WO2015020969A2 - Analysis of dna methylation in ultra-small clinical samples - Google Patents

Abstract

A method for determining a DNA methylation status is provided by this invention.

Description

Analysis of DNA Methylation in Ultra-Small Clinical Samples

GOVERNMENT INTEREST

[0001] Work described herein was supported by National Institutes of Health Grant RR024420. The United States Government has certain rights in the invention.

REFERENCE TO RELATED APPLICATIONS

[0002] The present application claims priority to U.S. Provisional Patent Application No. 61/862,884, filed on August 6, 2013 and U.S. Provisional Patent Application No. 61/876,050, filed on September 10, 2013, the entirety of which is hereby incorporated by reference.

BACKGROUND OF THE INVENTION

Field of the Invention

[0003] The invention disclosed herein relates generally to the fields of clinical testing in oncology, inflammatory, benign diseases, psychiatry, psychology, and other areas where epigenetic features play a role. Particularly, the invention provides methods for analysis of DNA methylation in ultra-small clinical samples.

Description of Related Art

[0004] Enzymes and, in particular, restriction enzymes have been invaluable assistants in the laboratory for decades, so it is not surprising that they have been instrumental in driving discoveries in epigenetics-related research as well. DNA methylation is one of the well-characterized DNA modifications in vertebrates. Because DNA methylation does not alter the sequence of DNA and is lost during PCR, studying DNA methylation in the genome, such as the presence, location and quantification of 5- methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) in different genomic DNA samples, has become the focus of assay and product development efforts (see Figure 1).

[0005] Approximately 60-70% of all human gene promoters overlap with CpG islands — regions with an elevated GC content and a high frequency of CpG dinucleotides. Gene silencing by means of DNA methylation of specific gene promoters is a well-known feature of neoplastic cells and plays an important role in normal cell differentiation and development. DNA methylation occurs mainly at CpG dinucleotides and involves the enzymatic addition of a methyl group to the cytosine residue, without changing the primary DNA sequences.

[0006] Such modifications at regulatory regions (in particular gene promoters), correlate well with the transcriptional state of a gene: DNA methylation represses transcription while DNA unmethylation can lead to increased transcription levels. DNA methylation is an essential mechanism for normal cellular development, imprinting, X- chromosome inactivation, and maintaining tissue specificity. It can also contribute significantly to the progression of various human diseases.

[0007] The profiling of tumor suppressor genes and other key genes allows the correlation of CpG island methylation status with transcriptional status, biological phenotypes, or disease outcomes. Therefore, the results can provide insights into the molecular mechanisms and biological pathways and aid in the discovery and development of biomarkers. However, microgram quantities of genomic DNA sample required is one of the key limitations for existing methods for analysis of DNA methylation. While DNA amplification can be readily used to increase the amount of DNA before genomic analysis of DNA, pre-analytical amplification is impossible for analysis of DNA methylation because methylation groups of the original sample are not reproduced in the amplification products. Thus, there is a need in the art to provide reliable, effective, and efficient methods for analysis of DNA methylation in clinical setting using ultra-small (300 pg or less) of genomic DNA.

SUMMARY OF THE INVENTION

[0008] It is against the above background that the present invention provides certain advantages and advancements over the prior art.

[0009] Although this invention is not limited to specific advantages or functionality, it is noted that the invention disclosed herein provides a method for determining a DNA methylation status, comprising: (a) obtaining a DNA sample from a subject; (b) digesting the DNA sample with a methylation-sensitive restriction enzyme in the presence of a glycol compound; (c) amplifying the digested sample of step (b); (d) quantifying amplification results from step (c) using a real-time quantitative PCR; and (e) determining the DNA methylation status within a recognition site of the methylation- sensitive restriction enzyme.

[0010] In one aspect of the invention, the subject in the disclosed method is a mammal, wherein the mammal is a human.

[0011] In another aspect of the invention, the DNA in the disclosed method comprises a genomic DNA.

[0012] In another aspect of the invention, the DNA sample in the disclosed method is between about 1 pg and about 1 ng.

[0013] In another aspect of the invention, the DNA sample in the disclosed method the DNA sample is 300 pg.

