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  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
  • C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
  • C12N15/1006—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers
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  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
  • C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
  • C12N15/1006—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers
  • C12N15/1013—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers by using magnetic beads
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  • C12Q2523/00—Reactions characterised by treatment of reaction samples
  • C12Q2523/10—Characterised by chemical treatment
  • C12Q2523/125—Bisulfite(s)
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  • C12Q2527/00—Reactions demanding special reaction conditions
  • C12Q2527/119—Reactions demanding special reaction conditions pH
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q2527/00—Reactions demanding special reaction conditions
  • C12Q2527/125—Specific component of sample, medium or buffer

Definitions

• the present disclosure relates to the purification of sulfonated DNA. Additionally, the disclosure is related to methods and systems for bisulfite conversion of DNA and improved purification of sulfonated DNA with supports, e.g., silica supports, and use of acidic conditions for binding sulfonated DNA to supports.
• supports e.g., silica supports
• DNA methylation is an epigenetic mechanism that occurs by the addition of a methyl group to DNA, specifically the cytosine ring of DNA, thereby modifying the function of the gene. Detecting and mapping sites of DNA methylation are essential for understanding epigenetic gene regulation and detecting aberrant DNA methylation involved in the development and progression of cancer and other disease states.
• mapping methylation sites is currently accomplished by the bisulfite method described by Frommer, et al. for the detection of 5-methylcytosines in DNA Proc. Natl. Acad. Sci. USA 89: 1827-31 (1992), incorporated herein by reference in its entirety for all purposes) or variations thereof.
• the bisulfite method of mapping 5-methylcytosines is based on the observation that cytosine, but not 5-methylcytosine, reacts with bisulfite.
• the reaction includes a series of steps, including: reacting cytosine with hydrogen sulfite to form sulfonated cytosine; spontaneous deamination of the sulfonated reaction intermediate which results in sulfonated uracil; and desulfonating uracil under alkaline conditions to form uracil.
• Base pair differences between uracil and 5-methylcytosine facilitate detection - uracil base pairs with adenine and 5-methylcytosine base pairs with guanine.
• a variety of methods are able to differentiate methylated cytosines from non-methylated cytosines, e.g., bisulfite genomic sequencing (Grigg G, & Clark S, Bioessays (1994) 16: 431-36; Grigg G, DNA Seq. (1996) 6: 189-98) or methylation-specific PCR (MSP) as is disclosed, e.g., in U.S. Patent No. 5,786,146.
• MSP methylation-specific PCR
• bisulfite treatment methods include washing steps and buffer changes to produce a purified, converted DNA sample for base pair analysis.
• a variety of approaches facilitate these steps, e.g., spin columns, ethanol purification, and solid supports.
• methods using silica spin columns or ethanol purification often result in sample losses that compromise the usefulness of the bisulfite method as a measure of cytosine methylation.
• improvements have been developed using solid supports, these methods require large amounts of DNA as input and also suffer from problems of sample loss and reproducibility.
• conventional methods often require days-long times to complete and do not provide an efficient conversion and recovery of the converted DNA. Consequently, conventional methods provide only qualitative measures of DNA methylation.
• the systems and methods disclosed herein increase bisulfite converted DNA recovery, especially for cytosine-rich targets, improve robustness to variations in the bisulfite treatment process, , and increase DNA recovery for a wide range of sample types.
• the methods include neutralizing DNA charge to promote silica-DNA interaction in addition to hydrogen bond disruption.
• the pH is less than 5.
• the pH is between 2.5 and 4.
• the acidic solution may further comprise a chaotropic salt or agent.
• the chaotropic salt is guanidine hydrochloride (GuHCl).
• the methods may further comprise incubating denatured, non-sulfonated DNA with a sulfonation reagent to produce the sulfonated DNA.
• a sulfonation reagent preferably refers to a reagent that selectively converts unmethylated cytosine nucleotides to uracil sulfonate nucleotides.
• the sulfonation reagent is ammonium bisulfite.
• the methods may further comprise at least one or all of: washing silica supports bound to sulfonated DNA; desulfonating the sulfonated DNA to form desulfonated DNA; and eluting the desulfonated DNA from the silica supports.
• the washing is done with an alcohol, e.g., ethanol.
• the desulfonating comprises incubation of sulfonated DNA at alkaline conditions and in some embodiments, sulfonated DNA is added directly to a detection assay, e.g., a polymerase chain reaction, such that the sulfonated DNA is desulfonated under the conditions of the conducting the detection assay.
• the elution comprises treatment with high heat.
• a method comprising combining sulfonated DNA and a silica support in an acidic binding solution, wherein the acidic binding solution has a pH less than or equal to an isoelectric point (pH(I)) of the sulfonated DNA, and wherein sulfonated DNA is bound to the silica support.
• silica support comprises at least one of a particle, a bead, and a fiber.
• chaotropic salt comprises at least one of guanidine hydrochloride (GuHCl) and guanidine isothiocyanate (GITC).
• the method further comprises incubating singlestranded non-sulfonated DNA or partially-sulfonated DNA with a sulfonation reagent to produce the sulfonated DNA.
• the acidic component comprises at least one of an acidic acetate, glycine, malate, formate, or citrate solution.
• chaotropic salt comprises at least one of GuHCl and GITC.
• a method of preparing desulfonated DNA comprising: i) incubating single-stranded non-sulfonated DNA or partially-sulfonated DNA with a sulfonation reagent to produce sulfonated DNA in a sulfonation reaction mixture; ii) combining the sulfonation reaction mixture with: a) an acidic component; and b) chaotropic salt; and c) a silica support to form an acidic binding solution, wherein the acidic binding solution has a pH less than or equal to an isoelectric point (pH(I)) of the sulfonated DNA, and wherein sulfonated DNA is bound to the silica support; iii) separating sulfonated DNA bound to the silica support from the acidic binding solution; iv) treating separated sulfonated DNA under conditions wherein the sulfonated DNA is desulfonated.
• non-sulfonated DNA comprises one or more unmethylated cytosine nucleotides
• desulfonated DNA comprises one or more deoxyuracil nucleotides
• treating separated sulfonated DNA under conditions wherein the sulfonated DNA is desulfonated comprises combining sulfonated DNA with a desulfonation solution.
• a composition comprising sulfonated DNA and a silica support in an acidic binding solution comprising a chaotropic salt, wherein the acidic binding solution has a pH less than or equal to a pH(I) of the sulfonated DNA, and wherein the sulfonated DNA is bound to the silica support.
• composition of embodiment 24, wherein the chaotropic salt comprises at least one of GuHCl and GITC.
• composition of embodiment 24, wherein the acidic binding solution comprises at least one of a acetate, glycine, malate, formate, or citrate buffer.
• composition of embodiment 26, wherein the acidic binding solution comprises a citrate buffer.
