skilled in the art in molecular biology, diagnostics, pharmacology, biochemistry, medicine, or related fields are intended to fall within the following claims. [Brief explanation of the drawing]

[0307] [Figure 1] This is an overview of one embodiment of a bisulfite treatment method, including the method disclosed herein. [Figure 2] This is a schematic diagram of the proposed coupling mechanism. [Figure 3]This provides graphs of the bound pH versus log strand recovered for six different marker DNAs. [Figure 4] This graph shows the percentage of converted cytosine versus the maximum binding pH. The percentage of converted cytosine (uracil sulfonate nucleotides / total nucleotides) for each DNA strand is plotted against the maximum estimated binding pH required for maximum strand recovery. A linear fitting of these points is shown. [Figure 5] This graph shows the percentage of converted cytosine versus maximum binding pH for DNA strands using the same primer / probe combination. Three of the DNA strands (BMP3, β-actin ("BTACT"), and NDRG4 strands) were modified to create sets of DNA that retain the same primer and probe binding sequences for each variant of a particular DNA, but contain different numbers of unmethylated C in other regions of the DNA strand. To further illustrate the strength of this correlation, lines were fitted to each set of sequences. [Figure 6] The graphs show the total DNA post-conversion and cleanup using either unadjusted binding pH (A) or low binding pH (B). [Figure 7] This graph shows the number of strands recovered per stool sample volume (mL) for four different DNA sequences (LASS4, PPP2R5C, LRRC4, and ZDHHC1) at either unadjusted binding pH (top panel) or low binding pH (bottom panel). ZDHHC1 DNA was tested twice. [Figure 8] Table A provides chain recovery from different lots of ammonium bisulfite from binding reactions carried out at low pH and unadjusted pH. Table B provides chain recovery from different concentrations of ammonium bisulfite from binding reactions carried out at low pH and unadjusted pH. [Figure 9] A table is provided showing the effects of contaminants present in binding reactions carried out at low pH and unadjusted pH. [Figure 10] A table is provided showing logs of recovered chains using various silica beads with an unadjusted pH-binding reaction. [Figure 11]A table is provided showing the logs of chains recovered using various silica beads with the low pH binding reaction described in Example 1. [Figure 12] A table is provided showing the chains recovered using a binding reaction adjusted to an acidic pH ("BNDpH") in the range of 4.85 to 2.13. [Figure 13] A table is provided showing the strands recovered for DNA molecules of different sizes and concentrations using unadjusted or low pH binding reactions. [Figure 14] A table comparing the reaction conditions provided by Protocol 1, Protocol 2, and Protocol 3 is shown. [Figure 15A] A table comparing chain recovery using Protocol 1, Protocol 2, and Protocol 3 is shown. [Figure 15B] A table comparing chain recovery using Protocol 1, Protocol 2, and Protocol 3 is shown. [Figure 16] Figures 15A-15B show a table with pairwise comparisons of strand retrieval for different methylated marker DNAs using Protocols 1, 2, and 3. [Figure 17] Figure 16 shows a graph comparing strand retrieval for different methylated marker DNAs, expressed as a ratio of retrieval using Protocol 2 versus retrieval using Protocol 1 or Protocol 3. “Protocol X” for each data point represents either Protocol 1 or Protocol 3 for that data point. [Figure 18] A table is shown comparing the recovery of different DNAs, using an unadjusted pH during the binding step, or reducing the pH during the binding of the indicated sulfonated DNA to silica beads using glycine, citrate, malic acid, or formate buffer.

Claims

[Claim 1] A method comprising combining sulfonated DNA and a silica support in an acidic binding solution, wherein the acidic binding solution has a pH below the isoelectric point (pH(I)) of the sulfonated DNA, and the sulfonated DNA is bound to the silica support.

Images