[0014] In another aspect of the invention, the methylation-sensitive restriction enzyme in the disclosed method comprises Hin6l.

[0015] In another aspect of the invention, the amplifying in the disclosed method comprises amplifying using phi29 polymerase.

[0016] In another aspect of the invention, the amplifying in the disclosed method further comprises amplifying using a single stranded DNA binding (SSB) protein of E.coli.

[0017] In another aspect of the invention, the real-time quantitative PCR in the disclosed method comprises TaqMan qPCR.

[0018] In another aspect of the invention, determining the DNA methylation status in the disclosed method comprises determining threshold cycle (C_T) values.

[0019] These and other features and advantages of the present invention will be more fully understood from the following detailed description of the invention taken together with the accompanying claims. It is noted that the scope of the claims is defined by the recitations therein and not by the specific discussion of features and advantages set forth in the present description. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] The following detailed description of the embodiments of the present invention can be best understood when read in conjunction with the following drawings, where like structure is indicated with like reference numerals and in which:

[0021] Figure 1 shows analysis of DNA methylation.

[0022] Figure 2 shows an example of the confirmatory PCR for an additive X. Lane 1 : concentration 1 (no digestion); Lane 2 and 3: concentrations 2 and 3 (partial digestion); Lane 4: concentration 4 (complete digestion).

[0023] Skilled artisans will appreciate that elements in the figures are illustrated for simplicity and clarity and have not necessarily been drawn to scale. For example, the dimensions of some of the elements in the figures can be exaggerated relative to other elements to help improve understanding of the embodiment(s) of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

[0024] All publications, patents and patent applications cited herein are hereby expressly incorporated by reference for all purposes.

[0025] Methods well known to those skilled in the art can be used to construct genetic expression constructs and recombinant cells according to this invention. These methods include in vitro recombinant DNA techniques, synthetic techniques, in vivo recombination techniques, and PCR techniques. See, for example, techniques as described in Maniatis et al., 1989, MOLECULAR CLONING: A LABORATORY MANUAL, Cold Spring Harbor Laboratory, New York; Ausubel et al., 1989, CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, Greene Publishing Associates and Wiley Interscience, New York, and PCR Protocols: A Guide to Methods and Applications (Innis er al., 1990, Academic Press, San Diego, CA).

Definitions

[0026] Before describing the present invention in detail, a number of terms will be defined. As used herein, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. For example, reference to a "nucleic acid" means one or more nucleic acids.

[0027] It is noted that terms like "preferably", "commonly", and "typically" are not utilized herein to limit the scope of the claimed invention or to imply that certain features are critical, essential, or even important to the structure or function of the claimed invention. Rather, these terms are merely intended to highlight alternative or additional features that can or cannot be utilized in a particular embodiment of the present invention.

[0028] For the purposes of describing and defining the present invention it is noted that the term "substantially" is utilized herein to represent the inherent degree of uncertainty that can be attributed to any quantitative comparison, value, measurement, or other representation. The term "substantially" is also utilized herein to represent the degree by which a quantitative representation can vary from a stated reference without resulting in a change in the basic function of the subject matter at issue.

[0029] As used herein, the terms "polynucleotide", "nucleotide", "oligonucleotide", and "nucleic acid" can be used interchangeably to refer to nucleic acid comprising DNA, RNA, derivatives thereof, or combinations thereof.

[0030] As used herein, the term "nucleic acid" refers to polynucleotides such as deoxyribonucleic acid (DNA), and, where appropriate, ribonucleic acid (RNA). The term should also be understood to include, as equivalents, analogs of either RNA or DNA made from nucleotide analogs, and, as applicable to the embodiment being described, single-stranded (such as sense or antisense) and double-stranded polynucleotides.

[0031] The terms "compound", "test compound," "agent", and "molecule" are used herein interchangeably and are meant to include, but are not limited to, peptides, nucleic acids, carbohydrates, small organic molecules, natural product extract libraries, and any other molecules (including, but not limited to, chemicals, metals, and organometallic compounds).

