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Multiplex detection of CpG methylation using microarray combining with target-selection-padlock probe
Clinica Chimica Acta 411 (2010) 1187–1194
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Clinica Chimica Acta j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / c l i n c h i m
Multiplex detection of CpG methylation using microarray combining with target-selection-padlock probe Xiaolong Shi a, Chao Tang b, Dequan Zhou c, Hong Zhao a, Zuhong Lu a,⁎ a b c
State Key Laboratory of Bioelectronics, Southeast University, Nanjing 210096, China School of Basic Medical Sciences, Southeast University, Nanjing 210096, China School of Life Sciences, Lanzhou University, Lanzhou 730000, China
a r t i c l e
i n f o
Article history: Received 15 October 2009 Received in revised form 17 March 2010 Accepted 19 March 2010 Available online 27 March 2010 Keywords: DNA methylation Microarray Padlock probe Single base extension (SBE) Polyacrylic acid-covered surface
a b s t r a c t Background: Microarray technology combining with bisulfite-PCR offers a high-throughput approach for detection of DNA methylation. However, the use of microarray-based DNA methylation analysis has been limited by the low throughput of sample preparation due to the difficulty in simultaneous amplification of multiple targets. Methods: A set of target-selection-padlock probes was designed to capture the target sequences containing the queried CpG sites from bisulfite-treated genomic DNA. Then all targets were simultaneously amplified by a pair of common primers. The methylation status of multiple targets was detected by single base extension (SBE) on oligonucleotide microarray based on polyacrylic acid-covered surface. Results: This assay has been successfully applied to analyze promoter methylation of 8 tumor suppressor genes in 12 colorectal cancer samples and 2 normal control samples. The target-selection-padlock probe exhibited both high specificity and high efficiency for the parallel amplification of multiple genes. The accurate and high-throughput detection for DNA methylation was achieved by a combination of targetselection-padlock probes and microarray. Conclusions: The present study provides a robust and accurate assay for DNA methylation status of multiple genes. This method may be useful for a large-scale screen of DNA methylation in cancer cell lines and clinical samples. © 2010 Published by Elsevier B.V.
1. Introduction Detection of aberrant methylation associated with cancer-related genes is a promising approach to assist cancer diagnosis and improve treatment options. Most of methylation assays identify the methylated cytosines in genomic DNA by bisulfite treatment, which effectively deaminates unmethylated cytosine residues into uracils, while the 5-methylcytosines are resistant to this treatment and remain unchanged. The target DNA is then amplified by PCR with specific primers to yield fragments in which all uracil residues are converted to thymine, whereas methylated cytosine residues are amplified as cytosine. A high-throughput assay of DNA methylation has been achieved by combining bisulfite-PCR with microarray technology [1–3]. This approach uses thousands of short oligonucleotides arrayed on glass slides for parallel evaluation of CpG methylation status at numerous CpG sites within multiple genes of interest. In most microarray-based approaches, large numbers of individual
Abbreviations: dNTP, deoxyribonucleoside triphosphate; EDC, 1-ethyl-3(3dimethylaminopropyl)-carbodiimide; MES, 2-(N-morpholino)-ethanesulfonic-acid; SBE, single base extension; SSC, saline–sodium citrate buffer. ⁎ Corresponding author. Tel./fax: +86 25 83793779. E-mail address: [email protected] (Z. Lu). 0009-8981/$ – see front matter © 2010 Published by Elsevier B.V. doi:10.1016/j.cca.2010.03.026
amplification reactions are required to achieve specific and sensitive detection when methylation status of multiple genes from multiple patients is analyzed. This low throughput of sample preparation does not match to the high-throughput capability of DNA microarray and has been a heavy obstruction in the automatization of the assay. Many efforts have been made to develop simultaneous amplification of multiple genes for DNA methylation detection. Cheng et al. [4] developed a multiplex PCR to improve the sample preparation for methylation detection, in which multiple specific primers tagged with a universal sequence were added into one tube to carry out PCR. In their study, five or six promoter regions have been successfully amplified in one PCR. However, because of the high redundancy of the bisulfite-treated genomic DNA, the PCR primer design for multiple PCR is very difficult. Besides, because of the T richness of the target sequences which often results in the mis-priming and non-specific amplification and the preferential amplification of unwanted primer– dimer, it is hard for parallel PCR reactions to exceed 10 amplicons in one PCR. Another approach for parallel amplification of multiple genes is based on oligonucleotide ligation assay (OLA), in which several pairs of oligonucleotide probes are annealed to the target DNA and then ligated into the longer oligonucleotides which are finally amplified with PCR. Only the probe pairs that are perfectly matched with the target DNA can be ligated and amplified. It is an attractive
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approach for a DNA methylation assay because the multiplex amplification of the ligated probes can be easily achieved by PCR using a pair of universal primers [5,6]. In this approach, the detection of methylation status of the queried CpG sites is based on the discriminative properties of the DNA ligase in joining 2 adjacent and perfectly matched DNA strands. However, the mismatch ligation would occur in an oligonucleotide ligation assay, which often reduces the accuracy of methylation detection [7,8]. A circularizing oligonucleotide probe, the so-called padlock probe, is an improved format of OLA which combines highly specific target sequence recognition with the potential for multiplexed analysis of large sets of target sequences [9,10]. Padlock probes have been used to detect gene mutations and single nucleotide polymorphisms by utilizing the ligase discrimination against single base mismatch at the nick. Compared to the ligation of 2 oligonucleotide probes, padlock probe can form a circle after ligation. The circularized DNA is resistant to the exonuclease digestion, while the linear templates and uncircularized padlock probes can be removed. Both the specificity and efficiency of amplification are greatly increased by the exonuclease treatment step. In principle, padlock probe could be used to directly detect the methylation status of CpG sites by utilizing the discriminative properties of DNA ligase. However, in our original experiments, we found that it is difficult to completely eliminate the mismatch ligation which often generates a false positive signal that disturbed the quantification of the methylation level. Recent reports described an extending format of padlock probe for mutation detection or capture of target sequences. These padlock probes hybridize to the target DNA sequences where the 3′ end of the probe is up to 500 bases apart from the 5′ end. The gap region can be filled and amplified for further analysis [11,12]. This approach has been successfully used to capture the target sequences from bisulfitetreated genomic DNA for methylation detection by massively parallel sequencing technology [13,14]. The current technology for massively parallel sequencing typically read the short sequences (25–35 bp) which only contain limited CpG sites. So it can only provide limited information on the methylation status of CpG sites. Additionally, the cost of massively parallel sequencing is expensive, particularly for the case of screening a large amount of samples in medical applications. In the present study, we adopted the target-selection-padlock probe to simultaneously amplify multiple promoter sequences for DNA methylation detection by microarray. To accurately measure the methylation status of queried CpG sites, we performed the single base extension reaction (SBE) on microarray to measure the methylation level of each CpG site [15,16]. Besides, a polyacrylic acid-covered surface was used to fabricate the oligonucleotide microarray, which has high immobilization capacity and hybridization efficiency of DNA [17]. These characters could be useful to improve the sensitivity of detection for the multiple targets amplified in one tube, since a reduction in absolute signal intensity was often observed in multiplex detection. We have used this approach to analyze the promoter methylation status of the tumor suppressor genes DAPK1, APC, MGMT, TIMP3, SHP-1, RASSF1, CDKN2A and ICSBP in 12 colorectal cancer samples and 2 normal control samples.