• composition of embodiment 24, wherein the acidic binding solution comprises a bisulfite salt.
• the bisulfite salt is ammonium bisulfite.
• composition of embodiment 24, wherein the pH of the acidic binding solution is less than 5.
• composition of embodiment 30, wherein the pH of the acidic binding solution is between 2.0 and 4.
• a kit comprising: i) a bisulfite reagent solution or components for preparing a bisulfite reagent solution; ii) a silica support; and iii) an acidic component having a pH less than or equal to a pH(I) of sulfonated DNA.
• the acidic component comprises at least one of an acetate, glycine, malate, formate, or citrate buffer.
• kit of embodiment 32 further comprising a chaotropic salt.
• kits of embodiment 36, wherein the chaotropic salt comprises at least one of GuHCl and GITC. 39.
• a method comprising combining sulfonated DNA and a silica support in an acidic binding solution, wherein the acidic binding solution has a pH less than or equal to a isoelectric point (pH(I)) of the sulfonated DNA, and wherein sulfonated DNA is bound to the silica support, wherein the silica support preferably comprises at least one of a particle, a bead, and a fiber, wherein the silica support is preferably magnetic.
• pH(I) isoelectric point
• the acidic binding solution comprises a chaotropic salt
• the chaotropic salt preferably comprises at least one of guanidine hydrochloride (GuHCl) and guanidine isothiocyanate (GITC).
• the method further comprises incubating single-stranded non-sulfonated DNA or partially-sulfonated DNA with a sulfonation reagent to produce the sulfonated DNA.
• the acidic component comprises at least one of an acidic acetate, glycine, malate, formate, or citrate solution.
• a method of preparing desulfonated DNA comprising: i) incubating single-stranded non-sulfonated DNA or partially-sulfonated DNA with a sulfonation reagent to produce sulfonated DNA in a sulfonation reaction mixture; ii) combining the sulfonation reaction mixture with: a) an acidic component; b) chaotropic salt; and c) a silica support to form an acidic binding solution, wherein the acidic binding solution has a pH less than or equal to an isoelectric point (pH(I)) of the sulfonated DNA, and wherein sulfonated DNA is bound to the silica support; iii) separating sulfonated DNA bound to
• treating separated sulfonated DNA under conditions wherein the sulfonated DNA is desulfonated comprises combining sulfonated DNA with a desulfonation solution.
• a composition comprising sulfonated DNA and a silica support in an acidic binding solution comprising a chaotropic salt, wherein the acidic binding solution has a pH less than or equal to the pH(I) of the sulfonated DNA, and wherein the sulfonated DNA is bound to the silica support, wherein the chaotropic salt preferably comprises at least one of GuHCl and GITC; and wherein the acidic binding solution preferably comprises at least one of a acetate, glycine, malate, formate, or citrate buffer, preferably a citrate buffer.
• composition of embodiment 60, wherein the acidic binding solution comprises a bisulfite salt, preferably ammonium bisulfite.
• composition of embodiment 60 or embodiment 61, wherein the pH of the acidic binding solution is less than 5, preferably between 2.0 and 4.
• a kit comprising: i) a bisulfite reagent solution or components for preparing a bisulfite reagent solution, wherein the bisulfite reagent solution preferably has a pH that is greater than a pH(I) of sulfonated DNA.; ii) a silica support; and iii) an acidic component, preferably an acidic solution, the acidic component having a pH less than or equal to the pH(I) of sulfonated DNA, wherein the acidic component preferably comprises at least one of an acetate, glycine, malate, formate, or citrate buffer, preferably citrate buffer.
• kit of embodiment 63 further comprising a chaotropic salt, wherein preferably the acidic component comprises a chaotropic salt, and wherein the chaotropic salt preferably comprises at least one of GuHCl and GITC.
• kits of embodiment 63 or 64, wherein the silica support comprises at least one of a particle, a bead, and a fiber.
• the pH of the acidic component is less than 5, preferably wherein the pH of the acidic component is between 2.0 and 4.
• the single-stranded DNA is isolated from a sample, e.g., a biological sample.
• the single-stranded DNA is isolated from a sample from a human subject, such as a subject having or suspected of having a cancer, e.g., from a plasma sample, a stool sample, or other sample from the subject.
• the single-stranded DNA is synthetic.
• single-stranded DNA is formed by exposing double-stranded DNA to denaturing conditions e.g., alkaline conditions, high temperature, etc.) under which the base-paired DNA strands separate to form single strands.
• the desulfonated DNA is measured, e.g., to measure one or more methylated DNA markers (MDMs) in the DNA.
• MDMs methylated DNA markers
• any of the DNAs named in herein e.g., in the Experimental section or in the Drawings, including MDMs disclosed in Tables 1-7, Figs. 3, 7-13, 15A and 15B, 16, and 18
• Fig. 1 is overview of one embodiment of a bisulfite treatment method comprising the methods disclosed herein.
• Fig. 2 is a schematic of the proposed binding mechanism.
• Fig. 3 provides graphs of binding pH vs. log strands recovered for six different marker DNAs.
• Fig. 4 is a graph of percent converted cytosines vs. maximum binding pH. The percent converted cytosines of each DNA strand (uracil sulfonate nucleotides /total nucleotides) was plotted against the highest estimated binding pH required for maximum strand recovery. A linear fit of these points is shown.
• Fig. 5 is a graph of percent converted cytosines vs. maximum binding pH of DNA strands that use the same primer/probe combinations.
• Three of the DNAs (BMP 3, p-actin (“ETACr”), and NDRG4 strands), were modified to produce sets of DNAs that retained the same primer and probe binding sequences for each variant of a particular DNA, but that contained different numbers of unmethylated Cs in other regions of that DNA strand. To further illustrate the strength of this correlation, lines were fit to each set of sequences.
• Figs.6A and 6B show graphs of total DNA post conversion and clean-up using a nonadjusted binding pH (Fig. 6A) or low binding pH (Fig. 6B).
• Fig. 7 are graphs of stool sample volume (mLs) versus strands recovered for a nonadjusted binding pH (top row) or low binding pH (bottom row) for four different DNA sequences (LASS4, PPP2R5C, LRRC4, and ZDHHC1)'.
• the ZDHHC1 DNAs were tested twice.
• Fig. 8A provides a table showing strand recovery from different lots of ammonium bisulfite from binding reactions performed at low pH and at non-adjusted pH.
• Fig. 8B provides a table showing strand recovery from different concentrations of ammonium bisulfite from binding reactions performed at low pH and at non-adjusted pH.
• Fig. 9 provides a table showing the effects of a contaminant present in binding reactions performed at low pH and at non-adjusted pH.
• Fig. 10 provides a table showing log of strands recovered using various silica beads using non-adjusted pH binding reactions.
