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Epiallele Quantification Using Molecular Inversion Probes Ramkumar Palanisamy, Ashley R. Connolly,* and Matt Trau* Centre for Biomarker Research and Development, Australian Institute for Bioengineering and Nanotechnology (AIBN), The University of Queensland, Brisbane Qld, 4072 Australia
bS Supporting Information ABSTRACT: The location and level of DNA methylation within a genome is emerging as an important biomarker for cancer diagnosis. Despite its potential, it is difficult to comprehensively analyze the epialleles that are often found in a biological sample. Therefore, an assay utilizing molecular inversion probes was designed and used to expose and quantify epialleles in heterogeneously methylated bisulphite treated genomic DNA. Different CpG dinucleotides were able to be rapidly quantified with high resolution, sensitivity and specificity over a large dynamic range using rapid flow cytometric readout of multiplexable microbead DNA biosensors.
M
ethylation of DNA is an epigenetic modification that is essential for normal cell function.1 The most common form of epigenetically modified DNA contains a methyl group at the C5 position of cytosine.2 Deregulation of cytosine methylation can result in tumor formation, and as a result, changes in cytosine methylation are emerging as a potential cancer biomarkers.39 Despite its potential, measuring changes in DNA methylation is difficult to analyze comprehensively. Several enzymes and antibodies have enabled the direct analysis of methylated DNA on a genome wide scale.1012 However, one of the most common analytical techniques involves the detection of a DNA fragment that has been chemically treated to convert cytosine (C) into uracil (U).13 Methylated cytosine remains unconverted, and this functional nucleotide change can be detected using a variety of novel methods to amplify the region of interest.1420 However, bisulphite treatment can fragment genomic DNA, which can retard amplification.21 Furthermore, a thorough understanding of the local methylation pattern of the region is required to enable efficient priming during amplification of bisulphite treated DNA. However, the extent of cytosine methylation often varies between patients, and biased polymerase chain reaction (PCR) priming often results in nonquantitative amplification of only one epiallele. This can confound analysis when methylation is used as a tumor biomarker.2225 To address this problem, we have developed a novel, multiplex assay capable of detecting and accurately quantifying both methylated and unmethylated epialleles in bisulfite treated DNA. The assay extends the application of molecular inversion probes (MIPs) from the analysis of single nucleotide polymorphisms and DNA mutations to the analysis of DNA methylation.26,27 MIPs have been used in the discovery of genome wide DNA methylation patterns.28 However, to enable quantitative analysis of methylation at specific CpG sites, a MIP assay was developed for applications that demand the rapid analysis of DNA methylation in large numbers of clinical samples. This was achieved by developing a modified multiplex MIP assay that r 2011 American Chemical Society
could be rapidly analyzed on microbead DNA biosensors using a flow cytometer. A DNA ligase was used to selectively circularize MIPs, and the amount of ligation was found to accurately portray the proportion of methylated and unmethylated cytosine at the site of interest. This enabled the different epialleles to be accurately quantified. The methylation profile of neighboring CpG sites could also be quantified with high resolution, and the ability to accurately quantify different epialleles over a large dynamic range was also evaluated. Multiple MIPs can be used to interrogate multiple CpG sites in a single multiplex reaction, and direct amplification of the MIPs circumvents many of the problems associated with amplifying bisulphite converted DNA. The assay can be used to accurately measure methylation profiles with a high level of specificity, sensitivity, and resolution using only a small amount (picograms) of bisulphite converted genomic DNA. The DNA methylation status of a CpG dinucleotide in heterogeneously methylated DNA can also be measured by strategically incorporating inosine nucleotides into the MIP. The assay incorporates a flow cytometric readout, which enables fast, accurate, and sensitive measurement of DNA methylation with multiplexable microbead DNA biosensors. In its current form, the assay has clinical potential for the rapid and quantitative analysis of DNA methylation profiles for biomarker research and development in a research or diagnostic setting.