(2 μg) was sheared to 300–1000 bp by Scientz-D sonifier (Scientz), with a setting at 85% power, sonication 8 times for 10 s, allowing the suspension to cool on ice for 40 s between pulses. After end-repair (Epicentre, Madison, WI), fragmented DNA was then ligated with the adapter pre-annealed from oligonucleotides 5′-GTCGA AGCTG AATGC CATGG GATC-3′ and 5′-GATCC CATGG CATTC AGCT-3′. Ligation was carried out at 20 °C for 1 h in a mixture comprising 2 μg of repaired DNA, 1 μmol/l of annealed linker, 1× ligase buffer and 10 U T4 DNA ligase (Fermentas, Glen Bernie, MD). The ligated DNA fragments were purified using the QIAquick PCR purification kit (Qiagen, Valencia, CA). Then PCR was carried out with 1 μmol/l of primer (5′-GTCGA AGCTG AATGC CATGG GATC-3′), at 72 °C for 10 min, and then at 95 °C for 2 min, 25 cycles of 94 °C for 30 s, 58 °C for 20 s, 72 °C for 45 s. These amplified genomic DNA were used as in vitro-unmethylated DNA. The methylated DNA was generated by in vitro methylation using the CpG Methyltransferase, M. SssI (New England BioLabs, Ipswich, MA). Genomic DNA (2 μg) was incubated in a reaction volume of 50 µl containing 16 U of M. SssI, 3.2 mmol/l of S-adenosyl-L-methionine and 1× NEB buffer 2 for 4 h at 37 °C. 2.3. Sodium bisulfite treatment of genomic DNA Typically, 1 μg of genomic DNA was denatured in 40 μl of 0.2 mol/ l NaOH by incubating for 10 min at 37 °C before addition of 30 μl of freshly prepared 10 mmol/l hydroquinone and 520 μl of 3 mol/ l sodium bisulfite. The reaction was incubated for 20 min at 50 °C, 15 s at 85 °C for 48 cycles (16 h) [4]. DNA was purified using the Wizard DNA-clean up kit (Promega, Madison, WI) following the supplier's instructions. Then 5.7 μl of 3 mol/l NaOH was added and incubated for 15–20 min at 37 °C. DNA was precipitated by adding a mixture of 1 μl glycogen solution (10 mg/ml), 17 μl of ammonium acetate solution and 450 μl of cold absolute ethanol. The mixture was incubated overnight at − 20 °C and then centrifuged at 4 °C for 20 min at maximum speed to pellet the DNA. The DNA pellet was washed once with 500 μl of cold 70% ethanol, then dried at room temperature and finally dissolved in 20 μl of H2O. 2.4. Target capturing by padlock probe Ten microliters of 1× Ampligase buffer containing 1 nmol/l of each padlock probes (listed in Supplementary Table 1) and 8 µl bisulfitemodified DNA (equivalent to 400 ng genomic DNA before bisulfite treatment) were denatured at 95 °C for 5 min, and then hybridized at 52 °C overnight. To perform gap-filling, 1 μl of 10 mmol/l dNTP, 1 μl of Stoffel Fragment DNA polymerase (Applied Biosystems, Foster City, CA) and 2 μl of Ampligase (Epicentre) were added, and the reaction of extension and ligation was performed at 52 °C for 2 h [11]. After circularization, 2 μl of exonuclease mixture (comprising 10 U/ml exonuclease I and 100 U/ml exonuclease III; NEB) was added to digest the linear DNA; the reaction was incubated at 37 °C for 2 h and then 95 °C for 5 min. 2.5. Circle amplification
2. Materials and methods 2.1. Cancer samples and DNA extraction Colorectal cancer samples and whole blood samples of healthy human were obtained from GuLou Hospital (Nanjing, China). The genomic DNA was extracted according to the standard methods using proteinase K digestion and phenol/chloroform extraction.
The circularization products were amplified by PCR reaction with 0.8 μmol/l of each primer (forward: 5′-CGTTTCCCCTGTGTGCCTTG-3′; reverse: 5′-CCATCTGTTGCGTGCGTGTC-3′). The PCR reaction was carried out at 95 °C for 2 min, followed by 38 cycles of 95 °C for 30 s, 58 °C for 20 s, and 72 °C for 45 s. The PCR products were then precipitated by cold ethanol and dried at room temperature. 2.6. Preparation of polyacrylic acid-covered slide
2.2. Unmethylated and methylated templates The unmethylated genomic DNA was generated by linker-PCR amplification from the genomic DNA of healthy human. Genomic DNA
Polyacrylic acid-covered slide was prepared according to a previously published protocol [17]. In brief, aminosilane-derived glass slides were immersed in polyacrylic acid solution at a
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concentration of 5 mg/ml (pH 8.0) for 10 min and then washed intensively with dH2O, and air dried. 2.7. Fabrication of microarray on the polyacrylic acid-covered surface The solution containing 10 μmol/l amino-modified SBE primer (listed in Supplementary Table 2), 10 mmol/l 2-(N-morpholino)ethanesulfonic-acid (MES, pH 5.1) and 20 mmol/l 1-ethyl-3(3dimethylaminopropyl)-carbodiimide (EDC) was printed onto the polyacrylic acid-covered slides by a contact-printing microarrayer (PixSys5500, Cartesian Technology, Irvine CA). After sprinting, the slides were kept in a chamber with 80% relative humidity at room temperature for 2 h. Then the slides were washed and air dried. 2.8. Single base extension (SBE) The precipitated PCR products were dissolved in a solution containing 2× SSC, 0.1% SDS and 50 mmol/l EDTA. Before annealing the PCR products to the oligonucleotide microarray, the solutions were heated at 95 °C for 2 min and then quickly cooled by ice-water bath. Then the solutions were added onto the slides, and the annealing step was performed at 45 °C for 4 h. Finally, the slides were rinsed with dH2O, and air dried. To perform SBE reaction, the reaction mixture containing 4 μmol/l of Cy3-dCTP, 4 μmol/l of Cy5-dUTP, 5 U of Therminator DNA polymerase (New England Biolabs) and 1× thermal DNA polymerase reaction buffer was preheated to 80 °C at thermal cycler and then quickly added to the preheated slides. The reaction was incubated at 60 °C for 4 min, then the slides were washed and air dried. The microarrays were scanned by a scanner (LuxScanner, CapitalBio, China). Cy3 was excited with a 532 nm laser and Cy5 with a 635 nm laser. Data were analyzed with SpotData Pro 2.0 software. 2.9. Methylation-specific PCR (MSP) and bisulfite sequencing Methylation-specific PCR (MSP) was performed using the HotStarTaq DNA polymerase (Tiangene) and methylation-specific primers (listed in Supplementary Table 3). The PCR program consisted of an initial melting step of 5 min at 95 °C, followed by 35 cycles of 30 s at 95 °C, 30 s at the annealing temperature listed in Supplementary Table 3, and 30 s at 68 °C and a final elongation step of 7 min at 68 °C. The PCR mixtures contained 1× HotStar buffer (Tiagene), 0.2 mmol/ l of each dNTP, 0.5 μmol/l of each primer, 1.5 U of HotStarTaq DNA polymerase (Tiagene), and 2 µl bisulfite-modified DNA (equivalent to 100 ng genomic DNA before bisulfite treatment) in 50 µl. PCR was preformed in a PTC-225 thermocycler (MJ Research, Watertown, MA). The PCR products were then analyzed on a 2% agarose gel. The
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promoter region of MGMT was amplified from bisulfite-treated genomic DNA with a pair of primers (forward: 5′-GGATA TGTTG GGATA GTT-3′; reverse: 5′-ACCCA AACAC TCACC AAAT-3′). The product was purified and sequenced with an ABI377A. 3. Results 3.1. The design of target-selection-padlock probe and the SBE primer Each target-selection-padlock probe has a common sequence flanked by 2 bisulfite-specific sequences for the target. The common sequence serve as primer binding sites for PCR amplification of the circularized padlock probes. The 2 bisulfite-specific sequences are designed to anneal to the CpG-less regions of the target sequences, which allow the unbiased capture for both methylated and unmethylated target DNA. The SBE primer is complementary to the bisulfitespecific sequences of the target, with the length of 25–30 bases, and its 3′ end was designed to be immediately adjacent to the queried CpG site. To establish an assay to analyze the promoter methylation status of tumor suppressor genes in colorectal cancer specimens, we designed a set of 8 target-selection-padlock probes specific for the promoter region of the tumor suppressor genes DAPK1, APC, MGMT, TIMP3, SHP-1, RASSF1, ICSBP and CDKN2A (listed in Supplementary Table 1). The promoter CpG islands of these genes have been shown to be frequently hypermethylated in colorectal carcinomas [1,4,18–23]. For each promoter region, three SBE primers were designed to evaluate the methylation status of three CpG sites, respectively (listed in Supplementary Table 2). 3.2. The outline of the assay The outline of the assay is illustrated in Fig. 1. Briefly, a set of target-selection-padlock probes is added to the bisulfite-treated genomic DNA, and these probes hybridize to the target DNA sequences where the 3′ end of the probe is 30–100 bases apart from the 5′ end. These gap regions contain the queried CpG sites. Then the gaps are filled and circularized by DNA polymerase and ligase. The linear genomic DNA and uncircularized padlock probes are removed by exonuclease, while the circularized ones may be amplified by using universal primers. Regions of interest were amplified by PCR converting originally unmethylated CpG dinucleotides to TG and conserving originally methylated CpG sites. The PCR products are then hybridized to the arrayed SBE primers which are designed to be extended by a DNA polymerase with Cy3-dCTP and Cy5-dUTP to detect the methylation status of each CpG site. The extension signal of
Fig. 1. The outline of the multiplex detection for CpG methylation using microarray combining with target-selection-padlock probe. (A) A set of target-selection-padlock probes is added to the bisulfite-treated genomic DNA. Each of these probes is hybridized to the target DNA sequences through the bisulfite-specific sequences on both ends, where the 3′ end of the probe is 30–100 bases apart from the 5′ end. These gap regions contain the queried CpG sites. Then the gaps are filled and circularized by DNA polymerase and ligase. (B) After the linear genomic DNA and uncircularized padlock probes are removed by exonuclease, the circularized ones are amplified by a pair of universal primers. (C) The amplification products are then hybridized to the arrayed SBE primers which are designed to be extended by a DNA polymerase with Cy3-dCTP and Cy5-dUTP to detect the methylation status of each CpG site. The extension signal of Cy3-dCTP and Cy5-dUTP reflects the methylated and unmethylated state of each CpG site, respectively.
