Chinese Chemical Letters  2014, Vol.25 Issue (12):1559-1564   PDF    
Detection of DNA methylation by hyperbranched rolling circle amplification and DNA microarray
Hong Zhaoa , Zu-Hong Lub    
aMianyang Normal University, Mianyang 621000, China;
bState Key Laboratory of Bioelectronics, School of Biological Science and Medical Engineering, Southeast University, Nanjing 210096, China
Abstract: Aberrant DNAmethylation of CpG sites has been confirmed to be closely associated with carcinogenesis. Based on the hyperbranched rolling circle amplification (HRCA) and microarray techniques, a new method for qualitative detection of methylation was developed. In the present study, padlock probes hybridize the sample DNA at the methylation site to form a probe-DNA complex which is ligated and digested simultaneously by methylation specific enzymes. Only at the methylated CpG site is the padlock probe ligated successfully to form a circle template for the HRCA reaction. Utilizing the method of 3-dimensional polyacrylamide gel-based microarray, the HRCA product will be immobilized on the slide to form a DNA microarray, which can universally hybridize the Cy3-labeled oligonucleotide probe to detect the methylation status of CpG sites. To control the false positive signals, DNA ligase and temperature of ligation/digestion are optimized. Methylation status of four CpG sites located in P15, Ecadherin, hMLH1 andMGMT genes were analyzed successfully with this method and all the results were compatible with that of methylation-specific PCR. Our research proves that this method is simple and inexpensive, and could be applied as a high-throughput tool to qualitatively determine the methylation status of CpG sites.
Key words: Methylation     Padlock probe     HRCA     Microarray    
1. Introduction

Alterations of DNA methylation patterns have been recognized as a common change in human cancer. Considerable evidences show that the alteration of the methylation status of the CpG site on the tumor suppressor gene may cause silencing of tumor suppressor genes to increase tumor growth [1, 2]. The methylation detection of a selected gene can be helpful in cancer diagnosis [3], and the potential of DNA methylation for early diagnosis,outcome prediction and therapy adjustments is well recognized [4]. Therefore,it is of interest to develop rapid and easy methods to detect such methylation.

With the improvement of analysis technologies,many methods have been developed for the DNA-detection [5, 6, 7, 8],especially for DNA methylation detection. However,many methods for the detection of methylation are based on the conversion of unmethylated cytosine residues into uracil after sodium bisulphite treatment,which are converted thymidine during subsequent PCR [9, 10, 11, 12, 13, 14, 15, 16]. However,for the reasons associated with the bisulphite treatment process,most of these methods are labor intensive. Based on ‘‘simultaneously-ligate-and-digest’’ strategy,Nygren et al. [17] have developed a rapid and easy method to detect changes in methylation status. In their study,a probe hybridized the genome DNA to form a probe-DNA complex which is ligated and digested simultaneously by ligase and methylation-specific enzymes. Through analysis of the PCR product,the methylation status of particular CpG sites can be detected. Rolling circle amplification (RCA) and hyperbranched rolling circle amplification (HRCA) were used for bio-chemical detection [18, 19],especially for detection of SNP (single nucleotide polymorphism) [20, 21, 22, 23, 24]. It is reported that the powerful HRCA is capable of 10 12 fold signal amplification [24, 25, 26]. Li et al.[27] have combined HRCA and 3-dimensional polyacrylamide gelbased microarray (3-D microarray) to develop a method for SNP detection. In their method,the products of HRCA could be immobilized on the slide to fabricate a microarray for SNP detecting. Their method shows high-throughput,high-sensitivity and low sample consumption.

Combining the ‘‘simultaneously-ligate-and-digest’’ strategy and HRCA-3-D microarray,we developed an approach to detect DNA methylation. In our approach,a padlock probe is designed to hybridize the sample DNA on the methylation site. The probe-DNA complex will be ligated and digested simultaneously by methylation specific enzymes. Only for the methylated CpG site,the padlock probe will be circularized to form a circle template for HRCA reaction. The HRCA product will be immobilized on the slide to fabricate a microarray,which can hybridize the fluorescent oligonucleotide probe to assess the methylation status of the CpG site. In the study,four CpG sites located in P15,E-cadherin,hMLH1 and MGMT gene were selected to analyze the methylation of samples and all the results were validated by methylation-specific PCR. The results show that this is both a rapid and easy method with the characteristics of high-throughput and high-sensitivity. 2. Experimental 2.1. Preparation of DNA targets