’ MATERIALS AND METHODS Fluorescently Encoded Microbeads. Preparation of 6 μm diameter silica microbeads has been previously described.29 Received: November 16, 2010 Accepted: March 1, 2011 Published: March 14, 2011 2631
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Table 1. Oligonucleotide Sequences oligonucleotide sequences 50 -30 universal forward primer
CGT TAT AGG ACT GAA GAG CCA T
fluorescent universal reverse primer
Alexa 647-CCA AAT GCT GTG TAG GTC ATC T
target: 80TC
CGT CTC TGA TTG TAC CTT GAT TTC GTA TTC TGA GAG GCT GCT GCG T
target: 80TT
CGT CTC TGA TTG TAC CTT GAT TTT GTA TTC TGA GAG GCT GCT GCG T
target: CytoTC
TTG CGT TTC GGG GTT TTT CGC GTC GTT TTT ATT GTC GTC GGT G
target: ThyTC
TTG TGT TTT GGG GTT TTT TGC GTT GTT TTT ATT GTT GTT GGT G
target: ThyTT
TTG TGT TTT GGG GTT TTT TGT GTT GTT TTT ATT GTT GTT GAT A
(80) methylated probe 80MIPG
50 P-AAA TCA AAA TAC AAT CAA AAA TGG CTC TTC AGT CCT ATA ACG UCC AAA TGC TGT GTA GGT CAT CTC TTC CTG GCC CTC AAC CAC TCA ACA ACC TCT CAA AAT ACG-30 OH
(80) unmethylated probe 80MIPA
50 P-AAA TCA AAA TAC AAT CAA AAA TGG CTC TTC AGT CCT ATA ACG UCC AAA TGC TGT GTA GGT CAT CTA GGA GCC TGG TAC ATT GGT GCA ACA ACC TCT CAA AAT ACA-30 OH 0
(78) cytosine probe 78MIPG
5 P-CAA AAT CAA AAT ACA ATC AAA TGG CTC TTC AGT CCT ATA ACG UCC AAA TGC TGT
(78) thymine probe 78MIPA
50 P-CAA AAT CAA AAT ACA ATC AAA TGG CTC TTC AGT CCT ATA ACG UCC AAA TGC TGT GTA
probe MIPG
50 P-CGA AAA ACC CCG AAA CGC AAA TGG CTC TTC AGT CCT ATA ACG UUU CCA AAT GCT GTG TAG GTC ATC TAG GAG CCT GGT ACA TTG GTG CGA CGA CAA TAA AAA CGA CG-30 OH
inosine probe iMIPG
50 P-CIA AAA ACC CCI AAA CIC AAA TGG CTC TTC AGT CCT ATA ACG UUU CCA AAT GCT GTG
MIPG and iMIPG bead barcode
NH3-TGT GGA CTG AAT TCT GTC TGC ACC AAT GTA CCA GGC TCC T
GTA GGT CAT CTA GTT GTG CAG GTG GTG GAT ACG CAA CAA CCT CTC AAA ATG-30 OH GGT CAT CTA AAG GCG TCT CCC TCT ATT GCG CAA CAA CCT CTC AAA ATA-30 OH
TAG GTC ATC TAG GAG CCT GGT ACA TTG GTG CIA CIA CAA TAA AAA CIA CG 30 OH 80MIPG bead barcode
NH3-TTG CTG GCA CAG GAG GTG ACA GTG GTT GAG GGC CAG GAA G
80MIPA bead barcode
NH3-TGT GGA CTG AAT TCT GTC TGC ACC AAT GTA CCA GGC TCC T
78MIPG bead barcode
NH3-CTC TCC TTG ATC TCC AAC CGT ATC CAC CAC CTG CAC AAC T
78MIPA bead barcode
NH3-GAA GTC CAG CAG AAT CAA TAC AAT AGA GGG AGA CGC CTT T
Unique populations of microbeads were prepared by covalently coupling different amounts of Attotec 488 and Attotec 550 to the interior of the microbeads, which were then coated with aminopropylsilane and functionalized with adipic acid. Coupling of Oligonucleotides to Microbeads. The 50 end of an amine derivitized oligonucleotide was covalently coupled to a unique population of adipic acid coated microbeads using 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) chemistry.30 The DNA coated microbeads were pelleted by centrifugation at 10 000g for 30 s and washed three times with 1 mL of 0.1 M 2-morpholinoethane sulfonic acid supplemented with 0.01% sodium dodecyl sulfate (SDS) before being stored at 4 °C. DNA Oligonucleotides. DNA oligonucleotides were purchased from GeneWorks (GeneWorks Pty. Ltd., South Australia, Australia). Fluorescently labeled oligonucleotides were purchased from Invitrogen (Invitrogen Corporation, California, USA). The oligonucleotides used in this study are listed in Table 1. DNA