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Fig. 2. The specificity and sensitivity of amplification by the padlock probes. (A) Multiplex amplification using the target-selection-padlock probes. Eight target-selection-padlock probes were designed to capture and amplify the corresponding targets from bisulfite-treated, in vitro-unmethylated genomic DNA. The PCR products were analyzed on a 2% agarose gel. The gel was loaded with a 50 bp marker. Lanes 1–8: the bands of PCR products from individual circularization and amplification reaction (lanes 1–8 represents APC, TIMP3, DAPK1, CDKN2A, ICSBP, MGMT, RASSF1, and SHP-1 gene, respectively); lane 9: the bands of PCR products from a multiplexed circularization and amplification reaction in one tube. (B) To estimate the sensitivity of amplification by the padlock probes, various amounts of genomic DNA from a whole blood sample of a healthy human were mixed with 500 ng of T7 phage DNA, treated with sodium bisulfite and used as the template for amplification by padlock probes. A set of 3 padlock probes (APC, CDKN2A, SHP-1) were simultaneously hybridized to the bisulfite-treated genomic DNA, followed by circularization and amplification. The PCR products were then analyzed on a 2% agarose gel.
Cy3-dCTP and Cy5-dUTP reflects the methylated and unmethylated state of each CpG site, respectively.
rise to a smear band of 120–200 bp. These results indicated that the target sequences could be specifically amplified by these targetselection-padlock probes.
3.3. The specificity and sensitivity of amplification by the padlock probes The sensitivity of assay for DNA methylation of multiple targets is influenced by the specificity of target amplification and the sensitivity of microarray. In this study, the target capture with padlock probe was mediated by 2 specific sequences and enzymatic reaction, which could provide a higher specificity of target amplification than that of single probe in principle. To estimate the specificity and sensitivity of amplification by the padlock probes, we firstly tested the specificity of the 8 padlock probes by hybridizing them individually or simultaneously to the bisulfite-treated, in vitrounmethylated DNA, followed by circularization and amplification. As shown in Fig. 2A, each of the eight probes gave rise to the main band with the expected size, and the mix of all eight probes gave
Fig. 3. The promoter region of MGMT was captured from bisulfite-treated in vitrounmethylated DNA and then amplified by a pair of universal primers. The promoter region of MGMT was also amplified from bisulfite-treated in vitro-unmethylated DNA with a pair of bisulfate specific primers (forward: 5′-GGATA TGTTG GGATA GTT-3′; reverse: 5′-ACCCA AACAC TCACC AAAT-3′). Both amplification products were purified and diluted to a concentration of 20 ng/μl. Then 20 μl of each amplification products were hybridized to the microarray respectively to carry out SBE reaction. The signal intensity was the average signal value taken from three times experiments with the same procedure. The error bars indicate the SD.
Fig. 4. (A) Comparison of different DNA polymerases in their ability to incorporate Cy5dUTP and Cy3-dCTP. The PCR product was amplified from bisulfite-treated in vitrounmethylated DNA with MGMT specific primers and then hybridized to the immobilized SBE primer on a polyacrylic acid slide. The SBE reactions were carried out with different DNA polymerases. (B) The effect of the preheating temperature of the reaction mixture on the signal intensity and discrimination of SBE. The reaction mixture containing 4 μM of Cy3-dCTP, 4 μM of Cy5-dUTP, 5 U of Therminator DNA polymerase (NEB) and 1× thermal DNA polymerase reaction buffer were preheated to 80 °C or 45 °C at thermal cycler and then added to the slides for SBE reaction.
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As the amount of DNA from clinical samples is often limited, we next estimated the amount of starting template needed for multiplex amplification by padlock probes. Various amounts of genomic DNA were mixed with 500 ng of T7 phage DNA, treated with sodium bisulfite and then subjected to amplification by padlock probes. A set of three padlock probes (APC, CDKN2A, SHP-1) were simultaneously hybridized to the bisulfite-treated genomic DNA, followed by circularization and amplification. As shown in Fig. 2B, the results were not influenced by the amount of input DNA within the range of 100–500 ng, whereas the APC amplification signals were lost for samples containing 50 ng DNA. These results indicated that the multiple target sequences could be effectively amplified by these target-selection-padlock probes even the amount of starting template is as low as 100 ng. To further address the specificity of padlock probe, the promoter region of MGMT was amplified from bisulfite-treated in vitrounmethylated DNA by both padlock probe and bisulfate specific PCR. Then the amplification products were hybridized to the microarrays for methylation detection by SBE reaction. As shown in Fig. 3, the signal intensities of both amplification methods were almost the same. The high resolution detections were achieved with both the amplification product by bisulfate specific PCR and the amplification product by padlock probe. These results indicated that the amplification by the padlock probes can achieve almost the same specificity as the bisulfate specific PCR, avoiding the difficulty in primer design for multiple PCR and facilitating the simultaneous amplification of multiple genes for DNA methylation detection. 3.4. The specificity and sensitivity of multiplex assay with the microarray combining with the target-selection-padlock probe Another critical factor that influenced the specificity and sensitivity of multiplex assay is the sensitivity of microarray. The sensitivity of microarray is influenced by efficiency of hybridization and extension of SBE on microarray. In this study, we employ a polyacrylic acidcovered slide to fabricate the microarray. The polyacrylic acid-covered slide has a low three-dimension surface and negative charge, which endue the slide with large DNA immobilization capacity, high DNA hybridization efficiency and low non-specific adsorption. These
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Fig. 6. The quantification curves of the assay. MGMT was used as an example to show the assay linearity measured at individual CpG dinucleotides. The in vitro-methylated and unmethylated DNA were mixed in 0%, 25%, 50%, 75%, and 100% ratios and subjected to bisulfite treatment-padlock capture-microarray analysis. Representative microarray images are shown scanned in both Cy3 and Cy5 channels. False color green (Cy3) and red (Cy5) represent the methylated and unmethylated alleles of CpG dinucleotides, respectively. Color composites of the two channels reflect the methylation levels. The plotted value at x-axis represents the fluorescence intensity Cy3/(Cy3 + Cy5) ratio. The value at y-axis represents the percentage of in vitro-methylated genomic DNA mixed with in vitro-unmethylated genomic DNA. The experiments were repeated three times with different sample preparations and array hybridizations. The error bars indicate the SD.
characters are advantageous to improve the specificity and sensitivity of the assay. To obtain the specific and sensitive extension signals, we first optimized the reaction conditions of SBE. In order to choose the optimal DNA polymerase for single base extension reaction,
Fig. 5. The sensitivity of microarray for multiplex detection. The capture products by 8 padlock probes from bisulfite-treated in vitro-unmethylated DNA were simultaneously amplified by a pair of universal primers. The amplification products were then purified and diluted to a concentration of 20 ng/μl or 2 ng/μl. Then 20 μl of amplification products at the concentration of 20 ng/μl or 2 ng/μl were hybridized to the microarray for SBE reaction, respectively. The SBE reaction was performed as described in the Materials and methods section.
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Therminator DNA Polymerase, Taq polymerase, Klenow polymerase (exo-) and Bst large fragment polymerase were compared for the ability to perform sequence-specific incorporation of Cy3-dCTP and Cy5-dUTP. The results showed that Therminator DNA Polymerase had the highest efficiency in those DNA polymerase (see Fig. 4A). Hence,
Therminator DNA Polymerase was adopted for SBE in following experiments. The optimal extension time was determined to be 4 min by testing 3, 4 and 5 min of reaction times. To reduce the negative signal which generates from the extension of self-priming of SBE primer or mismatched primer, the extension reaction mixture was
Fig. 7. (A) Representative microarray analysis of 8 promoter regions in colorectal cancer clinical samples using microarray combining with target selection of padlock probes; each SBE primer was spotted with three reduplications. (B) The arrangement of SBE primers in Fig. 7A. (C) DNA methylation profiles of clinical samples. 12 colorectal cancer samples (T1–T12) and 2 normal samples (N1 and N2) were analyzed. Three CpG sites per promoter region were analyzed in each sample. The color scale represents the percentage of methylation levels determined from the standard curves at each CpG site. The padlock probe and SBE primer sequences used in these experiments are listed in the Supplementary Tables 1 and 2, respectively.
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preheated before adding to the microarray. This step is also critical to increase the extension signal because the temperature can influence the activity of Therminator DNA polymerase intensively (see Fig. 4 B). The signal intensity of SBE is also affected by time of hybridization; usually, hybridization of 4 h could reach a good signal; however, an overnight hybridization gives a stronger signal. To investigate the specificity and sensitivity of the assay on a microarray, a set of padlock probes specific for eight tumor suppressor genes was used to simultaneously amplify the target genes from bisulfite-treated in vitro-unmethylated DNA. The amplification products were diluted to two concentrations and then hybridized to the microarray for SBE. The methylated and unmethylated states of each CpG site were reflected by the extension signal of Cy3-dCTP and Cy5dUTP, respectively. Because the in vitro-unmethylated DNA was used as the analyzed material, almost all of the original C residues should be converted to T residues by bisulfite treatment. As shown in Fig. 5, the strong signals were observed from Cy5-dUTP, while only a faint signal of Cy3-dCTP was observed. When the concentration of amplification products was 20 ng/μl, quantization of the signal intensities gave a relative value of N5400 for the Cy5-dUTP extension, whereas for the Cy3-dCTP extension, only a very thin signal intensity of b110 was observed. When the concentration of amplification products was decreased to 2 ng/μl, the signal intensities from Cy5-dUTP extension were N2400, with an average ratio (Cy5/Cy3) of 34. Although a decrease in signal intensity was observed with the decreasing concentrations of amplification products (from 20 to 2 ng/μl), the reduction in extension signal was relatively smaller than the decrease in the concentration of targets. The data indicated that over 30-folddynamic ranges were achieved by SBE on the microarray even at a relatively low target concentration (2 ng/μl), suggesting the microarray combining with the target-selection-padlock probe was sensitive for the multiplex assay.