[0032] The term "detection" is used herein to refer to any process of observing a marker, or a change in a marker (such as for example the change in the methylation state of the marker), in a biological sample, whether or not the marker or the change in the marker is actually detected. In other words, the act of probing a sample for a marker or a change in the marker, is a "detection" even if the marker is determined to be not present or below the level of sensitivity. Detection may be a quantitative, semiquantitative or non-quantitative observation.

[0033] The term "including" is used herein to mean, and is used interchangeably with, the phrase "including but not limited to."

[0034] The term "isolated" as used herein with respect to nucleic acids, such as DNA or RNA, refers to molecules in a form which does not occur in nature. Moreover, an "isolated nucleic acid" is meant to include nucleic acid fragments which are not naturally occurring as fragments and would not be found in the natural state.

[0035] The term "or" is used herein to mean, and is used interchangeably with, the term "and/or", unless context clearly indicates otherwise.

[0036] A "sample" includes any material that is obtained or prepared for detection of a molecular marker or a change in a molecular marker such as for example the methylation state, or any material that is contacted with a detection reagent or detection device for the purpose of detecting a molecular marker or a change in the molecular marker.

[0037] A "subject" is any organism of interest, generally a mammalian subject, such as a mouse, and preferably a human subject.

Overview

[0038] In certain aspects, the invention relates to methods for clinical testing of DNA methylation in genomic DNA samples extracted from subjects with various diseases, including but not limited to oncology, inflammatory, and psychiatry diseases.

[0039] Described herein is a method for analysis of DNA methylation in selected fragments using ultra-small samples (300 pg or less) of genomic DNA extractable from clinical samples. Analysis of DNA methylation in multiple fragments may also be performed according to the method described herein using 300 pg (or less) of DNA samples. An overview of the disclosed method is as follows. DNA samples obtained from a subject was divided into two parts, one part was treated with the methylation- sensitive restriction enzyme and/or methylation-dependent restriction enzyme in defined conditions, while the other part was incubated without the enzyme and serves as the control. Genomic DNA in both parts was amplified using genome-wide amplification with phi29 enzyme, and selected fragments were analyzed using TaqMan quantitative PCR. The ACt of the restriction enzyme-treated and control parts of the sample were compared to determine the methylation status of the recognition sites for the restriction enzyme within the selected fragments.

Digestion of ultra-small samples (300 pg or less) of genomic DNA with a methylation- sensitive restriction enzyme

[0040] In one embodiment, methylation-sensitive restriction enzyme Hin6l (ThermoScientific) was used for restriction digestion (see Example 1). This enzyme recognizes the site GCGC, and does not cut DNA if the second nucleotide (cytosine) is methylated. Importantly, the reaction conditions generally used for restriction digest with Hin6l (see e.g., http://www.thermoscientificbio.com/search/?term=Hin6n are not suitable for effective and efficient digestion of genomic DNA at ultra-low levels (300 pg or less).

[0041] Alternatively, a restriction digestion can be carried for example with the following, but not limiting, methylation-sensitive and methylation-dependent restriction enzymes and their isoschizomers as shown below in Tables 1 and 2.

[0042] Table 1. Methylation-sensitive restriction enzymes.

Sac II CCGCjGG Clontech: 1079A/B

Sai l GjTCGAC Clontech: 1080A/B/AH/BH

Sma 1 CCC|GGG Clontech: 1085A/B/AH/BH

SnaB 1 TACjGTA Clontech: 1245A/B

Dpnll IGATC NEB: R0543S

Hpall CJ.CGG NEB: R0171S

Mspl CjCGG NEB: R0106S

Sall-HF GjTCGAC NEB: R3138S

ScrFI CCjNGG NEB: R0110S

Wherein N = A or C or G or T; D = A or G or T; B = C or G or T; V = A or C or G; R = A or G; S = C or G; W = A or T; Y = C or T.

[0043] See e.g., http://www.clontech.com/takara/US/Products/Epigenetics/DNA_Preparation/MSRE_Over view) and https://www.neb.com/products/epigenetics/methylation-sensitive-restriction- enzymes.

[0044] Table 2. Methylation-dependent restriction enzymes.