Extraction and Bisulphite Treatment and Quantification. Genomic DNA was extracted from MCF7 cells using a QIAGEN DNeasy blood and tissue kit (QIAGEN Pty. Ltd., Venlo, Netherlands). Purified DNA was bisulphite treated using a MethylEasy Xceed kit (Human Genetic Signatures Pty. Ltd., New South Wales, Australia) according to the manufacturer's instructions. The amount of bisulphite converted DNA was quantified by measuring the level of the COL2A1 gene in each sample using real time PCR as previously outlined.31 DNA Ligation. MIP ligation was performed in a 10 μL reaction containing 200 pg of MIP, 2 ng of bisulphite treated genomic DNA, 1 U Ampligase (Epicenter Biotechnologies,
Wisconsin, USA), 1 ligase buffer (200 mM TrisHCl, 250 mM KCl, 100 mM MgCl2, 5 mM NAD, pH 8.3), and 0.1% Triton X-100. The reaction was heated to 95 °C for 1 min and then thermally cycled at 95 °C for 1 min and 65 °C for 2 min for 10 cycles. The MIP containing inosine (iMIPG) was ligated using identical conditions except the reaction was thermally cycled at 95 °C for 1 min and 45 °C for 2 min for 10 cycles. Polymerase Chain Reaction. One microliter of the ligation reaction was added to a 20 μL PCR containing 1 U AmpliTaq DNA polymerase (Applied Biosystems, California, USA), 2 mM MgCl2, 1 PCR buffer, 0.2 mM of each deoxynucleotide triphosphate, 0.1% Tween 20, and 125 nM of forward and reverse primer. The reaction was denatured at 94 °C for 10 min, and then, PCR was performed for 30 cycles of 94 °C for 30 s, 65 °C for 45 s, and 72 °C for 10 s. Asymmetric PCR was performed using the same conditions with 125 nM fluorescent Alexa 647 reverse primer (Invitrogen Corporation, California, USA) and 62.5 nM forward primer. DNA Hybridization. Four microliters of asymmetric PCR product was mixed with 1 104 optically encoded microbeads in hybridization buffer (3 M tetramethylammonium chloride, 50 mM TrisHCl, 0.1% SDS, and 2 mM ethylenediaminetetraacetic acid (EDTA), pH 8.0). The reaction was made to 30 μL with 18 Ω water, and hybridization was performed at 55 °C for 60 min while shaking at 1400 rpm in a thermal mixer (Eppendorf, Hamburg, Germany). Following hybridization, the microbeads were washed and suspended in 100 μL of 0.01% SDS in preparation for analysis using flow cytometry. Flow Cytometry. Fluorescence on the surface of each microbead was measured using a DakoCytomation Moflo flow 2632
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Figure 1. (A) MIPs designed to detect methylated (80MIPG) and unmethylated (80MIPA) DNA were mixed with bisulphite treated DNA and ligated in a multiplex assay. The MIP complementary to methylated DNA was circularized upon ligation while the noncomplementary MIP remained linear. (B) A fluorescently labeled primer was used to asymmetrically amplify the circularized MIP. (C) The extent of DNA amplification was determined by hybridizing the amplicon to optically encoded microbeads which were analyzed using flow cytometry.
cytometer. Optically encoded microbeads were excited with a 488 nm laser, and the emission of Attotec 488 and Attotec 550 was monitored at 530 and 580 nm, respectively. Alexa 647 labeled DNA was excited with a 635 nm laser, and emission was monitored at 670 nm. Data was collected on a logarithmic scale and analyzed using Summit V 4.0 software.