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N2), the strong Cy5-dUTP extension signals (the relative value of N11,400) were observed, while only a faint signal of Cy3-dCTP (the relative value of b140) was observed for these promoters. These collective data suggest that our strategy can be feasibly used to detect the methylation of multiplex samples with high sensitivity. DNA methylation profiles of clinical samples were shown in Fig. 7C. Hypermethylation (N40%) were observed in the promoter regions of DAPK1 (2 samples), APC (1 sample), MGMT (4 samples), TIMP3 (3 samples), SHP-1(1 sample), RASSF1 (3 samples) and CDKN2A (3 samples); the low degree of methylation (5%–40%) were observed in DAPK1 (2 samples), APC (1 sample), MGMT (2 samples), TIMP3 (2 samples), SHP-1 (2 samples), RASSF1 (3 samples), and CDKN2A (1 sample). No methylation was observed in ICSBP. 3.7. Verification by methylation-specific PCR (MSP) and bisulfite DNA sequencing To validate the allele-specific SBE findings, methylation-specific PCR (MSP) was conducted in five samples: two colorectal cancer samples T1 and T6, a normal sample N1, in vitro-methylated genomic DNA as a positive control and in vitro-unmethylated genomic DNA as a negative control. The MSP primers for the eight genes were designed to confirm the assays by microarrays (Supplementary Table 3). As shown in Fig. 8A, MSP results of the five samples were completely matched with the microarray results. To further confirm the methylation analysis with microarray, the promoter region of MGMT was amplified with bisulfite-specific primers from bisulfite-treated genomic DNA of clinical sample T1. The PCR product was sequenced by the Sanger method. The results
3.5. Quantification of SBE on the microarray As shown in Fig. 5, a strong signal and high discrimination were obtained by SBE on the microarray based on the polyacrylic acidcovered surface. This means that a widely dynamic range can be achieved by SBE. Next, we evaluated the quantitative capability of the microarray for DNA methylation assay. The in vitro-methylated and unmethylated DNA were mixed in the ratios 1:0, 3:1, 1:1, 1:3 and 0:1, representing methylation statuses of 100%, 75%, 50%, 25% and 0%, respectively. These mixtures were subjected to bisulfite treatment and amplification with padlock probes, followed by SBE detection. The average relative fluorescence intensity representing either methylated (Cy3-dCTP) or unmethylated (Cy5-dUTP) alleles from each spot were used to calculate the methylation ratio of Cy3/(Cy3 + Cy5). Quantization assay and quantification standard curves were showed in Fig. 6. There was a good correlation, with an R2 value of 0.9998. These results indicated that SBE on the polyacrylic acid-covered surface was sensitive and the quantization was proportional to the original DNA. 3.6. Multiplex detection of DNA methylation in colorectal cancer We investigated the promoter methylation status of the tumor suppressor genes DAPK1, APC, MGMT, TIMP3, SHP-1, RASSF1, CDKN2A and ICSBP in 12 colorectal cancer samples (T1–T12) and 2 normal control samples (N1 and N2) using microarray combining with target selection of padlock probes. These genes have demonstrated a high frequency of hypermethylation in cancer cell lines and tissues [1,4,18– 23]. Methylation statuses of these genes were simultaneously detected by SBE in each sample. Three CpG sites per promoter region were evaluated. The results were shown in Fig. 7. Statistical analysis indicated that the signal intensities (Cy3 + Cy5) of all spots were more than 7200, averaging 10,800. In the 2 normal control samples (N1 and
Fig. 8. (A) MSP analysis of the 8 genes in 5 samples: 2 colorectal cancer samples T1 and T6, a normal sample N1, in vitro-methylated genomic DNA as a positive control and in vitro-unmethylated genomic DNA as a negative control. M and U indicate amplification using methylated and unmethylated sequence-specific primers, respectively. Pos, positive control; Neg, negative control; Mr., DNA marker: from bottom to top, the bands are 100, 150, 200, 250 and 300 bp, respectively. (B) Sequencing result of PCR product with an ABI377A. The promoter of MGMT gene from sample T1 was amplified by bisulfite-specific primers and sequenced by the Sanger method. The queried CpG dinucleotides were underlined.
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from the Sanger sequencing indicated that the queried CpG sites and vicinal CpG sites were methylated on the MGMT gene in this sample (shown in Fig. 8B), which were consistent with the results detected by SBE on microarray (shown in Fig. 7).
Acknowledgements
4. Discussion
Appendix A. Supplementary data
In the current microarray-based methylation analysis, the target sequences were amplified from bisulfite-treated genomic DNA and then hybridized to probes immobilized on glass slide. Although these approaches were convenient for the detection of multiple CpG sites in one or several genes, they will be laborious and time-consuming when hundreds of genes in hundreds of different patient samples are analyzed. Additionally, because the bisulfite treatment renders genomic DNA into AT-rich sequences, it is difficult to design the bisulfite-specific primers and amounts of optimization of PCR that had to be done. As the same reason, multiplex PCR with several primer pairs in one tube may present a significant technical challenge. In the present study, we adopted the target-selection-padlock probes to simultaneously amplify multiple target sequences in one PCR, and then the methylation status of targets was detected by SBE on microarray. This strategy could improve the sample preparation for microarray-based DNA methylation detection for multiple genes. This target-selection-padlock probe has several advantages in realizing the parallel amplification of multiple genes for methylation detection. Firstly, the regions of 5′ and 3′ end of the padlock probe are designed to specifically hybridize to the bisulfite-treated target sequences, which provide a higher specificity than only a single sequence. The next enzymatic steps of extension and ligation additionally enhance the specificity of capture. Secondly, unlike the traditional padlock probe which is designed to detect only one site [9,10], the targetselection-padlock probe which leaves a gap of 30–100 bases allows the simultaneous detection of multiple CpG sites. After hybridizing amplified target sequences to the SBE primers immobilized on the slides, the methylation status could be detected by SBE reaction. In the assay with a traditional padlock probe, the detection was achieved through unique address sequences on the probes that can hybridize to universal arrays. Although this approach is convenient in operation, the non-specific ligation and unligated probes may give rise to spurious probe amplicons which would influence the accuracy of assay. In our method, the SBE probes were designed based on the target sequences rather than the sequence of padlock probe, avoiding the generation of background signal from padlock probes. Furthermore, SBE couples target hybridization with enzymatic primer extension reaction, so it could provide a high accuracy and discrimination power for variation of C and T. Our results also demonstrate that the target sequences amplified by these targetselection-padlock probes were successfully detected by SBE on microarray. In conclusion, the present study provides a new strategy to improve the assay for DNA methylation of multiplex genes by utilizing a set of target-selection-padlock probes and a sensitive microarray. It allows a robust and accurate assay for DNA methylation status of multiple genes that should be useful in the large-scale screen of DNA methylation from the cancer cell lines and cancer tissues.
Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.cca.2010.03.026.
This work was funded by the National 863 project of China (grant No. 2006 AA020702).