The samples of tumor tissues were collected from breast tumor patients during surgery at Dezhou Tumor Hospital (Shandong, China). All samples were snap-frozen in liquid nitrogen and stored at -70°C until genomic DNA preparation. Tissue samples (numbered from T1 to T6) were treated with proteinase K with a concentration of 1-2 mg/mL at 50-55°C overnight,or 24 h before the phenol-chloroform procedure. Genomic DNA was extracted by the standard phenol-chloroform procedure,precipitated by ethanol and dissolved in TE buffer. All the sample DNA concentrations were calculated according to their OD260 readings.

Whole blood cells of healthy human were obtained from Zhongda Hospital (Nanjing,China). Genomic DNA was extracted by standard phenol-chloroform procedure. The DNA from blood cells of healthy individuals was divided into two parts. One aliquot was treated with methylase SssI,as a positive control under the conditions recommended by the supplier (New England Biolabs, Ipswitch,Mass). The other aliquot was not treated with methylase SssI and represented the negative control. In this way the positive control had 100% methylated cytosine in the test CpG sites, whereas the negative control had all unmethylated cytosine in the test CpG sites. 2.2. Padlock probe ligation and probe-DNA complex digestion

A sample of 25 ng of genomic DNA and 1 nmol/L of the padlock probe (detailed description of padlock probe shown in Fig. 1 and in Table 1) in 5mL TE buffer [10 mmol/L Tris-HCl (pH 8.5) and 1 mmol/L EDTA] was denatured for 10 min at 95°C,followed by annealing for 30 min at 60°C. After hybridization,the mixture was diluted at room temperature with sterile water and 1.5mL ligase buffer A to 10 mL in tube.Amixture of 0.25mL ligase-65 (MRC-holland),5 U HhaI (Promega) and 1.5mLligasebufferBin total volume of 10mL was added to the tube. Simultaneous ligation and digestion was then performed by incubation for 60 min at 49°C,followed by inactivation of the enzyme at 98°C for 3 min.

Fig. 1. Schematic outlining the strategy of DNA methylation analysis.

Table 1
Sequences of oligonucleotide sequence used in the study. For each padlock probe,the target complementary sequences are in lower case. Italic upper case underlined indicates the segments where the acrylamide-modified universal primer R1 hybridizes. For the reason of acrylamide-modification,HRCA products generated from universal primer R1 can be immobilized on slides to form DNA microarray. The upper case underlined has the same sequence with universal fluorescent probes R3,and the lower case underlined indicates the sequences complementary to the reverse primers R2-x,x=1,2,3or4.
2.3. HRCA reaction and HRCA products arraying

The HRCA reaction was performed in 20mL volume containing 5mL of ligation mixture above,400mmol/L dNTP mix,1mmol/L each of the two primers,8mL of Bst polymerase (New England Biolabs,MA),and 1×modified ThermoPol reaction buffer containing 20 mmol/L Tris-HCl (pH 8.8),10 mmol/L KCl,10 mmol/L (NH4)2SO4 and 0.1% Triton X-100. The reaction mixture was incubated for 3 h at 55°C. Then the HRCA products was purified by dehydrated alcohol for 2 h and diluted with the 10mL ddH2O, preparing for the next step.

Acrylamide-modified glass slides were prepared in-house,then HRCA products would be arrayed and immobilized on slides to form DNA microarray,following the modified method of Li et al. [27] (described in Supporting information). 2.4. Signal detection and methylation-specific PCR

Cy3 labeled probes were suspended in hybridization solution (3:1 v/v; Telechem). Hybridization with fluorescent probes in a moist chamber was performed at 37°C for 2 h. Then the slide was subjected to electrophoresis under 10 V/cm in 1×TBE for 10 min at r.t. to remove non-specifically fluorescent labeled probes and eliminate the background. Then the slide images were captured by a scanner (LuxScan-10 K/A,CapitalBio,Beijing,China) and analyzed with Spot Data Pro 3.0 software. Analyses were conducted using Microsoft Excel.