Wherein N = A or C or G or T; D = A or G or T; B = C or G or T; V = A or C or G; R = A or G; S = C or G; W = A or T; Y = C or T.

[0045] See e.g., https://www.neb.com/products/epigenetics/methylation-dependent- restriction-enzymes; Kami et a/., PNAS (2011); Murray, Microbiology (2002); Sitaraman ef a/., Gene (2011).

[0046] In general, the cleavage by Hin6l or any other methylation-sensitive or Methylation-dependent enzyme can be detected, for example, but not limiting to, quantitative PCR (qPCR) and next generation sequencing (NGS). [0047] It is well established that the reaction rate (V) of a single substrate enzymatic reaction depends on the concentration of the substrate as described by the Michaelis- Menten model (see e.g., https://en.wikipedia.org/wiki/lv1ichaelis%E2%80%93Menten kinetics). The model takes the form of an equation describing the rate of enzymatic reactions, by relating reaction rate v to [*-*], the concentration of a substrate S. Its formula is given by

₌ d[P\ ₌ V_max[S\

V dt K_m + [S]

where, V_max represents the maximum rate achieved by the system, at maximum (saturating) substrate concentrations. The Michaelis constant K_m is the substrate concentration at which the reaction rate is half of V_max- Biochemical reactions involving a single substrate are often assumed to follow Michaelis-Menten kinetics, without regard to the model's underlying assumptions.

[0048] Thus, in order to accelerate digestion of a genomic DNA present at an ultra- low concentration (300 pg or less), the inventors have advantageously determined the "optimal conditions" for digestion with a methylation-sensitive restriction enzyme Hin6l (see Example 1).

[0049] The efficiency with which a restriction enzyme cuts its recognition sequence at different locations in a piece of DNA can vary 10 to 50-fold. This is may be due to influences of sequences bordering the recognition site, which perhaps can either enhance or inhibit enzyme binding or activity (see, e.g., http://www.vivo.colostate.edu/hbooks/genetics/biotech/enzymes/cuteffects.html).

[0050] "Optimal conditions" of the digestion reaction are defined as "complete digestion" of all unmethylated sites GCGC by the methylation-sensitive restriction enzyme Hin6l within an acceptable (<5 hr) timeframe. "Complete digestion" is defined as the absence of a specific PCR product from a target within the genome when the target contains an unmethylated site GCGC, and 40 cycles of PCR are performed. Additionally, the "complete" digestion is defined as the absence of a specific PCR product following 40 cycles of qPCR, when the undigested part of the sample demonstrates PCR product with C_T range of between 17 and 27.

[0051] Surprisingly and unexpectedly, the inventors have discovered that DNAzol Direct, a glycol compound previously used for storing and/or processing of biological samples for direct use in PCR, may be advantageously added to the restriction digestion of a genomic DNA with a methylation-sensitive restriction enzyme Hin6l. For example, as described in U.S. Patent No. 7,727,718 (incorporated herein by reference in its entirety) DNAzol Direct, a glycol compound, may comprise ethylene glycol, polyethylene glycols, polyglycol, propylene glycol, polypropylene glycol and glycol derivatives including polyoxyethylene lauryl ether, octylphenol-polyethylene glycol ether, and polyoxyethylene cetyl ether.

[0052] The glycol compounds of this invention may further comprise 1 ,2- propanediol, 1 ,3-butanediol, 1 ,4-butanediol, 1 ,4-cyclohexanedimethanol-, 1,6- hexanediol, butylene glycol, diethylene glycol, dipropylene glycol, ethylene and propylene glycol (including ethylene and propylene glycol monomers and polymers, e.g., low molecular weight (less than 600) polyethylene glycols and low molecular weight (less than 600) polypropylene glycols), glycerol, long chain PEG 8000 (about 180 ethylene monomers), methyl propanediol, methyl propylene glycol, neopentyl glycol, octylphenol-polyethylene glycol ether, PEG-4 through PEG-100 and PPG-9 through PPG-34, pentylene glycol, polyethylene glycol 200 (PEG 200 about 4 ethylene monomers), polyethylene glycols, polyglycol, polyoxyethylene cethyl ether, and octyl- polyethylene glycol ether, polyoxyethylene cetyl ether, polyoxyethylene lauryl ether, polypropylene glycols, tetraethylene glycol, triethylene glycol, trimethylpropanediol, tripropylene glycol.