References [1] Adorján P, Distler J, Lipscher E, et al. Tumour class prediction and discovery by microarray-based DNA methylation analysis. Nucleic Acids Res 2002;30:e21. [2] Mund C, Beier V, Bewerunge P, Dahms M, Lyko F, Hoheisel JD. Array-based analysis of genomic DNA methylation patterns of the tumour suppressor gene p16INK4A promoter in colon carcinoma cell lines. Nucleic Acids Res 2005;33:e73. [3] Gitan RS, Shi H, Chen CM, Yan PS, Huang TH. Methylation-specific oligonucleotide microarray: a new potential for high-throughput methylation analysis. Genome Res 2002;12:158. [4] Cheng YW, Shawber C, Notterman D, Paty P, Barany F. Multiplexed profiling of candidate genes for CpG island methylation status using a flexible PCR/LDR/ Universal Array assay. Genome Res 2006;16:282. [5] Dahl C, Guldberg P. A ligation assay for multiplex analysis of CpG methylation using bisulfite-treated DNA. Nucleic Acids Res 2007;35:e144. [6] Nygren AOH, Ameziane N, Duarte HMB, et al. Methylation-Specific MLPA (MSMLPA): simultaneous detection of CpG methylation and copy number changes of up to 40 sequences. Nucleic Acids Res 2005;33:e128. [7] Bhagwat AS, Sanderson RJ, Lindahl T. Delayed DNA joining at 3′ mismatches by human DNA ligases. Nucleic Acids Res 1999;27:4028. [8] Abravaya K, Carrino JJ, Muldoon S, Lee HH. Detection of point mutations with a modified ligase chain reaction (Gap-LCR). Nucleic Acids Res 1995;23:675. [9] Banér J, Nilsson M, Mendel-Hartvig M, Landegren U. Signal amplification of padlock probes by rolling circle replication. Nucleic Acids Res 1998;26:5073. [10] Nilsson M, Malmgren H, Samiotaki M, Kwiatkowski M, Chowdhary BP, Landegren U. Padlock probes: circularizing oligonucleotides for localized DNA detection. Science 1994;265:2085. [11] Li M, Diehl F, Dressman D, Vogelstein B, Kinzler KW. BEAMing up for detection and quantification of rare sequence variants. Nature Methods 2006;3:95. [12] Krishnakumar S, Zheng J, Wilhelmy J, Faham M, Mindrinos M, Davis R. A comprehensive assay for targeted multiplex amplification of human DNA sequences. Proc Natl Acad Sci 2008;105:9296. [13] Deng J, Shoemaker R, Xie B, et al. Targeted bisulfite sequencing reveals changes in DNA methylation associated with nuclear reprogramming. Nat Biotechnol 2009;27:353. [14] Ball MP, Li JB, Gao Y, et al. Targeted and genome-scale strategies reveal gene-body methylation signatures in human cells. Nat Biotechnol 2009;27:361. [15] Pastinen T, Kurg A, Metspalu A, Peltonen L, Syvänen AC. Minisequencing: a specific tool for DNA analysis and diagnostics on oligonucleotide arrays. Genome Res 1997;7:606. [16] Lindroos K, Sigurdsson S, Johansson K, Rönnblom L, Syvänen AC. Multiplex SNP genotyping in pooled DNA samples by a four-colour microarray system. Nucleic Acids Res 2002;30:e70. [17] Shi X, Tang C, Zhou D, Sun J, Lu Z. PCR-product microarray based on polyacrylic acid-modified surface for SNP genotyping. Electrophoresis 2009;30:1286. [18] Esteller M, Corn PG, Baylin SB, Herman JG. A gene hypermethylation profile of human cancer. Cancer Res 2001;61:3225. [19] Xu XL, Yu J, Zhang HY, et al. Methylation profile of the promoter CpG islands of 31 genes that may contribute to colorectal carcinogenesis. World J Gastroenterol 2004;10:3441. [20] Clark SJ, Harrison J, Paul CL, Frommer M. High sensitivity mapping of methylated cytosines. Nucleic Acids Res 1994;22:2990. [21] Lee S, Kim WH, Jung HY, Yang MH, Kang GH. Aberrant CpG island methylation of multiple genes in intrahepatic cholangiocarcinoma. Am J Pathol 2002;161:1015. [22] Schagdarsurengin U, Wilkens L, Steinemann D. Frequent epigenetic inactivation of the RASSF1A gene in hepatocellular carcinoma. Oncogene 2003;22:1866. [23] Möllemann M, Wolter M, Felsberg J, Collins VP, Reifenberger G. Frequent promoter hypermethylation and low expression of the MGMT gene in oligodendroglial tumors. Intl J Cancer 2005;113:379.
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Clinica Chimica Acta j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / c l i n c h i m
Multiplex detection of CpG methylation using microarray combining with target-selection-padlock probe Xiaolong Shi a, Chao Tang b, Dequan Zhou c, Hong Zhao a, Zuhong Lu a,⁎ a b c
State Key Laboratory of Bioelectronics, Southeast University, Nanjing 210096, China School of Basic Medical Sciences, Southeast University, Nanjing 210096, China School of Life Sciences, Lanzhou University, Lanzhou 730000, China
a r t i c l e
i n f o
Article history: Received 15 October 2009 Received in revised form 17 March 2010 Accepted 19 March 2010 Available online 27 March 2010 Keywords: DNA methylation Microarray Padlock probe Single base extension (SBE) Polyacrylic acid-covered surface
a b s t r a c t Background: Microarray technology combining with bisulfite-PCR offers a high-throughput approach for detection of DNA methylation. However, the use of microarray-based DNA methylation analysis has been limited by the low throughput of sample preparation due to the difficulty in simultaneous amplification of multiple targets. Methods: A set of target-selection-padlock probes was designed to capture the target sequences containing the queried CpG sites from bisulfite-treated genomic DNA. Then all targets were simultaneously amplified by a pair of common primers. The methylation status of multiple targets was detected by single base extension (SBE) on oligonucleotide microarray based on polyacrylic acid-covered surface. Results: This assay has been successfully applied to analyze promoter methylation of 8 tumor suppressor genes in 12 colorectal cancer samples and 2 normal control samples. The target-selection-padlock probe exhibited both high specificity and high efficiency for the parallel amplification of multiple genes. The accurate and high-throughput detection for DNA methylation was achieved by a combination of targetselection-padlock probes and microarray. Conclusions: The present study provides a robust and accurate assay for DNA methylation status of multiple genes. This method may be useful for a large-scale screen of DNA methylation in cancer cell lines and clinical samples. © 2010 Published by Elsevier B.V.
1. Introduction Detection of aberrant methylation associated with cancer-related genes is a promising approach to assist cancer diagnosis and improve treatment options. Most of methylation assays identify the methylated cytosines in genomic DNA by bisulfite treatment, which effectively deaminates unmethylated cytosine residues into uracils, while the 5-methylcytosines are resistant to this treatment and remain unchanged. The target DNA is then amplified by PCR with specific primers to yield fragments in which all uracil residues are converted to thymine, whereas methylated cytosine residues are amplified as cytosine. A high-throughput assay of DNA methylation has been achieved by combining bisulfite-PCR with microarray technology [1–3]. This approach uses thousands of short oligonucleotides arrayed on glass slides for parallel evaluation of CpG methylation status at numerous CpG sites within multiple genes of interest. In most microarray-based approaches, large numbers of individual
Abbreviations: dNTP, deoxyribonucleoside triphosphate; EDC, 1-ethyl-3(3dimethylaminopropyl)-carbodiimide; MES, 2-(N-morpholino)-ethanesulfonic-acid; SBE, single base extension; SSC, saline–sodium citrate buffer. ⁎ Corresponding author. Tel./fax: +86 25 83793779. E-mail address: [email protected] (Z. Lu). 0009-8981/$ – see front matter © 2010 Published by Elsevier B.V. doi:10.1016/j.cca.2010.03.026
amplification reactions are required to achieve specific and sensitive detection when methylation status of multiple genes from multiple patients is analyzed. This low throughput of sample preparation does not match to the high-throughput capability of DNA microarray and has been a heavy obstruction in the automatization of the assay. Many efforts have been made to develop simultaneous amplification of multiple genes for DNA methylation detection. Cheng et al. [4] developed a multiplex PCR to improve the sample preparation for methylation detection, in which multiple specific primers tagged with a universal sequence were added into one tube to carry out PCR. In their study, five or six promoter regions have been successfully amplified in one PCR. However, because of the high redundancy of the bisulfite-treated genomic DNA, the PCR primer design for multiple PCR is very difficult. Besides, because of the T richness of the target sequences which often results in the mis-priming and non-specific amplification and the preferential amplification of unwanted primer– dimer, it is hard for parallel PCR reactions to exceed 10 amplicons in one PCR. Another approach for parallel amplification of multiple genes is based on oligonucleotide ligation assay (OLA), in which several pairs of oligonucleotide probes are annealed to the target DNA and then ligated into the longer oligonucleotides which are finally amplified with PCR. Only the probe pairs that are perfectly matched with the target DNA can be ligated and amplified. It is an attractive
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approach for a DNA methylation assay because the multiplex amplification of the ligated probes can be easily achieved by PCR using a pair of universal primers [5,6]. In this approach, the detection of methylation status of the queried CpG sites is based on the discriminative properties of the DNA ligase in joining 2 adjacent and perfectly matched DNA strands. However, the mismatch ligation would occur in an oligonucleotide ligation assay, which often reduces the accuracy of methylation detection [7,8]. A circularizing oligonucleotide probe, the so-called padlock probe, is an improved format of OLA which combines highly specific target sequence recognition with the potential for multiplexed analysis of large sets of target sequences [9,10]. Padlock probes have been used to detect gene mutations and single nucleotide polymorphisms by utilizing the ligase discrimination against single base mismatch at the nick. Compared to the ligation of 2 oligonucleotide probes, padlock probe can form a circle after ligation. The circularized DNA is resistant to the exonuclease digestion, while the linear templates and uncircularized padlock probes can be removed. Both the specificity and efficiency of amplification are greatly increased by the exonuclease treatment step. In principle, padlock probe could be used to directly detect the methylation status of CpG sites by utilizing the discriminative properties of DNA ligase. However, in our original experiments, we found that it is difficult to completely eliminate the mismatch ligation which often generates a false positive signal that disturbed the quantification of the methylation level. Recent reports described an extending format of padlock probe for mutation detection or capture of target sequences. These padlock probes hybridize to the target DNA sequences where the 3′ end of the probe is up to 500 bases apart from the 5′ end. The gap region can be filled and amplified for further analysis [11,12]. This approach has been successfully used to capture the target sequences from bisulfitetreated genomic DNA for methylation detection by massively parallel sequencing technology [13,14]. The current technology for massively parallel sequencing typically read the short sequences (25–35 bp) which only contain limited CpG sites. So it can only provide limited information on the methylation status of CpG sites. Additionally, the cost of massively parallel sequencing is expensive, particularly for the case of screening a large amount of samples in medical applications. In the present study, we adopted the target-selection-padlock probe to simultaneously amplify multiple promoter sequences for DNA methylation detection by microarray. To accurately measure the methylation status of queried CpG sites, we performed the single base extension reaction (SBE) on microarray to measure the methylation level of each CpG site [15,16]. Besides, a polyacrylic acid-covered surface was used to fabricate the oligonucleotide microarray, which has high immobilization capacity and hybridization efficiency of DNA [17]. These characters could be useful to improve the sensitivity of detection for the multiple targets amplified in one tube, since a reduction in absolute signal intensity was often observed in multiplex detection. We have used this approach to analyze the promoter methylation status of the tumor suppressor genes DAPK1, APC, MGMT, TIMP3, SHP-1, RASSF1, CDKN2A and ICSBP in 12 colorectal cancer samples and 2 normal control samples.