• Fig. 11 provides a table showing log of strands recovered using various silica beads using low pH binding reactions described in Example 1.
• Fig. 12 provides a table showing strands recovered using binding reactions adjusted to acidic pHs (“BND pH”) ranging from 4.85 down to 2.13.
• Fig. 13 provides a table showing strands recovered for different sizes DNA molecules, and for different concentrations of DNA molecules, using non-adjusted or low pH binding reactions.
• Fig. 14 shows a table comparing reaction conditions provided by Protocol 1, Protocol 2, and Protocol 3.
• Figs 15A and 15B show tables comparing strand recoveries using Protocol 1, Protocol
• Fig. 16 shows a table with pairwise comparisons of strand recoveries shown in Figs. 15A-15B for different methylation marker DNAs using Protocol 1, Protocol 2, and Protocol
• Fig. 17 shows a graph comparing strand recoveries shown in Figs. 16 for different methylation marker DNAs, shown as the ratios of Protocol 2 recovery vs. recovery using Protocol 1 or Protocol 3.
• Protocol X for each datapoint represents either Protocol 1 or 3 for that datapoint.
• Fig. 18 shows a table comparing recoveries of different DNAs using unadjusted pH during the binding step, or using glycine, citrate, malate, or formate buffer to reduce the pH during binding the indicated sulfonated DNAs to silica beads.
• composition “consisting essentially of’ recited elements may contain an unrecited contaminant at a level such that, though present, the contaminant does not alter the function of the recited composition as compared to a pure composition, i.e., a composition “consisting of’ the recited components.
• each intervening number there between with the same degree of precision is explicitly contemplated.
• the numbers 7 and 8 are contemplated in addition to 6 and 9, and for the range 6.0-7.0, the number 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 contemplated.
• a “DNA fragment” or “small DNA” or “short DNA” means a DNA that consists of no more than approximately 200 base pairs or nucleotides in length.
• “methylation” or “methylated,” as used in reference to the methylation status of a cytosine, e.g., in a CpG locus, 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 a 5-methyl cytosine).
• Methylation may be determined directly, e.g., as evidenced by routine methods for analysis of methylation status of cytosines, e.g., by determining the sensitivity (or lack thereof) of a particular C-residue to conversion to uracil by treatment with bisulfite. For example, a cytosine residue in a sample that is not converted to uracil when the sample is treated with bisulfite in a manner that would be expected to convert that residue if non-methylated (e.g., under conditions in which a majority or all of the non-methylated cytosines in the sample are converted to uracils) may generally be deemed “methylated”.
• methyl cytosine As used herein, the terms “methyl cytosine,” “methyl C,” “methylated cytosine,” “methylated C,” and “meC” are used interchangeably and encompass both 5-methylcytosine (5mC) and 5-hydroxymethyl cytosine (5hmC).
• bisulfite reagent refers to a reagent comprising bisulfite, disulfite, hydrogen sulfite, or combinations thereof, useful as disclosed herein to distinguish between methylated and unmethylated CpG dinucleotide sequences.
• Methods of said treatment are known in the art (e.g., PCT/EP2004/011715 and WO 2013/116375, each of which is incorporated by reference in its entirety).
• bisulfite treatment is conducted in the presence of denaturing solvents such as but not limited to n-alkyleneglycol or diethylene glycol dimethyl ether (DME), or in the presence of dioxane or dioxane derivatives.
• the denaturing solvents are used in concentrations between 1% and 35% (v/v).
• the bisulfite reaction is carried out in the presence of scavengers such as but not limited to chromane derivatives, e.g., 6-hydroxy-2, 5,7,8, - tetramethylchromane 2-carboxylic acid or trihydroxybenzone acid and derivatives thereof, e.g., Gallic acid (see: PCT/EP2004/011715, which is incorporated by reference in its entirety).
• the bisulfite reaction comprises treatment with ammonium hydrogen sulfite, also referred to as ammonium bisulfite, e.g., as described in WO 2013/116375.
• sulfonated DNA refers to a DNA comprising cytosines or uracils that have been sulfonated as a result of treatment with a sulfonation reagent, e.g. a bisulfite reagent.
• Sulfonated DNA may be partially sulfonated, such that the DNA comprises unmethylated cytosine(s) that have not been sulfonated to form uracil sulfonate, or the DNA may be completely sulfonated, such that every unmethylated (or otherwise unprotected) cytosine has been sulfonated to form uracil sulfonate.
• Sulfonated DNA may also be DNA synthesized, e.g., using standard nucleic acids synthesis chemistries, to mimic DNA that has been treated with a bisulfite reagent.
• a strand of DNA may be synthesized from nucleotide monomers to form a strand that comprises one or more uracil sulfonate nucleotides,
• unsulfonated DNA refers to DNA that has not been treated with a sulfonation reagent, e.g., a bisulfite reagent, under conditions in which cytosines are converted to uracil sulfonate nucleotides.
• Unsulfonated DNA may comprise one or more unmethylated cytosine nucleotides.
• unsulfonated DNA is free of uracil sulfonate nucleotides.
• a low pH refers to a binding solution or condition that has a pH at or below the pH(I) of a solid support to which DNA, e.g., sulfonated DNA, is to be bound.
• a low pH binding condition is provided by use of a binding solution comprising a binding agent, e.g., a chaotropic salt such as guanidine hydrochloride, in combination with an acidic component, e.g., a buffer having an acidic pH.
• an acidic component is premixed with a binding agent before the binding mixture is combined with sulfonated DNA, while in some embodiments, an acidic component is combined with sulfonated DNA before or after the binding agent, is combined with the sulfonated DNA.
• non-adjusted pH As used herein in reference to solutions or conditions for binding sulfonated DNA to a solid support, the terms “non-adjusted pH,” “standard pH,” and “high pH” are used interchangeably, and refer to binding reaction solutions or conditions that do not include an acidic component to reduce the pH of the binding solution or condition relative to the pH of binding solutions comprising only the sulfonated DNA and the binding agent, e.g., GuHCl.
• non-adjusted pH binding solutions have in a pH at or above the pH(I) of the solid support, especially a silica support.
• a non-adjusted pH binding solution is a mixture comprising some or all of the sulfonation reagent used to sulfonate the sample of DNA, the sulfonated DNA, and the binding agent, e.g., GuHCl.
• the pH of the non-adjusted binding solution is at or near the pH of the sulfonation reagent alone.
• a sample suspected of containing a human gene or chromosome or sequences associated with a human chromosome may comprise a cell, chromosomes isolated from a cell (e.g, a spread of metaphase chromosomes), genomic DNA (in solution or bound to a solid support such as for Southern blot analysis), RNA (in solution or bound to a solid support such as for Northern blot analysis), cDNA (in solution or bound to a solid support), cell-free DNA (e.g, circulating cell free DNA from plasma; fragmented DNA from a bodily fluid, such as plasma, urine, stool, etc.); DNA isolated from exosomes or other microvesicles, e.g, from a body fluid, and the like.