[0053] The glycol compounds of this invention may yet further comprise ethylene glycol, propylene glycol, polyethylene glycols, polypropylene glycols, polyglycol and glycol derivatives including polyoxyethylene lauryl ether, octylphenol-polyethylene glycol ether, and polyoxyethylene cetyl ether. The preferred organic solvents of this invention are polyethylene glycols and glycols derivatives. The most preferred solvents are polyethylene glycols.

[0054] Polyalkylene glycols comprise polyethylene glycol (PEG) and polypropylene glycol. PEGs are generally commercially available diols having a molecular weight of from 200 to 10,000 daltons, more preferably about 200-300 daltons. Suitable PEGs can be obtained from Spectrum Laboratory Products, Inc, (Gardena, Calif., Molecular weight 200, Cat. # PO 107). The molecular weight of the polyethylene glycol (PEG) can range from about 200 to about 10,000. Generally, the polyalkylene concentration will depend on the polyalkylene used. Depending on the weight range of polyethylene glycol used, the concentration can be adjusted. The PEG at a concentration from about 0.1% to about 100% and PPG, when added to a PCR mix, have been shown to inhibit the effect of impurities on PCR.

[0055] Surprisingly and unexpectedly, the inventors have discovered that addition of DNAzol Direct (Molecular Research Center, Inc.; Cat. # DN 131) to the reaction mix (see Example 1) resulted in an accelerated and complete digestion of a genomic DNA present at an ultra-low concentration (300 pg or less). In a typical reaction described herein, acceleration of a complete restriction digestion with a methylation-sensitive restriction enzyme Hin6l was achieved with a genomic DNA sample at a concentration of 2.33 ng/ml.

Efficient amplification of genomic DNA using phi29 Pol in the presence of the E.coli ssb protein

[0056] Several useful methods have been developed that permit amplification of nucleic acids. Most were designed around the amplification of selected DNA targets and/or probes, including the polymerase chain reaction (PCR), ligase chain reaction (LCR), self-sustained sequence replication (3SR), nucleic acid sequence based amplification (NASBA), strand displacement amplification (SDA), and amplification with Ο,β replicase (Birkenmeyer and Mushahwar, J. Virological Methods, 35:117-126 (1991); Landegren, Trends Genetics, 9:199-202 (1993)).

[0057] An exemplary method is known as primer extension preamplification (PEP). This technique uses random primers in combination with a thermostable DNA polymerase to replicate copies throughout the genome. Exemplary conditions that can be used for PEP-PCR are described in Zhang et a/., Proc. Natl. Acad. Sci. USA, 89:5847-51 (1992); Casas et al., Biotechniques 20:219-25 (1996); Snabes ef a/., Proc. Natl. Acad. Sci. USA, 91 :6181-85 (1994,); or Barrett et al., Nucleic Acids Res., 23:3488- 92 (1995).

[0058] Further amplification methods may include, but not limited to, isothermal strand displacement nucleic acid amplification as described in U.S. Pat. NOs. 6,214,587 or 5,043,272.

[0059] Other non-PCR-based methods that can be used in the invention include, for example, strand displacement amplification (SDA) which is described in Walker et al., Molecular Methods for Virus Detection, Academic Press, Inc., 1995; U.S. Pat. Nos. 5,455,166, and 5,130,238, and Walker et al., Nucl. Acids Res. 20:1691-96 (1992) or hyperbranched strand displacement amplification which is described in Lage et al., Genome Research 13:294-307 (2003).

[0060] Other methods may include, but not limited to, are Nicking Enzyme Amplification Reaction (NEAR) as described in http://www.envirologix.com/artman/publish/article_314.shtml; nucleic acid sequence- based amplification (NASBA) as described in http://www.premierbiosoft.com/tech_notes/NASBA.html; and Cross Priming Amplification as described in http://www.readcube.eom/articles/10.1038/srep00246?locale=en.