’ RESULTS Assay Design. A standard assay was designed, and several variables were evaluated. The assay was designed to measure the methylation status of the 80 CpG site of the BRCA1 promoter, which has been implicated in breast cancer.32 A MIP (80MIPG) was designed to detect methylated C, and a second MIP (80MIPA) was designed to detect unmethylated C at this site (Figure 1A). Each MIP was composed of multiple domains. The 50 and 30 end of each MIP contained a 20 nucleotide (nt) binding domain complementary to the region of interest. This enabled each MIP to form a loop containing 40 bp of double stranded (ds) DNA with the CpG interrogation site in the center. When the interrogation site complements the region of interest, DNA ligase covalently couples the 50 end of the MIP to the adjacent 30 end to form circular DNA. The circularized MIP is a suitable substrate for PCR amplification with a fluorescently labeled primer (Figure 1B). However, when the interrogation site does not complement the region of interest, the MIP is not ligated and amplification fails. All MIPs were designed with identical primer binding domains so the methylation status of multiple CpG sites could be interrogated and amplified using a single pair of primers in a multiplex reaction. To deconvolute the amplicons, each MIP contained a unique DNA barcode addressed to complementary oligonucleotides immobilized on the surface of optically unique
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microbeads (Figure 1C). This design enables multiple CpG sites to be interrogated using multiple MIPs in a single reaction with fast flow cytometric readout. Ligation Optimization. Several parameters likely to affect the assay were optimized to ensure CpG sites could be measured with accuracy and specificity. In particular, the concentration of MIP used in the assay was optimized to minimize nonspecific ligation, while ensuring there was a sufficient amount available for amplification and detection. Various amounts of 80MIPG (200 fg to 2000 pg) were added to replicate reactions containing bisulphite treated genomic DNA. Each reaction was ligated, amplified, and analyzed using gel electrophoresis. An optimal amount of PCR product was obtained when 200 pg of the MIP was added to the assay and all subsequent assays, to ensure a large signal was produced with the highest level of specificity (Figure S-1, Supporting Information). The ligation reaction can be thermally cycled to increase the amount of circularized MIP produced prior to PCR amplification. The effect of ligation cycling varied with the amount of DNA added to the reaction. However, when 500 pg of bisulphite treated DNA and 200 pg of 80MIPG were added to the assay, 10 ligation cycles were sufficient to produce a detectable product after DNA amplification (Figure S-2, Supporting Information). Additional cycles of ligation produced no substantial increase in the amount of product produced. Detection Limit. Suspect tumors are often sampled by needle biopsy, which only provides a small amount of tissue for diagnosis. The tissue is often subjected to multiple tests to ensure accurate diagnosis. As a result, analytical techniques that can be performed on minute amounts of DNA are sought after. Therefore, the minimum amount of genomic DNA required to perform the assay was assessed. The 80 CpG of the BRCA1 promoter has been reported to be unmethylated in MCF7 cells.33 Therefore, a MIP (80MIPA) was designed to detect uracil (U) at the 80 CpG of the BRCA1 promoter. Replicate reactions containing a different amount of bisulphite treated genomic DNA were prepared and analyzed. The amount of amplicon generated in each reaction was measured by flow cytometry and is depicted in Figure 2. Each sample produced a fluorescent signal that was found to increase as the amount of