Bisulfite modification of DNA samples was performed by the modified method of Frommeret al.[9]. The CpG island regions of the target gene were amplified with sequence-specific primers for methylated and unmethylated DNA. The methylation-specific PCR is described in Supporting information. 3. Results and discussion 3.1. Experiment strategy

Fig. 1 outlines our strategy of the DNA methylation analysis. Padlock probe anneal to the target sequence and the 5' and 3' arms are adjacent to each other. This kind of probe-DNA complex can be simultaneously ligated and digested by methylation-specific enzymes (here the recognition site of HhaI is GCGC). If the CpG site is methylated,the padlock probe will be circularized to form a circle template for the HRCA reaction. If CpG site is not methylated, the padlock probe-DNA complex will be digested by the methylation specific enzyme and no circle template is formed. Ligated circles templates are then hybridized with two primers and amplified by the HRCA reaction. For one primer has been modified with acrylamide,the products of HRCA reaction can be immobilized on the slide to form 3-dimensional polyacrylamide gel-based microarray. After immobilization,the slide is processed by electrophoresis in a NaOH solution to obtain single strand DNA. DNA microarray will hybridize with Cy3-labeled universal probe, and only for the methylated sample,the gel spot can produce an intense fluorescent signal on the slide.

We examined the feasibility of our strategy by assessing the methylation status of four CpG sites located in P15,E-cadherin, hMLH1 and MGMT gene (shown in Fig. 2). All padlock probes used in the study include three fragments. The first fragment is a targetcomplementary sequence at both the 5' and 3' end of the padlock probe; the second fragment produces more hybrid sites for the universal fluorescent labeled probe and the third fragment hybridizes the universal acryl-modified primer for all amplification (detailed description is shown in Table 1).

Fig. 2. Target nucleotide sequences used in the study. Four CpG sites located in first exon regions of P15,E-cadherin,hMLH1 and MGMT gene (only part of the four genes sequence shown in the figure). CpG test site 1,2,3 and 4 in four genes are underlined and marked with 1,2,3 and 4 respectively.
3.2. False positive and determination limit

Incomplete methylation-sensitive digestion of the probe-DNA complex may be occurring during the sample preparation and may result in the false-positive detection of methylation after HRCA reaction. Choice of ligase and temperature of ligation-digestion may cause incomplete methylation-sensitive digestion. Here the negative control (unmethylated CpG site) and the positive control (methylated CpG site) are used to analyze the false positive signal. Under normal conditions,the positive control is amplified to produce the positive signal,while the negative control should be digested by HhaI and not be amplified by HRCA. If the negative control is digested incompletely,the HRCA for negative control will take place to produce false positive signal.

Ampligase is widely used in RCA or HRCA to detect SNP. Here, the experimental group containing a positive control and a negative control was used. The quantity of sample DNA in both positive control and negative control is about 25 ng. One group was digested and ligated by HhaI and Ampligase; the other was treated by HhaI and ligase-65. The final product of digestion and ligation was amplified by HRCA reaction. From result shown in the Fig. 3A, it was found that the product of negative control treated by HhaI and Ampligase had been amplified to produce false positive signals. Ampligase is hardly inactive completely at 98°C while the ligase-65 and HhaI is inactive completely at the same temperature. When the negative control (unmethylated) is treated by HhaI and Ampligase,the incompletely inactive Ampligase will religate the digested DNA fragment to form template for HRCA in the subsequent experimental step,which will cause false positive signals.

Fig. 3. False positive and determination limit. (A) HRCA products were determined by 2% agarose gel. 1 is positive control treated by Ampligase; 2 is positivecontrol treated by ligase-65; 3 is negative control treated by Ampligase; 4 is negative control treated by ligase-65. In lane 3 the digested negative control was amplified by HRCA,which is false positive. (B) Changing the reaction temperature of ligation and digestion versus false positive signal intensity. (C) The determination limit was considered with the series changes of the sample concentration.

Six experimental groups were treated by HhaI and ligase-65 at 35,40,45,50,55,60°C,respectively,in which each experimental group contains a positive control and a negative control,with the quantity of DNA in each control at about 25 ng. Based on the protocol described above,all experimental groups were tested by HRCA-3-D microarray. The result shown in the Fig. 3B demonstrated that if the reaction temperature was no more than 50°C, the false positive signals were at an acceptable level. However, when the temperature was too low,the true positive signal intensities became low. According to the result,the optimal temperature range was selected between 45°C and 50°C. The possible explanation for this result is that higher temperatures will inhibit the activity of HhaI which will cause incomplete digestion of probe-DNA complex,and lower temperatures may inhibit the activity of ligase-65,which may cause decrease of the true positive signals.