[0061] The use of the following polymerases has been previously described: Bst and Klenow fragment (https://www.neb.com/applications/dna-amplification-and- pcr/isothermal-amplification; http://www.neb-online.de/isothermal_amp.pdf); RPA (http://alere-technologies.com/en/products/lab-solutions/isothermal-amplification.html); thermophilic Helicase-Dependent Amplification (tHDA)

(http://www.biohelix.com/products/isoampiii_enzyme_mix.asp;

en.wikipedia.org/wiki/Helicase-dependent_amplification).

[0062] Phi29 DNA polymerase, for example, has proved useful in several amplification methods, such as for example, but not limited to Multiple Displacement Amplification (MDA). MDA can be used to amplify linear DNA, especially genomic DNA.

[0063] It has been previously shown that inclusion of E. coli SSB in reaction mixtures comprising linear DNA molecules leads to a much increased yield of amplified DNA products (see e.g., U.S. Publication No.: 20110065151 , PCT/EP2009/056235, published as WO2009141430 A1 , Joneja et al., 2011 ; incorporated herein by reference in its entirety). Other E. coli SSB may include, but not limited to, ET SSB (NEW England Biolabs; Cat. No.: M0249S), RecA (New England Biolabs; Cat. No.: M0249S), T4 gene 32 protein (NEW England Biolabs; Cat. No.: M0300S), and Tth RecA (New England Biolabs; Cat. No.: M2402S).

[0064] "Efficient amplification" as described herein is defined as the ability to amplify 0.35 ng of cell-free DNA (one half of the 0.7 ng is used for the digestion with Hin6l, and one half - as control) to no less than 10 μg of product. Importantly, the cell-free fetal DNA (cfDNA) is generally severely fragmented, which makes the amplification reaction using phi29 polymerase very inefficient. The two-step approach described herein (see Example 2) allows efficient amplification of highly fragmented DNA, which is difficult to achieve by other means.

Quantification ofPCR fragments using TaqMan quantitative PCR

[0065] Quantification of methylated or unmethylated CpG sites within amplified PCR fragments was carried out using TaqMan probe-based real-time PCR method as previously described (see e.g., 2011 MethyLight PCR Handbook, Qiagen; Zeschnigk et a/., 32(16) Nucleic Acids Research (2004)).

[0066] In general, TaqMan probe-based real-time PCR method allows the direct quantification of the degree of methylation in a sample by using the threshold cycle values (C_T) determined by qPCR. In general, the PCR reaction exploits the 5^' nuclease activity of a DNA polymerase to cleave a TaqMan probe during PCR. The TaqMan probe contains a reporter dye at the 5' end of the probe and a quencher dye at the 3' end of the probe. During the reaction, cleavage of the probe separates the reporter dye and the quencher dye, which results in increased fluorescence of the reporter. Accumulation of PCR products is detected directly by monitoring the increase in fluorescence of the reporter dye (see e.g., TaqMan Universal PCR Master Mix Protocol, Applied Biosystems).

[0067] Selection of informative fragments for TaqMan probe-based real-time PCR method was done based on (a) the highest difference in R=Cy5/Cy3 ratio between test and control samples, which has been determined previously in microarray-based discovery experiments; (b) consistent difference between test and control samples; and confirmed by (c) Fisher's Exact test (see e.g., Handbook on Biological Statistics found at http://udel.edu/~mcdonald/statfishers.html). At the end of the Discovery phase up to 48 fragments for confirmation by qPCR are selected.

Determinination of the DNA methylation status

[0068] Determination of the DNA methylation status within the recognition site of the methylation-sensitive or methylation-dependent restriction enzyme was based on the comparison of C_T points in the amplification plots of restriction enzyme-treated and control parts of the same sample using AC_t method (including the scoring protocol) (see e.g., 2012 EpiTect Methyl II PCR Array Handbook, Qiagen).

EXAMPLES [0069] The Examples that follow are illustrative of specific embodiments of the invention, and various uses thereof. They are set forth for explanatory purposes only, and are not to be taken as limiting the invention.