• a cell-free DNA e.g, circulating cell free DNA from plasma; fragmented DNA from a bodily fluid, such as plasma, urine, stool, etc.
• a sample may include a specimen of synthetic origin. Samples may be unpurified or may be partially or completely purified or otherwise processed. A sample “suspected of containing” a nucleic acid may contain or not contain the target nucleic acid molecule.
• the present technology is not limited by the type of biological sample used or analyzed.
• the present technology is useful with a variety of biological samples including, but not limited to, tissue (e.g, organ (e.g, heart, liver, brain, lung, stomach, intestine, spleen, kidney, pancreas, and reproductive organs), glandular, skin, and muscle), cell (e.g, blood cell (e.g, lymphocyte or erythrocyte), muscle cell, tumor cell, and skin cell), gas, bodily fluid (e.g, blood or portion thereof, serum, plasma, urine, semen, saliva, etc.), or solid (e.g., stool) samples obtained from a human (e.g., adult, infant, or embryo) or animal (e.g, cattle, poultry, mouse, rat, dog, pig, cat, horse, and the like).
• tissue e.g, organ (e.g, heart, liver, brain, lung, stomach, intestine, spleen, kidney, pancreas, and reproductive organs), gland
• biological samples may be solid food and/or feed products and/or ingredients such as dairy items, vegetables, meat and meat by-products, and waste.
• Biological samples may be obtained from all of the various families of domestic animals, as well as feral or wild animals, including, but not limited to, such animals as ungulates, bear, fish, lagomorphs, rodents, pinnipeds, etc.
• Bio samples also include biopsies and tissue sections (e.g., biopsy or section of tumor, growth, rash, infection, or paraffin-embedded sections), medical or hospital samples (e.g, including, but not limited to, blood samples, saliva, buccal swab, cerebrospinal fluid, pleural fluid, milk, colostrum, lymph, sputum, vomitus, bile, semen, oocytes, cervical cells, amniotic fluid, urine, stool, hair, and sweat), laboratory samples (e.g, subcellular fractions), and forensic samples (e.g., blood or tissue (e.g., spatter or residue), hair and skin cells containing nucleic acids), and archeological samples (e.g., fossilized organisms, tissue, or cells).
• medical or hospital samples e.g, including, but not limited to, blood samples, saliva, buccal swab, cerebrospinal fluid, pleural fluid, milk, colostrum, lymph, sputum
• Environmental samples include, but are not limited to, environmental material such as surface matter, soil, water (e.g, freshwater or seawater), algae, lichens, geological samples, air containing materials containing nucleic acids, crystals, and industrial samples, as well as samples obtained from food and dairy processing instruments, apparatus, equipment, utensils, disposable and non-disposable items.
• environmental material such as surface matter, soil, water (e.g, freshwater or seawater), algae, lichens, geological samples, air containing materials containing nucleic acids, crystals, and industrial samples, as well as samples obtained from food and dairy processing instruments, apparatus, equipment, utensils, disposable and non-disposable items.
• Samples may be prepared by any desired or suitable method.
• nucleic acids are analyzed directly from bodily fluids, stool, or other samples using the methods and systems described in U.S. Pat. Nos. 9,000,146; and 10,047,390, each of which is herein incorporated by reference in its entirety for all purposes.
• sample e.g., suspected of comprising a target sequence, gene or template (e.g, the presence or absence of which can be determined using the compositions and methods of the present technology)
• template e.g., the presence or absence of which can be determined using the compositions and methods of the present technology
• target when used in reference to a nucleic acid detection or analysis method, refers to a nucleic acid having a particular sequence of nucleotides to be detected or analyzed, e.g, in a sample suspected of containing the target nucleic acid.
• a target is a nucleic acid having a particular sequence for which it is desirable to determine a methylation status.
• target When used in reference to the polymerase chain reaction, “target” generally refers to the region of nucleic acid bounded by the primers used for polymerase chain reaction. Thus, the “target” is sought to be sorted out from other nucleic acid sequences that may be present in a sample.
• a “segment” is defined as a region of nucleic acid within the target sequence.
• sample template refers to nucleic acid originating from a sample that is analyzed for the presence of a target.
• nucleic acid molecule refers to any nucleic acid containing molecule, including but not limited to, DNA or RNA.
• the term encompasses sequences that include any of the known base analogs of DNA and RNA including, but not limited to, 4 acetylcytosine, 8-hydroxy-N6-methyladenosine, aziridinylcytosine, pseudoisocytosine, 5- (carboxyhydroxyl-methyl) uracil, 5-fluorouracil, 5-bromouracil, 5- carboxymethylaminomethyl-2-thiouracil, 5-carboxymethyl-aminomethyluracil, dihydrouracil, inosine, N6-isopentenyladenine, 1 -methyladenine, 1 -methylpseudo-uracil, 1- methylguanine, 1 -methylinosine, 2,2-dimethyl-guanine, 2-methyladenine, 2-methylguanine, 3-methyl-cytosine, 5-methylcytosine, 5- hydroxymethylcytosine, 5-carboxylcytosine, 5- formylcytosine, N6-methyladenine, 7
• a nucleic acid sequence or molecule may be DNA or RNA, of either genomic or synthetic origin, that may be single or double stranded, and represent the sense or antisense strand.
• nucleic acid sequence may be dsDNA, ssDNA, mixed ssDNA, mixed dsDNA, dsDNA made into ssDNA (e.g., through melting, denaturing, helicases, etc.), A-, B-, or Z- DNA, triple-stranded DNA, RNA, ssRNA, dsRNA, mixed ss and dsRNA, dsRNA made into ssRNA e.g., via melting, denaturing, helicases, etc.), messenger RNA (mRNA), ribosomal RNA (rRNA), transfer RNA (tRNA), catalytic RNA, snRNA, microRNA, or protein nucleic acid (PNA).
• mRNA messenger RNA
• rRNA ribosomal RNA
• tRNA transfer
• nucleic acid e.g., sequence or molecule (e.g., target sequence and/or oligonucleotide)
• the nucleic acid sequence may be amplified or created sequence (e.g., amplification or creation of nucleic acid sequence via synthesis (e.g., polymerization (e.g., primer extension (e.g., RNA-DNA hybrid primer technology)) and reverse transcription (e.g., 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).
• PCR polymerase chain reaction
• RCA rolling circle amplification
• NASBA nucleic acid sequence
• process control refers to an exogenous molecule, e.g., an exogenous nucleic acid added to a sample prior to extraction of target DNA that can be measured post-extraction to assess the efficiency of the process and be able to determine success or failure modes.