(2 μg) was sheared to 300–1000 bp by Scientz-D sonifier (Scientz), with a setting at 85% power, sonication 8 times for 10 s, allowing the suspension to cool on ice for 40 s between pulses. After end-repair (Epicentre, Madison, WI), fragmented DNA was then ligated with the adapter pre-annealed from oligonucleotides 5′-GTCGA AGCTG AATGC CATGG GATC-3′ and 5′-GATCC CATGG CATTC AGCT-3′. Ligation was carried out at 20 °C for 1 h in a mixture comprising 2 μg of repaired DNA, 1 μmol/l of annealed linker, 1× ligase buffer and 10 U T4 DNA ligase (Fermentas, Glen Bernie, MD). The ligated DNA fragments were purified using the QIAquick PCR purification kit (Qiagen, Valencia, CA). Then PCR was carried out with 1 μmol/l of primer (5′-GTCGA AGCTG AATGC CATGG GATC-3′), at 72 °C for 10 min, and then at 95 °C for 2 min, 25 cycles of 94 °C for 30 s, 58 °C for 20 s, 72 °C for 45 s. These amplified genomic DNA were used as in vitro-unmethylated DNA. The methylated DNA was generated by in vitro methylation using the CpG Methyltransferase, M. SssI (New England BioLabs, Ipswich, MA). Genomic DNA (2 μg) was incubated in a reaction volume of 50 µl containing 16 U of M. SssI, 3.2 mmol/l of S-adenosyl-L-methionine and 1× NEB buffer 2 for 4 h at 37 °C. 2.3. Sodium bisulfite treatment of genomic DNA Typically, 1 μg of genomic DNA was denatured in 40 μl of 0.2 mol/ l NaOH by incubating for 10 min at 37 °C before addition of 30 μl of freshly prepared 10 mmol/l hydroquinone and 520 μl of 3 mol/ l sodium bisulfite. The reaction was incubated for 20 min at 50 °C, 15 s at 85 °C for 48 cycles (16 h) [4]. DNA was purified using the Wizard DNA-clean up kit (Promega, Madison, WI) following the supplier's instructions. Then 5.7 μl of 3 mol/l NaOH was added and incubated for 15–20 min at 37 °C. DNA was precipitated by adding a mixture of 1 μl glycogen solution (10 mg/ml), 17 μl of ammonium acetate solution and 450 μl of cold absolute ethanol. The mixture was incubated overnight at − 20 °C and then centrifuged at 4 °C for 20 min at maximum speed to pellet the DNA. The DNA pellet was washed once with 500 μl of cold 70% ethanol, then dried at room temperature and finally dissolved in 20 μl of H2O. 2.4. Target capturing by padlock probe Ten microliters of 1× Ampligase buffer containing 1 nmol/l of each padlock probes (listed in Supplementary Table 1) and 8 µl bisulfitemodified DNA (equivalent to 400 ng genomic DNA before bisulfite treatment) were denatured at 95 °C for 5 min, and then hybridized at 52 °C overnight. To perform gap-filling, 1 μl of 10 mmol/l dNTP, 1 μl of Stoffel Fragment DNA polymerase (Applied Biosystems, Foster City, CA) and 2 μl of Ampligase (Epicentre) were added, and the reaction of extension and ligation was performed at 52 °C for 2 h [11]. After circularization, 2 μl of exonuclease mixture (comprising 10 U/ml exonuclease I and 100 U/ml exonuclease III; NEB) was added to digest the linear DNA; the reaction was incubated at 37 °C for 2 h and then 95 °C for 5 min. 2.5. Circle amplification
2. Materials and methods 2.1. Cancer samples and DNA extraction Colorectal cancer samples and whole blood samples of healthy human were obtained from GuLou Hospital (Nanjing, China). The genomic DNA was extracted according to the standard methods using proteinase K digestion and phenol/chloroform extraction.
The circularization products were amplified by PCR reaction with 0.8 μmol/l of each primer (forward: 5′-CGTTTCCCCTGTGTGCCTTG-3′; reverse: 5′-CCATCTGTTGCGTGCGTGTC-3′). The PCR reaction was carried out at 95 °C for 2 min, followed by 38 cycles of 95 °C for 30 s, 58 °C for 20 s, and 72 °C for 45 s. The PCR products were then precipitated by cold ethanol and dried at room temperature. 2.6. Preparation of polyacrylic acid-covered slide
2.2. Unmethylated and methylated templates The unmethylated genomic DNA was generated by linker-PCR amplification from the genomic DNA of healthy human. Genomic DNA
Polyacrylic acid-covered slide was prepared according to a previously published protocol [17]. In brief, aminosilane-derived glass slides were immersed in polyacrylic acid solution at a
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concentration of 5 mg/ml (pH 8.0) for 10 min and then washed intensively with dH2O, and air dried. 2.7. Fabrication of microarray on the polyacrylic acid-covered surface The solution containing 10 μmol/l amino-modified SBE primer (listed in Supplementary Table 2), 10 mmol/l 2-(N-morpholino)ethanesulfonic-acid (MES, pH 5.1) and 20 mmol/l 1-ethyl-3(3dimethylaminopropyl)-carbodiimide (EDC) was printed onto the polyacrylic acid-covered slides by a contact-printing microarrayer (PixSys5500, Cartesian Technology, Irvine CA). After sprinting, the slides were kept in a chamber with 80% relative humidity at room temperature for 2 h. Then the slides were washed and air dried. 2.8. Single base extension (SBE) The precipitated PCR products were dissolved in a solution containing 2× SSC, 0.1% SDS and 50 mmol/l EDTA. Before annealing the PCR products to the oligonucleotide microarray, the solutions were heated at 95 °C for 2 min and then quickly cooled by ice-water bath. Then the solutions were added onto the slides, and the annealing step was performed at 45 °C for 4 h. Finally, the slides were rinsed with dH2O, and air dried. To perform SBE reaction, the reaction mixture containing 4 μmol/l of Cy3-dCTP, 4 μmol/l of Cy5-dUTP, 5 U of Therminator DNA polymerase (New England Biolabs) and 1× thermal DNA polymerase reaction buffer was preheated to 80 °C at thermal cycler and then quickly added to the preheated slides. The reaction was incubated at 60 °C for 4 min, then the slides were washed and air dried. The microarrays were scanned by a scanner (LuxScanner, CapitalBio, China). Cy3 was excited with a 532 nm laser and Cy5 with a 635 nm laser. Data were analyzed with SpotData Pro 2.0 software. 2.9. Methylation-specific PCR (MSP) and bisulfite sequencing Methylation-specific PCR (MSP) was performed using the HotStarTaq DNA polymerase (Tiangene) and methylation-specific primers (listed in Supplementary Table 3). The PCR program consisted of an initial melting step of 5 min at 95 °C, followed by 35 cycles of 30 s at 95 °C, 30 s at the annealing temperature listed in Supplementary Table 3, and 30 s at 68 °C and a final elongation step of 7 min at 68 °C. The PCR mixtures contained 1× HotStar buffer (Tiagene), 0.2 mmol/ l of each dNTP, 0.5 μmol/l of each primer, 1.5 U of HotStarTaq DNA polymerase (Tiagene), and 2 µl bisulfite-modified DNA (equivalent to 100 ng genomic DNA before bisulfite treatment) in 50 µl. PCR was preformed in a PTC-225 thermocycler (MJ Research, Watertown, MA). The PCR products were then analyzed on a 2% agarose gel. The
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promoter region of MGMT was amplified from bisulfite-treated genomic DNA with a pair of primers (forward: 5′-GGATA TGTTG GGATA GTT-3′; reverse: 5′-ACCCA AACAC TCACC AAAT-3′). The product was purified and sequenced with an ABI377A. 3. Results 3.1. The design of target-selection-padlock probe and the SBE primer Each target-selection-padlock probe has a common sequence flanked by 2 bisulfite-specific sequences for the target. The common sequence serve as primer binding sites for PCR amplification of the circularized padlock probes. The 2 bisulfite-specific sequences are designed to anneal to the CpG-less regions of the target sequences, which allow the unbiased capture for both methylated and unmethylated target DNA. The SBE primer is complementary to the bisulfitespecific sequences of the target, with the length of 25–30 bases, and its 3′ end was designed to be immediately adjacent to the queried CpG site. To establish an assay to analyze the promoter methylation status of tumor suppressor genes in colorectal cancer specimens, we designed a set of 8 target-selection-padlock probes specific for the promoter region of the tumor suppressor genes DAPK1, APC, MGMT, TIMP3, SHP-1, RASSF1, ICSBP and CDKN2A (listed in Supplementary Table 1). The promoter CpG islands of these genes have been shown to be frequently hypermethylated in colorectal carcinomas [1,4,18–23]. For each promoter region, three SBE primers were designed to evaluate the methylation status of three CpG sites, respectively (listed in Supplementary Table 2). 3.2. The outline of the assay The outline of the assay is illustrated in Fig. 1. Briefly, a set of target-selection-padlock probes is added to the bisulfite-treated genomic DNA, and these probes hybridize to the target DNA sequences where the 3′ end of the probe is 30–100 bases apart from the 5′ end. These gap regions contain the queried CpG sites. Then the gaps are filled and circularized by DNA polymerase and ligase. The linear genomic DNA and uncircularized padlock probes are removed by exonuclease, while the circularized ones may be amplified by using universal primers. Regions of interest were amplified by PCR converting originally unmethylated CpG dinucleotides to TG and conserving originally methylated CpG sites. The PCR products are then hybridized to the arrayed SBE primers which are designed to be extended by a DNA polymerase with Cy3-dCTP and Cy5-dUTP to detect the methylation status of each CpG site. The extension signal of
Fig. 1. The outline of the multiplex detection for CpG methylation using microarray combining with target-selection-padlock probe. (A) A set of target-selection-padlock probes is added to the bisulfite-treated genomic DNA. Each of these probes is hybridized to the target DNA sequences through the bisulfite-specific sequences on both ends, where the 3′ end of the probe is 30–100 bases apart from the 5′ end. These gap regions contain the queried CpG sites. Then the gaps are filled and circularized by DNA polymerase and ligase. (B) After the linear genomic DNA and uncircularized padlock probes are removed by exonuclease, the circularized ones are amplified by a pair of universal primers. (C) The amplification products are then hybridized to the arrayed SBE primers which are designed to be extended by a DNA polymerase with Cy3-dCTP and Cy5-dUTP to detect the methylation status of each CpG site. The extension signal of Cy3-dCTP and Cy5-dUTP reflects the methylated and unmethylated state of each CpG site, respectively.