Example 1: Restriction digestion of ultra-small genomic DNA samples with Hin6l methylation-sensitive restriction enzyme

[0070] Genomic DNA preparation

[0071] High-quality genomic DNA is a prerequisite for a successful digestion reaction. Therefore, sample handling and genomic DNA isolation procedures are crucial to the success of the experiment. Residual traces of proteins, salts, or other contaminants will either degrade the DNA or decrease the restriction enzyme activities necessary for optimal DNA digestion. Genomic DNA was isolated using DNeasy Blood and Tissue Kit (Qiagen). Genomic DNA samples were diluted or resuspended in DNase-free water, or alternatively, in DNase-free 10 mM Tris buffer pH 8.0 without EDTA. The measurement of concentration of genomic DNA and calculation of the genomic DNA amount isolated was done with a PicoGreen reagent as described by Life Technologies, Invitrogen; (cat. # P7581).

Determining "optimal conditions" of the digestion reaction

[0072] To determine the optimal conditions of the digestion reaction serial dilutions of lambda DNA were incubated for 210 min in selected conditions and subjected to 40 cycles of PCR with primers flanking one of the GCGC sites in lambda genome. The lowest dilution of lambda DNA that produced a specific product was noted.

[0073] The following parameters were evaluated: pH of the reaction; temperature of the reaction; concentration of divalent cations; and additives (e.g., dimethylsulfoxide (DMSO), formamide, dithiotreitol (DTT), Glycerol, Mg²⁺, Ca²⁺, Zn²⁺, bovine serum albumin (BSA), DNAzol Direct, and molecular crowders such as, but not limited to betain, PEG-1000, PEG-400, dextran sulphate. Additives were tested at different concentrations and in various combinations.

[0074] The following, but not limiting, reaction conditions were tested: ratio DNA:enzyme (15U/ng to 200U/ng), temperature (20°C; 30°C , 37°C, 42°C, 48°C, 56°C), pH (from 7.0 to 9.0); time (from 1hr to 144hr). [0075] The following, but not limiting, buffers were tested: FastDigest buffer (Fermentas-ThermoFisher).

[0076] The results are summarized in Table 3 and Figure 2.

[0077] Table 3. Example of the optimization reaction for efficiency of digestion.

Selected fragment is methylated in MCF7 cells and unmethylated in T47D cells. Expected result is an amplified product for tubes #1 , #3, #5, #7, and #9, and no amplified product in tubes #2, #4, #6, #8, and #10.

Measurement of DNA concentration and purity by UV spectrophotometry

[0078] The measurement of concentration of genomic DNA and calculation of the genomic DNA amount isolated was done with a PicoGreen reagent as described by Life Technologies, Invitrogen; (cat. # P7581).

Restriction Digestion Protocol

[0079] The complete restriction digest was carried out according to the following protocol.

Reaction Mix:

H₂0 50%

DNAzol Direct (Molecular Research Center, Inc.; Cat. # DN 131) 35% 10^x Buffer Tango (Fermentas; Cat. # BY5) 10% Hin6l (Thermo Scientific; Cat. # ER0481 5%

[0080] The reaction mix was pipetted up and down to gently, but thoroughly mix the components and the tubes containing the reaction mix were briefly centrifuged in a microcentrifuge. Incubation of the complete restriction digest was carried out for 210 min at 42°C in a thermal cycler. The digested sample was used in the subsequent amplification reaction (see Example 2).

Example 2: Amplification of genomic DNA using phi29 Polymerase in the presence of the E.coli SSB protein

[0081] Mix the digested samples thoroughly by vortexing before use. Centrifuge the samples briefly in a microcentrifuge and proceed to step 1 of the amplification reaction.

[0082] Amplification was done as described below.

[0083] 85 μΙ of the Master Mix was added to a PCR tube, followed by addition of 10 μΙ of digested genomic DNA. The sample was slowly vortexed and spun down before incubation. The sample was subsequently incubated for 2 min at 95°C in a thermal cycler. After incubation, the sample was kept on ice for 30 seconds.