• the nature of the process control nucleic acid used is usually dependent on the assay type and the material that is being measured. For example, if the assay being used is for detection and/or quantification of double stranded DNA or mutations in it, then double stranded DNA process controls are typically spiked into the samples before extraction. Similarly, for assays that monitor mRNA or microRNAs, the process controls used are typically either RNA transcripts or synthetic RNA. Process controls typically aid in assessment of the efficiency of a process and in determination of success or failure of processes and process steps.
• exogenous refers to nontarget DNA that is isolated and purified from a source other than the source or sample containing the target DNA.
• purified fish DNA is exogenous DNA with respect to a sample comprising human target DNA, e.g., as described in U.S. Patent No.9,212,392, which is incorporated herein by reference.
• Exogenous DN A need not be from a different organism than the target DNA.
• purified fish DNA obtained commercially would be exogenous if added to a reaction configured to detect a target nucleic acid in a sample from a particular fish.
• exogenous DNA is selected to be undetected by an assay configured to detect and/or quantify the target nucleic acid in the reaction into which the exogenous DNA is added.
• fish DNA refers to bulk e.g., genomic DNA isolated from fish, e.g., as described in U.S. Patent No.9,212,392.
• Bulk purified fish DNA is commercially available, e.g., provided in the form of cod and/or herring sperm DNA (Roche Applied Science, Mannheim, Germany) or salmon DNA (USB/Affymetrix).
• “Fish DNA” is distinct from any particular gene from a fish that is in isolated form, e.g., that has been separately synthesized or that has been separated from the other DNA of the fish genome.
• the term “zebrafish DNA” refers to DNA isolated from Danio rerio, or created in vitro (e.g., enzymatically, synthetically) to have a sequence of nucleotides found in DNA from Danio rerio.
• the zebrafish DNA is a methylated DNA added as a detectable control DNA, e.g., a process control for verifying DNA recovery through sample processing steps.
• zebrafish DNA comprising at least a portion of the RASSF1 gene finds use as a process control, e.g., for human samples, as described in WO 2018/017710A 1 , which is incorporated herein by reference, and which describes use of zebrafish DNA as a process control for human samples.
• ZFRASSF1 refers to a process control comprising at least a portion of the zebrafish RASSF1 gene.
• the terms “recovery” and “recovered” as used in reference to strands of nucleic acid refers to the amount or number of strands of DNA measured in 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).
• the strands recovered are compared to a reference value, e.g., an amount or number of strands added or expected to be present in a sample prior to processing, or an amount measured in or expected to be present in a reference sample.
• kits refers to any delivery system for delivering materials.
• delivery systems include systems that allow for the storage, transport, or delivery of reagents and devices (e.g., chaotropic salts, particles, buffers, denaturants, oligonucleotides, filters etc. in the appropriate containers) and/or supporting materials (e.g., sample processing or sample storage vessels, written instructions for performing a procedure, etc.) from one location to another.
• reagents and devices e.g., chaotropic salts, particles, buffers, denaturants, oligonucleotides, filters etc. in the appropriate containers
• supporting materials e.g., sample processing or sample storage vessels, written instructions for performing a procedure, etc.
• enclosures e.g., boxes
• fragmented kit refers to a delivery system comprising two or more separate containers that each contains a subportion of the total kit components.
• the containers may be delivered to the intended recipient together or separately.
• a first container may contain materials for sample collection and a buffer, while a second container contains capture oligonucleotides and denaturant.
• fragmented kit is intended to encompass kits containing Analyte specific reagents (ASR's) regulated under section 520(e) of the Federal Food, Drug, and Cosmetic Act, but are not limited thereto.
• ASR's Analyte specific reagents
• any delivery system comprising two or more separate containers that each contains a subportion of the total kit components are included in the term “fragmented kit.”
• a “combined kit” refers to a delivery system containing all of the components of a reaction assay in a single container (e.g., in a single box housing each of the desired components).
• kit includes both fragmented and combined kits.
• system refers to a collection of articles for use for a particular purpose.
• the articles comprise instructions for use, as information supplied on e.g., an article, on paper, online (e.g, at a website or web address) on recordable media (e.g., diskette, CD, DVD, flash drive, etc.).
• instructions direct a user to an online location, e.g., a website for viewing, hearing, and/or downloading instructions.
• instructions or other information are provided as an application (“app”), e.g., for a computer or for a mobile device, such as a smart phone.
• the term “information” refers to any collection of facts or data. In reference to information stored or processed using a computer system(s), including but not limited to internets, the term refers to any data stored in any format (e.g., analog, digital, optical, etc.).
• the term “information related to a subject” refers to facts or data pertaining to a subject (e.g., a human, plant, or animal).
• the term “genomic information” refers to information pertaining to a genome including, but not limited to, nucleic acid sequences, genes, allele frequencies, RNA expression levels, protein expression, phenotypes correlating to genotypes, etc.
• Allele frequency information refers to facts or data pertaining to allele frequencies, including, but not limited to, allele identities, statistical correlations between the presence of an allele and a characteristic of a subject (e.g., a human subject), the presence or absence of an allele in an individual or population, the percentage likelihood of an allele being present in an individual having one or more particular characteristics, etc.
• the methods provide conditions that promote a highly stable binding of the sulfonated DNA to the beads. This facilitates the efficient recovery of bisulfite-treated DNA despite the highly basic reaction conditions of desulfonation that one of skill in the art would expect to disrupt the interaction of the DNA with the beads.
• the technology provides methods for preparing bisulfite-converted DNA with improved recovery of the input DNA.
• the technology is related to the experimental findings described below and developed in the experimental examples. These examples describe the development and testing of reagents used for the analysis of the methylation state of a nucleic acid.
• the technology is related to binding solutions that have 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 binding of the sulfonated DNA to silica.
• Common sulfonation reagents include sodium bisulfite and ammonium bisulfite.
• ammonium bisulfite is used but the principles of the technology are readily applicable to any DNA treated in a manner that creates a charge, e.g., a highly negative charge, across the molecule.
• DNA treated with other sulfonation reagents, e.g., sodium bisulfite find use with the present technology.
• the pH of a sulfonation reaction is largely determined by the ammonium bisulfite, which typically has a pH that is above the pH(I) of silica (as shown schematically in Fig. 2), and at or above the pH(I) of sulfonated DNA.
• the pH of a binding reaction is above the pH(I) of the silica (or other support material) and/or the DNA, electrostatic repulsion may prevent complete binding of the DNA to the silica.
• conducting the binding reaction at an acidic pH e.g., by addition of acid to the sulfonated DNA-silica bead mixture, may neutralize the silica and protonate the uracil sulfonate groups of the DNA, thereby reducing electrostatic repulsion and promoting silica-sulfonated DNA interaction.