genomic DNA increased. As the concentration of the bisulphite treated DNA increased from 125 pg to 5 ng, there was a 5-fold increase in fluorescence. The signal from the sample containing 125 pg of DNA was not statistically significantly different from a replicate reaction containing no DNA. Therefore, the assay could be used to reliably detect DNA methylation using only 250 pg of bisulphite treated DNA with a coefficient of variation of 2.9%. Specificity. The accuracy of the assay is determined by the ability of DNA ligase to accurately discriminate a single base pair mismatch between the MIP and the CpG site of interest. Since nonspecific ligation of a MIP will limit the specificity of methylation detection, the fidelity of the DNA ligase was assessed. The extent of nonspecific DNA ligation was measured by assaying genomic DNA with two MIPs that contain a single base mismatch at the CpG interrogation site. The BRCA1 gene in MCF7 cells is reported to be unmethylated.33 80MIPA was designed to detect unmethylated cytosine at the 80 CpG of the BRCA1 promoter while 80MIPG was designed to detect methylated cytosine at the same position. An assay containing both MIPs and bisulphite treated genomic DNA was ligated and amplified. Each MIP contained a unique DNA barcode, which 2633
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Figure 2. Different amounts of bisulphite treated DNA from MCF7 cells were mixed with a MIP (80MIPA) designed to detect unmethylated cytosine at the 80 CpG of the BRCA1 promoter. Each reaction was ligated, and a fluorescently labeled primer was used to asymmetrically amplify the circularized DNA. The amplicons were hybridized to microbeads and analyzed by flow cytometry. The level of fluorescence on each microbead revealed the extent of DNA ligation for the different amounts of DNA. The fluorescence values represent the mean ( standard deviation of triplicate assays.
Figure 3. MIPs designed to detect methylated (80MIPG) and unmethylated cytosine (80MIPA) at the 80 CpG of the BRCA1 promoter were mixed with bisulphite treated DNA from MCF7 cells and ligated in a duplex assay. A fluorescently labeled primer was used to asymmetrically amplify the circularized DNA, which was hybridized to optically encoded microbeads. The level of fluorescence on each microbead was measured using flow cytometry. The products of the assay, 80MIPA and 80MIPG, were also analyzed by gel electrophoresis to verify the expected size of the amplicon (inset).
enabled the amplicons to be deconvoluted after hybridization to their complementary counterpart on optically encoded microbeads. This enabled the extent of DNA ligation and amplification to be measured using flow cytometry. The fluorescence of each amplicon is depicted in Figure 3. The signal from 80MIPA was 9-fold greater than that of 80MIPG, which confirms the
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Figure 4. Replicate reactions containing 1 pg of synthetic DNA target representing unmethylated cytosine (80TT) at the 80 CpG of the BRCA1 promoter were mixed with different amounts of synthetic target representing the methylated counterpart (80TC). Each reaction also contained two MIPs designed to detect the methylated (80MIPG) and unmethylated cytosine (80MIPA). MIP ligation was performed in 10 μL (2), 20 μL (0), or 30 μL () following which the ligated DNA was amplified and hybridized to microbeads to quantify the extent of amplification. The relative amount of fluorescence from each amplicon (y-axis) was correlated linearly to the relative amount of each target added to a reaction (x-axis). When the relative amount of target added to a reaction became excessive, the linear correlation diminished and the assay was inaccurate. This represents the dynamic range of the assay which varied with the ligation volume (inset).