[0084] 4 μΙ phi29 DNA Polymerase (New England Biolabs; cat. # M0269L) and 1 μΙ E. coli SSB-protein (10-20 ng/ml, Epicenter Technologies, an lllumina company; cat. # SSB02200) were added to the sample. The sample was then briefly vortexed, kept on ice 5 min, and spun down in a microcentrifuge. The sample was subsequently incubated for 16h at 30°C in a thermal cycler.

Example 3: Quantification of PCR fragments using TaqMan quantitative PCR

[0085] Quantification of methylated or unmethylated CpG sites within amplified PCR fragments was carried out using TaqMan probe-based real-time PCR method as previously described (see e.g., 2011 MethyLight PCR Handbook, Qiagen; TaqMan Universal PCR Master Mix Protocol, Applied Biosystems; Zeschnigk et al., 32(16) Nucleic Acids Research (2004)).

Example 4: Determinination of the DNA methylation status

[0086] After the cycling program was completed, the C_T values were determined according to the following protocol. C_T was calculated separately for control and test parts of the sample, and then the difference was calculated (ACt). ACt >8 was considered significant and indicates unmethylated fragment (value 0). 2<ACt <8 was considered undefined and the fragment is not scored. 0<ACt<2 was considered significant, and the fragment was scored as methylated (value 1).

[0087] Each potentially informative fragment determined in the Discovery phase by microarray analysis is tested via qPCR in >=30 samples for each group and the frequencies of unmethylated score and methylated score were recorded. Fragments with differences >0.75 were combined into the composite biomarker. The fragments with higher differences were preferentially selected in order to determine the minimal number of fragments and to bring the probability of error to less than 0.001%. For example, if each fragment has probability of error <0.25, so that 0.25*0.25*0.25*0.25*0.25*0.25=0.000244 or 0.02%, so six fragments are insufficient and three additional are added: 0.000244*0.25*0.25*0.25=0.000003815 or 0.00015%. Considering that some of the fragments may fail in the reaction, the actual number of components in the composite biomarker was no less than 12 with the cumulative error less than 0.00000006 or less than 0.000006%.

[0088] References

U.S. Publication No.: 2012/038930

U.S. Patent No.: 7,727,718

U.S. Patent No.: 5,945,515

U.S. Patent No.: 5,001 ,050

U.S. Patent No.: 4,683,202

U.S. Publication No.: 2006/0134650 U.S. Patent No.: 6,214,587

U.S. Patent No.: 5,043,272

U.S. Patent No.: 5,455,166

U.S. Patent No.: 5,130,238

Walker et a/., Molecular Methods for Virus Detection, Academic Press, Inc., 1995.

[0089] Having described the invention in detail and by reference to specific embodiments thereof, it will be apparent that modifications and variations are possible without departing from the scope of the invention defined in the appended claims. More specifically, although some aspects of the present invention are identified herein as particularly advantageous, it is contemplated that the present invention is not necessarily limited to these particular aspects of the invention.

Claims

WHAT IS CLAIMED IS:

1. A method for determining a DNA methylation status, comprising:

(a) obtaining a DNA sample from a subject;

(b) digesting the DNA sample with a methylation-sensitive restriction enzyme in the presence of a glycol compound;

(c) amplifying the digested sample of step (b);

(d) quantifying amplification results from step (c) using a real-time quantitative PCR; and

(e) determining the DNA methylation status within a recognition site of the methylation-sensitive restriction enzyme.

2. The method of claim 1, wherein the subject is a mammal, wherein the mammal is a human.

3. The method of claim 1 , wherein the DNA comprises a genomic DNA.

4. The method of claim 1 , wherein the DNA sample is between about 1 pg and about 1 ng.

5. The method of claim 4, wherein the DNA sample is about 300 pg.

6. The method of claim 1 , wherein the methylation-sensitive restriction enzyme comprises Hin6l.

7. The method of claim 1 , wherein the amplifying comprises amplifying using phi29 DNA polymerase.

8. The method of claim 7, wherein amplifying further comprises amplifying using a single stranded DNA binding protein of E.coli.

9. The method of claim 1 , wherein the real-time quantitative PCR comprises TaqMan qPCR.

10. The method of claim 1 , wherein determining the DNA methylation status comprises determining threshold cycle (C_T) values.