• DNA suitable for use with the technology is not limited to DNA isolated by any particular method, and suitable DNA may be prepared in a number of ways, including by the use of commercial kits, columns, and the like. Exemplary methods of isolating DNA from samples, e.g., from a subject, are described below.
• DNA may be isolated from a stool sample.
• An exemplary embodiment of isolating DNA from stool that is suitable for use with the present technology is found in U.S. Patent No. 9,000,146, which is incorporated herein by reference. Briefly, the stool sample is homogenized with a buffer, the solids are removed, e.g., by centrifugation, and specific target DNAs are captured from the resulting clarified supernatant using particles or beads comprising oligonucleotides complementary to the target DNA(s).
• Target DNAs bound to the capture oligonucleotides are separated from the solution, e.g., by use of magnetic field to collect magnetic beads, and the captured DNAs are optionally washed with buffer prior to elution from the capture oligonucleotides, e.g., using denaturing conditions.
• cell-free genomic DNA may be isolated from cell-conditioned media using, for example, the “Maxwell® RSC ccfDNA Plasma Kit (Promega Corp., Madison, WI). Following the kit protocol, 1 mL of cell conditioned media (CCM) is used in place of plasma, and processed according to the kit procedure. The elution volume is 100 pL, of which 70 pL are generally used for bisulfite conversion.
• CCM cell conditioned media
• An exemplary procedure for isolating DNA from a 4 mL sample of plasma, e.g., from a human blood sample, is as follows:
• Plasma lysis buffer is:
• IGEPAL CA-630 Olethylphenoxy poly(ethyleneoxy)ethanol, branched
• sulfonated DNA samples may be alcohol precipitated or isolated, e.g., by gel filtration.
• Sulfonated DNA may also be bound to a support such as silica, e.g., silica-coated surfaces of reaction vessels, silica particles or fibers, paramagnetic silica beads, etc., by addition of chaotropic salt, e.g., a guanidine salt such as guanidine hydrochloride (GuHCl) or guanidine isothiocyanate (GITC), at about, e.g., 6 to 7 M concentration.
• chaotropic salt e.g., a guanidine salt such as guanidine hydrochloride (GuHCl) or guanidine isothiocyanate (GITC)
• a guanidine salt such as guanidine hydrochloride (GuHCl) or guanidine isothiocyanate (GITC)
• a guanidine salt such as guanidine hydrochloride (GuHCl) or guanidine isothiocyanate (GITC)
• a typical method of bisulfite treatment of DNA (without a low pH binding step of the present technology) is as follows:
• each tube combine 64 pL DNA, 7 pL 1 N NaOH, and 9 pL of carrier solution containing 0.2 mg/mL BSA and 0.25 mg/mL of fish DNA.
• Magnetic beads Promega MagneSil Paramagnetic Particles, Promega catalogue number AS 1050, 16 pg/pL).
• Binding buffer 6.5-7 M guanidine hydrochoride.
• Post-conversion Wash buffer 80% ethanol with 10 mM Tris HC1 (pH 8.0).
• Desulfonation buffer 70% isopropyl alcohol, 0.1 N NaOH was selected for the desulfonation buffer.
• Samples are mixed using any appropriate device or technology to mix or incubate samples at the temperatures and mixing speeds essentially as described below.
• a Thermomixer Eppendorf
• An exemplary desulfonation is as follows:
• the converted DNA is then used in a detection assay, e.g., a pre-am pl ifi cation and/or flap endonuclease assays, as described below.
• a detection assay e.g., a pre-am pl ifi cation and/or flap endonuclease assays, as described below.
• magnetic beads are used for the treatment and isolation of DNA, e.g., beads comprising a magnetic core and a silica coating.
• the silica coating binds DNA and the magnetic core provides an efficient way to concentrate and isolate the beads (and bound DNA) using a magnet.
• the silica-coated magnetic beads are MagneSil Paramagnetic Particles (Promega, Madison, WI; catalogue number AS 1220 or AS640A, promega.com).
• the technology is not limited to any particular type of magnetic bead.
• Embodiments of the technology described herein make use of any magnetic beads (e.g., paramagnetic beads) that have an affinity for nucleic acids.
• the magnetic beads have a magnetite (e.g., FesC ) core and a coating comprising silicon dioxide (SiCh).
• the bead structure e.g., size, porosity, shape
• composition of the solution in which a nucleic acid is bound to the bead can be altered to bind different types (e.g., DNA or RNA in single stranded, double stranded, or other forms or conformations; nucleic acids derived from a natural source, synthesized chemically, synthesized enzymatically (e.g., by PCR)) and sizes of nucleic acids (e.g., small oligomers, primers, genomic, plasmids, fragments, e.g., consisting of 200 or fewer bases) selectively.
• nucleic acids e.g., small oligomers, primers, genomic, plasmids, fragments, e.g., consisting of 200 or fewer bases
• the technology is not limited to a particular size of magnetic bead. Accordingly, embodiments of the technology use magnetic beads of a number of different sizes. Smaller beads provide more surface area (per weight unit basis) for adsorption, but smaller beads are limited in the amount of magnetic material that can be incorporated in the bead core relative to a larger bead.
• the particles are distributed over a range of sizes with a defined average or median size appropriate for the technology for which the beads are used. In some embodiments, the particles are of a relatively narrow monomodal particle size distribution.
• the beads that find use in the present technology have pores that are accessible from the exterior of the particle. Such pores have a controlled size range that is sufficiently large to admit a nucleic acid, e.g., a DNA fragment, into the interior of the particle and to bind to the interior surface of the pores.
• the pores are designed to provide a large surface area that is capable of binding a nucleic acid.
• the technology is not limited to any particular method of nucleic acid ⁇ e.g., DNA) binding and/or isolation.
• aspects of the technology relating to the bisulfite reaction are combined with other suitable methods of DNA isolation ⁇ e.g., precipitation, column chromatography ⁇ e.g., a spin column), etc.
• the beads (and bound material) are removed from a mixture using a magnetic field.
• other forms of external force in addition to a magnetic field are used to isolate the biological target substance according to the present technology.
• suitable additional forms of external force include, but are not limited to, gravity filtration, vacuum filtration, and centrifugation.
• Embodiments of the technology apply an external magnetic field to remove the complex from the medium.
• a magnetic field can be suitably generated in the medium using any one of a number of different known means. For example, one can position a magnet on the outer surface of a container of a solution containing the beads, causing the particles to migrate through the solution and collect on the inner surface of the container adjacent to the magnet. The magnet can then be held in position on the outer surface of the container such that the particles are held in the container by the magnetic field generated by the magnet, while the solution is decanted out of the container and discarded. A second solution can then be added to the container, and the magnet removed so that the particles migrate into the second solution.