cytosine at 80 in the BRCA1 promoter of MCF7 cells is unmethylated and demonstrates the specificity of DNA ligation. Quantification of CpG Methylation. Biopsies are often a heterogeneous mixture of tumor and normal cells, and the tumor itself is often present in only trace amounts. Therefore, sensitive analytical techniques capable of detecting methylation of a specific CpG site in a minority of tumor cells are required. Low levels of CpG methylation can be detected using various techniques, some of which are also capable of quantifying CpG methylation.34,35 The assay can detect CpG methylation with a high level of specificity, and since multiple MIPs can be used to interrogate a single methylation site, the assay can also be used to quantify CpG methylation. The accuracy and dynamic range of CpG quantification was investigated by preparing replicate reactions containing two different MIPs (80MIPG and 80MIPA) and different amounts of two synthetic DNA targets. One target (80TT) contained a TpG site representative of unmethylated DNA. The second target (80TC) was identical, except it contained a CpG site representative of methylated DNA. Different ratios of the two targets were prepared, and the extent of ligation and amplification of each MIP was measured. The extent of MIP amplification at each different target concentration is depicted in Figure 4, which demonstrates a linear correlation between the amount of MIP amplification and target concentration (R2 = 0.99). However, when methylated DNA was present in 200-fold excess, amplification did not accurately portray the amount of each target added to the assay (Figure 4). When ligation was performed in a larger volume, the linear correlation between MIP amplification and target concentration was maintained and the sensitivity of the assay increased from 200- to 600-fold (Figure 4). There was a linear correlation between the sensitivity of the assay and the ligation volume (R2 = 0.99, inset Figure 4). In principle, when DNA ligation is 2634
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Figure 5. Four MIPs were designed to interrogate the nucleotide sequence of the BRCA1 promotor at positions 78 and 80. MIPs were designed to detect either methylated (80MIPG) or unmethylated (80MIPA) cytosine at the 80, and MIPs were also designed to detect either cytosine (78MIPG) or thymine (78MIPA) at 78. The MIPs were mixed with 5 ng of bisulphite treated DNA and ligated in a multiplex assay. A fluorescently labeled primer was used to asymmetrically amplify the circularized DNA. The amplicons were hybridized to microbeads and analyzed by flow cytometry. The level of fluorescence on each microbead revealed the extent of ligation of each MIP. The fluorescence values represent the mean ( standard deviation of triplicate assays.
Figure 6. (A) A MIP containing six inosines (iMIPG) was used to detect a synthetic DNA target that contained cytosine in the interrogation site which was flanked with thymines (ThyTC). (B) The assay was replicated using the same MIP with a different synthetic DNA target that contained a cytosine interrogation site flanked by cytosines (CytoTC). (C) The MIP was also used to detect a synthetic target that contained a thymine in the interrogation site flanked by thymines (ThyTT) (D) The assay was performed using a MIP containing no inosines (MIPG), that was complementary to the synthetic DNA target containing a cytosine interrogation site and flanking cytosines (CytoTC). Ligation of iMIPG was performed at 45 °C. The fluorescence values represent the mean ( standard deviation of triplicate assays.
performed in 30 μL, the assay can accurately quantify the methylation status of a specific CpG site in tumor cells among 600-fold excess of normal cells. Resolution of CpG Analysis. A large number of potential CpG methylation biomarkers have been associated with several different cancers. Detecting methylation biomarkers in distinct CpG islands can be achieved using several techniques.12,36 However, monitoring methylation at neighboring CpG sites has the potential to be clinically relevant but remains largely unexplored due to a variety of technical limitations. The assay can be used to quantitatively analyze a single CpG site and may also be useful for analyzing methylation at neighboring CpG sites. This was investigated by interrogating two sites in genomic DNA that are separated by 2 nt. 80MIPA was designed to detect unmethylated cytosine at the 80 CpG of the BRCA1 promoter. Since there is no methylation site adjacent to the 80 CpG, 78MIPA was designed to detect the neighboring thymine at 78 of the BRCA1 promoter. Replicate reactions containing the MIPs and bisulphite treated genomic DNA were ligated and amplified. The unique DNA barcode on each MIP enabled the amplicons to be hybridized to cDNA on encoded microbeads, so the extent of ligation and amplification could be measured. The resultant fluorescence of each MIP is depicted in Figure 5, which demonstrates the signal attributed to unmethylated cytosine at 80 was similar to the 78 thymine. As expected, the signal from the MIPs designed to detect methylated cytosine at 80 was comparable to background levels of fluorescence, as was the mismatch 78MIPG at the 78 interrogation site (Figure 5). This demonstrates the assay can be used on genomic DNA to accurately evaluate nucleotides that are two bases apart, which in principle suggests it can be used to accurately evaluate DNA methylation at adjacent CpG sites.