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Fig. 2. The specificity and sensitivity of amplification by the padlock probes. (A) Multiplex amplification using the target-selection-padlock probes. Eight target-selection-padlock probes were designed to capture and amplify the corresponding targets from bisulfite-treated, in vitro-unmethylated genomic DNA. The PCR products were analyzed on a 2% agarose gel. The gel was loaded with a 50 bp marker. Lanes 1–8: the bands of PCR products from individual circularization and amplification reaction (lanes 1–8 represents APC, TIMP3, DAPK1, CDKN2A, ICSBP, MGMT, RASSF1, and SHP-1 gene, respectively); lane 9: the bands of PCR products from a multiplexed circularization and amplification reaction in one tube. (B) To estimate the sensitivity of amplification by the padlock probes, various amounts of genomic DNA from a whole blood sample of a healthy human were mixed with 500 ng of T7 phage DNA, treated with sodium bisulfite and used as the template for amplification by padlock probes. A set of 3 padlock probes (APC, CDKN2A, SHP-1) were simultaneously hybridized to the bisulfite-treated genomic DNA, followed by circularization and amplification. The PCR products were then analyzed on a 2% agarose gel.
Cy3-dCTP and Cy5-dUTP reflects the methylated and unmethylated state of each CpG site, respectively.
rise to a smear band of 120–200 bp. These results indicated that the target sequences could be specifically amplified by these targetselection-padlock probes.
3.3. The specificity and sensitivity of amplification by the padlock probes The sensitivity of assay for DNA methylation of multiple targets is influenced by the specificity of target amplification and the sensitivity of microarray. In this study, the target capture with padlock probe was mediated by 2 specific sequences and enzymatic reaction, which could provide a higher specificity of target amplification than that of single probe in principle. To estimate the specificity and sensitivity of amplification by the padlock probes, we firstly tested the specificity of the 8 padlock probes by hybridizing them individually or simultaneously to the bisulfite-treated, in vitrounmethylated DNA, followed by circularization and amplification. As shown in Fig. 2A, each of the eight probes gave rise to the main band with the expected size, and the mix of all eight probes gave
Fig. 3. The promoter region of MGMT was captured from bisulfite-treated in vitrounmethylated DNA and then amplified by a pair of universal primers. The promoter region of MGMT was also amplified from bisulfite-treated in vitro-unmethylated DNA with a pair of bisulfate specific primers (forward: 5′-GGATA TGTTG GGATA GTT-3′; reverse: 5′-ACCCA AACAC TCACC AAAT-3′). Both amplification products were purified and diluted to a concentration of 20 ng/μl. Then 20 μl of each amplification products were hybridized to the microarray respectively to carry out SBE reaction. The signal intensity was the average signal value taken from three times experiments with the same procedure. The error bars indicate the SD.
Fig. 4. (A) Comparison of different DNA polymerases in their ability to incorporate Cy5dUTP and Cy3-dCTP. The PCR product was amplified from bisulfite-treated in vitrounmethylated DNA with MGMT specific primers and then hybridized to the immobilized SBE primer on a polyacrylic acid slide. The SBE reactions were carried out with different DNA polymerases. (B) The effect of the preheating temperature of the reaction mixture on the signal intensity and discrimination of SBE. The reaction mixture containing 4 μM of Cy3-dCTP, 4 μM of Cy5-dUTP, 5 U of Therminator DNA polymerase (NEB) and 1× thermal DNA polymerase reaction buffer were preheated to 80 °C or 45 °C at thermal cycler and then added to the slides for SBE reaction.
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As the amount of DNA from clinical samples is often limited, we next estimated the amount of starting template needed for multiplex amplification by padlock probes. Various amounts of genomic DNA were mixed with 500 ng of T7 phage DNA, treated with sodium bisulfite and then subjected to amplification by padlock probes. A set of three padlock probes (APC, CDKN2A, SHP-1) were simultaneously hybridized to the bisulfite-treated genomic DNA, followed by circularization and amplification. As shown in Fig. 2B, the results were not influenced by the amount of input DNA within the range of 100–500 ng, whereas the APC amplification signals were lost for samples containing 50 ng DNA. These results indicated that the multiple target sequences could be effectively amplified by these target-selection-padlock probes even the amount of starting template is as low as 100 ng. To further address the specificity of padlock probe, the promoter region of MGMT was amplified from bisulfite-treated in vitrounmethylated DNA by both padlock probe and bisulfate specific PCR. Then the amplification products were hybridized to the microarrays for methylation detection by SBE reaction. As shown in Fig. 3, the signal intensities of both amplification methods were almost the same. The high resolution detections were achieved with both the amplification product by bisulfate specific PCR and the amplification product by padlock probe. These results indicated that the amplification by the padlock probes can achieve almost the same specificity as the bisulfate specific PCR, avoiding the difficulty in primer design for multiple PCR and facilitating the simultaneous amplification of multiple genes for DNA methylation detection. 3.4. The specificity and sensitivity of multiplex assay with the microarray combining with the target-selection-padlock probe Another critical factor that influenced the specificity and sensitivity of multiplex assay is the sensitivity of microarray. The sensitivity of microarray is influenced by efficiency of hybridization and extension of SBE on microarray. In this study, we employ a polyacrylic acidcovered slide to fabricate the microarray. The polyacrylic acid-covered slide has a low three-dimension surface and negative charge, which endue the slide with large DNA immobilization capacity, high DNA hybridization efficiency and low non-specific adsorption. These
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Fig. 6. The quantification curves of the assay. MGMT was used as an example to show the assay linearity measured at individual CpG dinucleotides. The in vitro-methylated and unmethylated DNA were mixed in 0%, 25%, 50%, 75%, and 100% ratios and subjected to bisulfite treatment-padlock capture-microarray analysis. Representative microarray images are shown scanned in both Cy3 and Cy5 channels. False color green (Cy3) and red (Cy5) represent the methylated and unmethylated alleles of CpG dinucleotides, respectively. Color composites of the two channels reflect the methylation levels. The plotted value at x-axis represents the fluorescence intensity Cy3/(Cy3 + Cy5) ratio. The value at y-axis represents the percentage of in vitro-methylated genomic DNA mixed with in vitro-unmethylated genomic DNA. The experiments were repeated three times with different sample preparations and array hybridizations. The error bars indicate the SD.
characters are advantageous to improve the specificity and sensitivity of the assay. To obtain the specific and sensitive extension signals, we first optimized the reaction conditions of SBE. In order to choose the optimal DNA polymerase for single base extension reaction,
Fig. 5. The sensitivity of microarray for multiplex detection. The capture products by 8 padlock probes from bisulfite-treated in vitro-unmethylated DNA were simultaneously amplified by a pair of universal primers. The amplification products were then purified and diluted to a concentration of 20 ng/μl or 2 ng/μl. Then 20 μl of amplification products at the concentration of 20 ng/μl or 2 ng/μl were hybridized to the microarray for SBE reaction, respectively. The SBE reaction was performed as described in the Materials and methods section.
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Therminator DNA Polymerase, Taq polymerase, Klenow polymerase (exo-) and Bst large fragment polymerase were compared for the ability to perform sequence-specific incorporation of Cy3-dCTP and Cy5-dUTP. The results showed that Therminator DNA Polymerase had the highest efficiency in those DNA polymerase (see Fig. 4A). Hence,
Therminator DNA Polymerase was adopted for SBE in following experiments. The optimal extension time was determined to be 4 min by testing 3, 4 and 5 min of reaction times. To reduce the negative signal which generates from the extension of self-priming of SBE primer or mismatched primer, the extension reaction mixture was
Fig. 7. (A) Representative microarray analysis of 8 promoter regions in colorectal cancer clinical samples using microarray combining with target selection of padlock probes; each SBE primer was spotted with three reduplications. (B) The arrangement of SBE primers in Fig. 7A. (C) DNA methylation profiles of clinical samples. 12 colorectal cancer samples (T1–T12) and 2 normal samples (N1 and N2) were analyzed. Three CpG sites per promoter region were analyzed in each sample. The color scale represents the percentage of methylation levels determined from the standard curves at each CpG site. The padlock probe and SBE primer sequences used in these experiments are listed in the Supplementary Tables 1 and 2, respectively.