• a magnetizable probe could be inserted into the solution and the probe magnetized, such that the particles deposit on the end of the probe immersed in the solution. The probe could then be removed from the solution, while remaining magnetized, immersed into a second solution, and the magnetic field discontinued permitting the particles go into the second solution.
• magnets designed to be used in both types of magnetic removal and transfer techniques described in general terms above. See, e.g., MagneSphere Technology Magnetic Separation Stand or the PolyATract Series 9600TM Multi-Magnet, both available from Promega Corporation; Magnetight Separation Stand (Novagen, Madison, Wis.); or Dynal Magnetic Particle Concentrator (Dynal, Oslo, Norway).
• Some embodiments comprise use of a magnetic device according to U.S. Pat. AppL Ser. No. 13/089116, which is incorporated herein by reference in its entirety for all purposes. Furthermore, some embodiments contemplate the use of a “jet channel” or pipet tip magnet separation (e.g., as described in U.S. Pat. Nos. 5,647,994 and 5,702,950). Some embodiments contemplate the use of an immersed probe approach (e.gively as described in U.S. Pat. Nos. 6,447,729 and 6,448,092), e.g., as exemplified by the KingFisher systems commercially available from Thermo Scientific.
• strand recovery was measured using flap endonuclease assays.
• Exemplary methods for extracting sample nucleic acids, e.g., from blood, and for quantitating strands of bisulfite-converted DNA and unconverted DNA are described, for example, in US Pat. No. 10,648,025, which is incorporated herein by reference for all purposes. QuARTS assay
• An exemplary method of quantifying DNA strands produced according to the technology is the QuARTS flap assay technology.
• the QuARTS technology combines a polymerase-based target DNA amplification process with an invasive cleavage-based signal amplification process.
• the technology is described, e.g., in U.S. Pat. Nos. 8,361,720; 8,715,937; 8,916,344; and 9,212,392, and U.S. Pat. AppL No. 15/841,006, each of which is incorporated herein by reference.
• Fluorescence signal generated by the QuARTS reaction is monitored in a fashion similar to real-time PCR and permits quantitation of the amount of a target nucleic acid in a sample.
• An exemplary QuARTS reaction typically comprises approximately 400-600 nmol/L (e.g., 500 nmol/L) of each primer and detection probe, approximately 100 nmol/L of the invasive oligonucleotide, approximately 600-700 nmol/L of each FRET cassette (FAM, e.g., as supplied commercially by Hologic, Inc.; HEX, e.g., as supplied commercially by BioSearch Technologies; and Quasar 670, e.g., as supplied commercially by BioSearch Technologies), 6.675 ng/pL FEN-1 endonuclease (e.g., Cleavase® 2.0, Hologic, Inc.), 1 unit Taq DNA polymerase in a 30 .L reaction volume (e.g., GoTaq® DNA polymerase, Promega Corp., Madison ,WI), 10 mmol/L 3-(n-morpholino) propanesulfonic acid (MOPS), 7.5 mmol/L MgCh, and 250
• a large volume of the treated DNA may be used in a single, large-volume multiplex amplification reaction.
• DNA is extracted from a cell line (e.g., DFCI032 cell line (adenocarcinoma); H1755 cell line (neuroendocrine)), using, for example, the Maxwell Promega blood kit # AS 1400, as described above.
• the DNA is bisulfite converted, e.g., as described above.
• a pre-amplification is conducted, for example, in a reaction mixture containing 7.5 mM MgCh, 10 mM MOPS, 0.3 mM Tris-HCl, pH 8.0, 0.8 mM KC1, 0.1 pg/pL BSA, 0.0001% Tween-20, 0.
• oligonucleotide primers e.g., for 12 targets, 12 primer pairs/24 primers, in equimolar amounts (including but not limited to the ranges of, e.g, 200-500 nM each primer), or with individual primer concentrations adjusted to balance amplification efficiencies of the different target regions), 0.025 units/pL HotStart GoTaq concentration, and 20 to 50% by volume of bisulfite-treated target DNA (e.g., 10 pL of target DNA into a 50 pL reaction mixture, or 50 pL of target DNA into a 125 pL reaction mixture).
• Thermal cycling times and temperatures are selected to be appropriate for the volume of the reaction and the amplification vessel. For example, the reactions may be cycled as follows
• aliquots of the pre-amplification reaction are diluted to 500 pL in 10 mM Tris, 0.1 mM EDTA, with or without fish DNA. Aliquots of the diluted pre-amplified DNA (e.g, 10 pL) are used in a QuARTS PCR-flap assay, e.g., as described above. See also U.S. Patent Appl. Ser. No. 62/249,097, filed October 30, 2015; Appl. Ser No. 15/335,096, filed October 26, 2016, and PCT/US16/58875, filed October 26, 2016, and U.S. Patent No. US 10,648,025, each of which is incorporated herein by reference in its entirety for all purposes.
• the present technology is not limited to use of flap endonuclease assays, and in different embodiments, any method of analyzing the bisulfite-converted DNA may be used.
• the analysis comprises direct sequencing, pyrosequencing, methylation-sensitive single-strand conformation analysis (MS-SSCA), high resolution melting analysis, methylation-sensitive single-nucleotide primer extension (MS- SnuPE), base-specific cleavage/mass spectrometry (e.g, by MALDI-TOF), methylationspecific PCR (MSP), microarray analysis, restriction digest analysis), INVADER assay, combined bisulfite restriction analysis, or methylated DNA immunoprecipitation (MeDIP).
• single stranded DNA template was sulfonated with ammonium bisulfite (ABS) as described in the exemplary Sulfonation steps, above.
• ABS ammonium bisulfite
• 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+), middle (228 mM K+) and high (474 mM K+) concentrations.
• KOAc potassium acetate
• a portion of the sulfonated DNA template was returned to a bisulfite solution instead of potassium acetate.
• Each sample was subsequently incubated with chaotrope and silica beads to promote binding.
• Table 1 below demonstrates that bisulfite solution inhibited the binding reaction due to high salt concentration for each template. Binding pH was 4.8 to 4.9.
• Results are shown in Fig. 3.
• the pH required for maximum log strand recovery varied for each marker and appears to correlate with percent converted cytosine (z.e., the percent of total nucleotides in a strand that are unmethylated cytosine nucleotides prior to treatment, and that are uracil sulfonate nucleotides at the binding step) of the sequence.
• a total of seventeen different DNA sequences were tested in the same manner above.
• the percent converted cytosines of each DNA was plotted against the highest estimated binding pH required for maximum strand recovery. A linear fit of these points (Fig. 4) had an R 2 of 0.76, suggesting the relationship between percent converted cytosines and binding pH was strong.