Analysis of Heterogeneous CpG Methylation. Monitoring the DNA methylation status of individual CpG dinucleotides within a CpG island has the potential to produce clinically relevant biomarkers but remains largely unexplored due to a variety of technical limitations. Nevertheless, this has been addressed using several techniques, with an accuracy that is reliant on a detailed understanding of CpG methylation at neighboring sites.31 As a result, heterogeneous DNA methylation can potentially bias CpG analysis, producing misleading results and discouraging its application as a cancer biomarker.37 In an attempt to overcome some of these limitations, DNA methylation was interrogated using a MIP containing a universal nucleotide positioned at neighboring sites of ambiguous methylation. iMIPG was designed to interrogate a unique CpG methylation site and also contained several inosines in both DNA binding domains. A synthetic DNA target (ThyTC) that contained a C interrogation site flanked by several thymines was added to the MIP, and the extent of ligation and amplification was measured. The resultant fluorescence is depicted in Figure 6, which demonstrates inosine did not retard hybridization, ligation, or amplification of the MIP. When the assay was replicated with a synthetic DNA target that contained a C interrogation site flanked by several cytosines (CytoTC), a larger fluorescent signal was measured. The magnitude of this signal was similar to that obtained when the same target was assayed using MIPG, which did not contain inosine (Figure 6). This suggests inosine can serve as a universal nucleotide when strategically positioned in the DNA binding domain of the MIP. When positioned at a site of ambiguous methylation, it may serve as a universal nucleotide to facilitate the analysis of a specific CpG site in heterogeneously methylated DNA. 2635
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’ DISCUSSION High-throughput DNA sequencing technologies are currently well suited for the discovery of genome wide methylation patterns. However, there is a major requirement for analyzing discrete methylation sites in research and diagnostic applications that involve a large number of patient samples. A variety of techniques can be used for the focused analysis of discrete CpG sites, but many have limited sensitivity and are not quantitative. We have developed and characterized a reproducible and costeffective method for high-resolution analysis of DNA methylation. The method employs MIPs that can be easily tailored to accurately quantify the methylation status of any unique CpG site in bisulfite treated genomic DNA. The assay uses a flow cytometry readout, which enables multiple methylation sites to be rapidly analyzed in a multiplex microbead DNA biosensor assay. Methylated cytosines are readily identified after bisulphite treatment of genomic DNA. This treatment can also fragment DNA, making it difficult to amplify and detect CpG sites directly from the bisulphite treated DNA template.21 This assay uses MIPs to interrogate a CpG site in bisulfite treated DNA. The circularized MIP then serves as the template for efficient PCR amplification, and as a result, this obviates some of the technical limitations of directly amplifying fragmented DNA resulting from bisulfite treatment. Nevertheless, the analysis of bisulfite treated DNA is mandatory in this assay, and highly fragmented DNA is likely to discourage circularization of the MIP, which will retard amplification and limit the accuracy of the assay. However, each MIP contained two 20 nt DNA binding domains which enable the assay to be performed on fragments that are only 40 bases long. In principle, this will enable the assay to accurately measure CpG methylation on fragmented DNA that may originate from bisulphite treatment or archival tissue.21,38,39 This design feature also obviates additional amplification complications that can arise if the PCR primers anneal to DNA containing unidentified or novel CpG sites, which will also retard PCR amplification and confound methylation analysis. “Indirect” amplification of a ligated MIP is likely to be sensitive and more reproducible than “direct” amplification of a fragmented genomic DNA template. As a result, 250 pg of bisulphite treated genomic DNA could be reproducibly amplified to de-tectable levels (CV = 2.9%). However, when excessive amounts of synthetic DNA were used, the accuracy of