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preheated before adding to the microarray. This step is also critical to increase the extension signal because the temperature can influence the activity of Therminator DNA polymerase intensively (see Fig. 4 B). The signal intensity of SBE is also affected by time of hybridization; usually, hybridization of 4 h could reach a good signal; however, an overnight hybridization gives a stronger signal. To investigate the specificity and sensitivity of the assay on a microarray, a set of padlock probes specific for eight tumor suppressor genes was used to simultaneously amplify the target genes from bisulfite-treated in vitro-unmethylated DNA. The amplification products were diluted to two concentrations and then hybridized to the microarray for SBE. The methylated and unmethylated states of each CpG site were reflected by the extension signal of Cy3-dCTP and Cy5dUTP, respectively. Because the in vitro-unmethylated DNA was used as the analyzed material, almost all of the original C residues should be converted to T residues by bisulfite treatment. As shown in Fig. 5, the strong signals were observed from Cy5-dUTP, while only a faint signal of Cy3-dCTP was observed. When the concentration of amplification products was 20 ng/μl, quantization of the signal intensities gave a relative value of N5400 for the Cy5-dUTP extension, whereas for the Cy3-dCTP extension, only a very thin signal intensity of b110 was observed. When the concentration of amplification products was decreased to 2 ng/μl, the signal intensities from Cy5-dUTP extension were N2400, with an average ratio (Cy5/Cy3) of 34. Although a decrease in signal intensity was observed with the decreasing concentrations of amplification products (from 20 to 2 ng/μl), the reduction in extension signal was relatively smaller than the decrease in the concentration of targets. The data indicated that over 30-folddynamic ranges were achieved by SBE on the microarray even at a relatively low target concentration (2 ng/μl), suggesting the microarray combining with the target-selection-padlock probe was sensitive for the multiplex assay.
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N2), the strong Cy5-dUTP extension signals (the relative value of N11,400) were observed, while only a faint signal of Cy3-dCTP (the relative value of b140) was observed for these promoters. These collective data suggest that our strategy can be feasibly used to detect the methylation of multiplex samples with high sensitivity. DNA methylation profiles of clinical samples were shown in Fig. 7C. Hypermethylation (N40%) were observed in the promoter regions of DAPK1 (2 samples), APC (1 sample), MGMT (4 samples), TIMP3 (3 samples), SHP-1(1 sample), RASSF1 (3 samples) and CDKN2A (3 samples); the low degree of methylation (5%–40%) were observed in DAPK1 (2 samples), APC (1 sample), MGMT (2 samples), TIMP3 (2 samples), SHP-1 (2 samples), RASSF1 (3 samples), and CDKN2A (1 sample). No methylation was observed in ICSBP. 3.7. Verification by methylation-specific PCR (MSP) and bisulfite DNA sequencing To validate the allele-specific SBE findings, methylation-specific PCR (MSP) was conducted in five samples: two colorectal cancer samples T1 and T6, a normal sample N1, in vitro-methylated genomic DNA as a positive control and in vitro-unmethylated genomic DNA as a negative control. The MSP primers for the eight genes were designed to confirm the assays by microarrays (Supplementary Table 3). As shown in Fig. 8A, MSP results of the five samples were completely matched with the microarray results. To further confirm the methylation analysis with microarray, the promoter region of MGMT was amplified with bisulfite-specific primers from bisulfite-treated genomic DNA of clinical sample T1. The PCR product was sequenced by the Sanger method. The results
3.5. Quantification of SBE on the microarray As shown in Fig. 5, a strong signal and high discrimination were obtained by SBE on the microarray based on the polyacrylic acidcovered surface. This means that a widely dynamic range can be achieved by SBE. Next, we evaluated the quantitative capability of the microarray for DNA methylation assay. The in vitro-methylated and unmethylated DNA were mixed in the ratios 1:0, 3:1, 1:1, 1:3 and 0:1, representing methylation statuses of 100%, 75%, 50%, 25% and 0%, respectively. These mixtures were subjected to bisulfite treatment and amplification with padlock probes, followed by SBE detection. The average relative fluorescence intensity representing either methylated (Cy3-dCTP) or unmethylated (Cy5-dUTP) alleles from each spot were used to calculate the methylation ratio of Cy3/(Cy3 + Cy5). Quantization assay and quantification standard curves were showed in Fig. 6. There was a good correlation, with an R2 value of 0.9998. These results indicated that SBE on the polyacrylic acid-covered surface was sensitive and the quantization was proportional to the original DNA. 3.6. Multiplex detection of DNA methylation in colorectal cancer We investigated the promoter methylation status of the tumor suppressor genes DAPK1, APC, MGMT, TIMP3, SHP-1, RASSF1, CDKN2A and ICSBP in 12 colorectal cancer samples (T1–T12) and 2 normal control samples (N1 and N2) using microarray combining with target selection of padlock probes. These genes have demonstrated a high frequency of hypermethylation in cancer cell lines and tissues [1,4,18– 23]. Methylation statuses of these genes were simultaneously detected by SBE in each sample. Three CpG sites per promoter region were evaluated. The results were shown in Fig. 7. Statistical analysis indicated that the signal intensities (Cy3 + Cy5) of all spots were more than 7200, averaging 10,800. In the 2 normal control samples (N1 and
Fig. 8. (A) MSP analysis of the 8 genes in 5 samples: 2 colorectal cancer samples T1 and T6, a normal sample N1, in vitro-methylated genomic DNA as a positive control and in vitro-unmethylated genomic DNA as a negative control. M and U indicate amplification using methylated and unmethylated sequence-specific primers, respectively. Pos, positive control; Neg, negative control; Mr., DNA marker: from bottom to top, the bands are 100, 150, 200, 250 and 300 bp, respectively. (B) Sequencing result of PCR product with an ABI377A. The promoter of MGMT gene from sample T1 was amplified by bisulfite-specific primers and sequenced by the Sanger method. The queried CpG dinucleotides were underlined.
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from the Sanger sequencing indicated that the queried CpG sites and vicinal CpG sites were methylated on the MGMT gene in this sample (shown in Fig. 8B), which were consistent with the results detected by SBE on microarray (shown in Fig. 7).
Acknowledgements
4. Discussion
Appendix A. Supplementary data
In the current microarray-based methylation analysis, the target sequences were amplified from bisulfite-treated genomic DNA and then hybridized to probes immobilized on glass slide. Although these approaches were convenient for the detection of multiple CpG sites in one or several genes, they will be laborious and time-consuming when hundreds of genes in hundreds of different patient samples are analyzed. Additionally, because the bisulfite treatment renders genomic DNA into AT-rich sequences, it is difficult to design the bisulfite-specific primers and amounts of optimization of PCR that had to be done. As the same reason, multiplex PCR with several primer pairs in one tube may present a significant technical challenge. In the present study, we adopted the target-selection-padlock probes to simultaneously amplify multiple target sequences in one PCR, and then the methylation status of targets was detected by SBE on microarray. This strategy could improve the sample preparation for microarray-based DNA methylation detection for multiple genes. This target-selection-padlock probe has several advantages in realizing the parallel amplification of multiple genes for methylation detection. Firstly, the regions of 5′ and 3′ end of the padlock probe are designed to specifically hybridize to the bisulfite-treated target sequences, which provide a higher specificity than only a single sequence. The next enzymatic steps of extension and ligation additionally enhance the specificity of capture. Secondly, unlike the traditional padlock probe which is designed to detect only one site [9,10], the targetselection-padlock probe which leaves a gap of 30–100 bases allows the simultaneous detection of multiple CpG sites. After hybridizing amplified target sequences to the SBE primers immobilized on the slides, the methylation status could be detected by SBE reaction. In the assay with a traditional padlock probe, the detection was achieved through unique address sequences on the probes that can hybridize to universal arrays. Although this approach is convenient in operation, the non-specific ligation and unligated probes may give rise to spurious probe amplicons which would influence the accuracy of assay. In our method, the SBE probes were designed based on the target sequences rather than the sequence of padlock probe, avoiding the generation of background signal from padlock probes. Furthermore, SBE couples target hybridization with enzymatic primer extension reaction, so it could provide a high accuracy and discrimination power for variation of C and T. Our results also demonstrate that the target sequences amplified by these targetselection-padlock probes were successfully detected by SBE on microarray. In conclusion, the present study provides a new strategy to improve the assay for DNA methylation of multiplex genes by utilizing a set of target-selection-padlock probes and a sensitive microarray. It allows a robust and accurate assay for DNA methylation status of multiple genes that should be useful in the large-scale screen of DNA methylation from the cancer cell lines and cancer tissues.
Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.cca.2010.03.026.
This work was funded by the National 863 project of China (grant No. 2006 AA020702).
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