• Strand recovery from binding reactions conducted at low pH (citrate buffer) and at non-adjusted pH (standard conditions) were compared for multiple sample types: singlestranded 126-mer strands of synthetic DNA (“126”), DNA isolated from stool samples spiked with purified cell line DNA (“ECLD”),and DNA isolated from stool samples previously characterized as giving high signal for methylated DNA (“HMS”), with 6 replicates for each combination of sample type and binding condition, with Table 3, below, showing strand recovery results. All DNA from stool samples was isolated with the capture process described above, and the synthetic 126-mer DNA strands were combined with exogenous fish DNA as carrier.
• 126 singlestranded 126-mer strands of synthetic DNA
• ECLD purified cell line DNA
• HMS high signal for methylated DNA
• nucleosomal DNA was prepared from HCT116 cells using the Active Motif Nucleosome Kit (Active Motif, catalog number 53504.) To mimic methylated DNA marker (MDM)-positive plasma samples, nucleosomal DNA was spiked into pooled plasma collected from healthy donors using LBgard® blood tubes (Exact Sciences, Inc.) or was spiked into SERACON negative diluent. Use of the nucleosome matrix preparation ensures that the MDM-positive DNA has a similar size distribution as that which would be expected in a sample from a cancer-positive patient.
• MDM methylated DNA marker
• the samples were treated to extract DNA using the plasma extraction procedure described above, or using QIASYMPHONY DSP Circulating DNA kit (Qiagen, Inc.).
• the extracted DNA samples were then bisulfite-treated and bound at standard (non-adjusted pH) binding reactions as described above (7 M GuHCl binding buffer added to the ABS reaction), and in low pH binding reactions (7 M GuHCl with 133 mM citrate buffer, pH 2.2 binding buffer).
• Fig. 6A Cell line DNA-spiked stool samples with sample input volumes ranging from 4 to 14 mLs (standard) were treated to capture the cell line DNA, which was then bisulfite-treated and isolated using standard pH (Fig. 6A)and low binding pH binding (Fig. 6B) solutions. Comparison of Figs. 6A and 6B shows improved linearity of total DNA and strand recovery with low binding pH. The samples used for non-adjusted (standard) and low binding pH had differing amounts of DNA templates spiked in thus, total strand recoveries shown should not be directly compared across binding conditions.
• Ammonium bisulfite was titrated from 57 to 43% concentration, then tested with synthetic DNA templates bound with low binding pH (citric acid buffer) or with no pH adjustment. Using the 56.6% as the reference value, the % difference in binding for the lower ABS concentrations were calculated for each of the indicated markers DNA. Results are shown in the table in Fig. 8B. Strand recovery alignment across ABS concentrations improved with low binding pH.
• Silica beads from multiple vendors were tested for use in bisulfite conversion with non-adjusted pH at binding or with low binding pH, with results are shown in Figs. 10 and 11, respectively. Some beads showed no strand recovery at unadjusted binding pH (Fig. 10), but showed improved recovery with lowered binding pH (Fig. 1 1). Bead concentrations were not adjusted to be equal for initial screenings in Fig. 10, but were aligned for the testing shown in Fig. 11.
• Acidic conditions can cause loss in strand recovery due to acid hydrolysis of DNA template.
• strand recovery was most consistent from about pH 3.9 to around 2.5 (see Fig. 12).
• DNA size and concentration can vary across and within sample types such as tissue, blood, and stool.
• calf thymus DNA was titrated to 300, 2700, and 5400 ng/ran at sizes of 200 bp, 3.6 reaction, and 20 kbp.
• DNA samples were spiked with 126 bp synthetic target molecules, and were treated with bisulfite using with non-adjusted binding pH and low binding pH for capture of the sulfonated DNA. Results are shown in Fig. 13.
• Protocol 1 is a standard protocol in which the sulfonation reaction is combined with a binding solution of 7M guanidine hydrochloride (GuHCl), but no acid is added to reduce the pH.
• GuHCl guanidine hydrochloride
• Protocol 2 the sulfonation reaction mixture is combined with a binding solution of 7M GuHCl, 133 mM Citrate, pH 2.2.
• Protocol 3 the sulfonation reaction mixture is combined with a binding solution of 7M GuHCl, but no acid is added to reduce the pH.
• the bead-bound sulfonated DNA is desulfonated on the solid support, as described, e.g., in U.S. Patent No. 9,315,853, which is incorporated herein by reference in its entirety.
• Protocol 1 Conversion protocols are described below for performance on Hamilton STARlet automatic pipetting system, but can be performed manually with equivalent equipment. Protocol 1
• This procedure describes processing of DNA captured from a sample, e.g., a stool sample, using sequence-specific capture beads that comprise a capture oligonucleotide complementary to the target DNA.
• ABS ammonium bisulfite
• This procedure describes processing of DNA captured from a sample, e.g., a stool sample, using sequence-specific capture beads that comprise a capture oligonucleotide complementary to the target DNA.
• This procedure describes processing of DNA extracted. from a sample, e.g., from plasma, using chaotrope-mediated binding to a silica support.
• the DNA is eluted from the support prior to the denaturation step below.
• genomic DNA was extracted from cell line OE33 cells (esophageal carcinoma) using the MAXWELL system (Promega Corp., Fitchburg, WI), and RSC Blood DNA Kit (Promega Corp., Cat # ASB1400), both according to manufacturer’s instructions.
• the prepared DNA was used in all 3 protocols, and was added directly to the Denaturation step of each protocol, with no intervening DNA capture step.
• Strand recoveries for each of Protocols 1, 2, and 3 were tested on DNA at concentrations of 26.7, 17.8, 11.87, 7.91, 5.27, 3.52 ng/pL. Four replicates of each condition were tested.
• the sequences tested in the prepared DNAs were methylation marker genes ZNF568, BMP 3, B3GALT6, NDRG4, VAV3, and ZNF682. Detailed data is shown in Figs. 15A and 15B.
• Fig. 16 shows a table with pairwise comparisons of strand recoveries from Protocol 2 (using low pH binding), compared to Protocol 1 and to Protocol 3 (both with no pH adjustment for binding.), based on the data shown in Figs. 15A-15B for the different methylation marker DNAs.
• Fig. 17 shows a graph showing the percentage change using Protocol 2 vs. Protocol 1 or Protocol 3 (collectively grouped as “Protocol X”) at the indicated DNA concentrations.
• the dashed line indicates 100%, i.e., equal recovery using Protocol 2 and Protocol X.
• Increased strand recovery levels were seen with the Protocol 2 method (which used low pH binding conditions) compared to both of the other two protocols (which both used standard, unadjusted pH binding conditions) with the exception of theVAV3 marker DNA at lower DNA concentrations tested, for which Protocol 1 gave higher strand recovery.
• Protocol 2 was used as described above, with the Sulfonated DNA Binding step modified to use glycine, citrate, malate, or formate buffer in the GuHCl binding solution.
• An unbuffered control reaction used GuHCl with no added buffer at the binding step. The results are shown in Fig. 18.

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