the assay was limited by the promiscuity of the DNA ligase.40 When a large amount of synthetic DNA target was used in the assay, the ligase covalently coupled the 50 phosphate of a MIP to the 30 hydroxyl, irrespective of the nucleotide at the interrogation site. However, when reduced amounts of synthetic DNA were assayed, the ligase re-tained specificity and only MIPs complementary to the targeted DNA were successfully ligated. The accuracy of the assay was limited by the ligase, which maintained specificity only when the concentration of the synthetic DNA target was below 20 pg/μL. Measuring cytosine methylation in DNA can be problematic, particularly when there are adjacent single nucleotide polymorphisms (SNPs) or neighboring methylation sites. Such heterogeneous methylation may produce an assay bias which can result in an inaccurate measurement of methylation status. To limit this bias, inosine nucleotides were incorporated into the DNA binding domains of a MIP at sites of ambiguious methylation. Insoine serves as a universal nucleotide and can hydrogen bond to each nucleotide in DNA.41,42 Incorporation of inosine
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into the DNA binding domain of the MIP promotes hydrogen bonding to cytosine or uracil with equal affinity in methylated or unmethylated DNA, respectively.43 However, inosine forms a less stable hydrogen bond with thymine, and as a result, ligation of the inosine MIP to a DNA target containing thymine was approximately 20% lower than that of cytosine (Figure 6).41,44 The promiscuity of inosine in hydrogen bond formation may also decrease the specificity of the MIP. However, incorporating six inosines in the DNA binding domains of the MIP and lowering the ligation temperature by 30% resulted in a level of DNA target specificity similar to that obtained from a MIP containing no inosines. This encourages the use of strategically positioned inosines in the DNA binding domain of MIPs and enables accurate detection of CpG methylation among SNPs or regions of heterogeneously methylated DNA. The assay was used to detect and quantify low levels of CpG methylation among unmethylated DNA. In its current form, the assay has the sensitivity to detect CpG methylation in the presence of 600-fold excess of unmethylated DNA. This represents a methylation sensitivity of 0.17%. With this level of sensitivity, it is anticipated that accurate quantification of CpG methylation will be achievable in extracts containing a mixture of normal and tumor cells, such as tissue biopsies and blood samples. This flexible, low cost assay can be easily tailored to quantify DNA methylation at multiple CpG sites in a genome using picogram amounts of bisulphite treated DNA. The flexibility of ligation based assays for high-throughput analysis of DNA methylation has been established.36 The flexibility of this assay extends the analysis to neighboring CpG sites and regions of heterogeneously methylated DNA, which is technically challenging but potentially clinically relevant. Direct amplification of a MIP may also limit the amplification bias which may occur when PCR primers bind directly to heterogeneously methylated sites in bisulfite treated genomic DNA.37 As a result, we anticipate this assay may be used to provide additional information about epiallele methylation patterns that may not be exposed by conventional amplification and sequencing methods. The assay employs a DNA barcode readout that can be utilized in array based applications or for rapid readout using flow cytometry. In its current form, we anticipate this assay will be used for multiplex quantitative analysis of DNA methylation in a research or diagnostic setting.
’ ASSOCIATED CONTENT
bS
Supporting Information. Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION Corresponding Author
*E-mail:
[email protected] (M.T.);
[email protected] (A.R. C.). Phone: þ61 7 3346 4173 (M.T.); þ61 3346 4172 (A.R.C.). Fax: þ61 7 3346 3973 (M.T.; A.R.C.).
’ ACKNOWLEDGMENT R.P. and A.R.C. contributed equally to this work. We gratefully acknowledge funding and support from the NBCF through the National Collaborative Breast Cancer Research Grant Program and are very grateful to Mr. James Bates, Dr. Darby Kozak, 2636
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Analytical Chemistry and Dr. Peter Surawski for preparing optically encoded microbeads.
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