The determination limit was considered with five experimental groups at different sample concentrations and each group included a positive control (methylated),a negative control (unmethylated) and a blank control in which an equal volume of water displaced the sample DNA. Both positive control and negative control in each group had an equal quantity of sample DNA,which was adjusted from 100 ng to 0.01 ng. According to the protocol described above,five experimental groups were tested by HRCA-3-D microarray. The result,shown in the Fig. 3C,demonstrated significant signal differences between the positive and negative control still existed when the quantity of sample DNA was as low as 0.1 ng,and in each group the negative control and blank control have no obvious signal difference. In order to achieve the best signals, the recommended quantity of target DNA should be more than 1 ng. 3.3. Microarray analysis using clinical sample

MethylationstatusoffourCpGsiteslocatedinfourgenesof six breast tumor tissues were tested by HRCA-3-D microarray. The results (shown in the Fig. 4A) indicate test site 1 in T2 and T6 and test site 2 in T3 are unmethylated,since the fluorescence signal of the sample and that of the negative control are very weak; while test site 1 and site 2 in T1,with strong fluorescence signals,are determined to be methylated. Summaries of the microarray results for CpG sites of six breast tumors are shown in Fig. 4B.

Fig. 4. Methylation analysis of CpG sites by HRCA-3-D microarray and methylation-specific PCR. T1,T2,T3,T4,T5 and T6 represent six tissue samples,respectively. (A) The experimental results on HRCA-3-D microarray for methylation status of for CpG sites in six samples and two controls; (P) positive control; (N) negative control. (B) Summaries of the microarray results are shown for six samples. Gray indicates the CpG site is methylated while white indicates the CpG site is unmethylated. (C) MSP analysis of CpG sites in six samples and two controls. M and U indicate amplification using methylated and unmethylated sequence-specific primers,respectively. (Pos) positive control; (Neg) negative control; (Mr) DNA marker.
3.4. Verification by methylation specific PCR (MSP)

To further validate the microarray analysis findings,MSP was conducted on these samples. A representation of the MSP analysis is shown in Fig. 4C. Six tissue samples were analyzed by MSP and the results of microarray analysis compared with that of MSP. The results of the two methods were fully compatible for the unmethylated site,which are shown at the test site 1 in T2,T3 and T6,or test site 3 in T4 and T6. For the methylated site,the result of MSP shows that the status of the site might be partially methylation or completely methylation. These results indicated this kind of microarray assay could provide effective qualitative detection for some specific CpG sites. 3.5. Discussion

Padlock probe and HRCA have been proved that it can be used in SNP typing by many groups. In this study; we attempted to use this method in DNA methylation analysis. The results showed that this method can be successfully used to test methylated CpG sites within the four tumor-related genes in samples. The derived methylation information for samples was assessed qualitatively and independently validated by classic MSP.

At the present time,most methylation assays are restricted in high-throughput analysis. For early diagnosis,outcome prediction and therapy adjustments,a rapid and easy methylation detection assay with high-throughput and low labor requirements is necessary. Microarray chip technology makes high-throughput analysis possible. Gitan et al. [13] have developed a novel technique called methylation-specific oligonucleotide (MSO) microarray that combines bisulfite DNA assay and oligonucleotides microarray for DNA methylation analysis. The MSO microarray potentially allows rapid screening of CpG sites in many genes. But cross-hybridization between imperfectly matched probes and targets can be observed in this method. On the other hand for each site to be detected,an oligonucleotide (as primer or probe) labeled with fluorescence has to be designed,which is so expensive.

In this study,we demonstrate that HRCA and 3-D microarray methods are simple,low-cost and offer high-throughput. Universal fluorescent probe used in this method means one probe is sufficient for detecting large numbers of multiple CpG sites simultaneously,which will reduce the experimental cost. Furthermore,with the powerful amplification ability of HRCA and high immobilization density of polyacrylamide microarray,this method has low consumption of sample DNA

If the suppression of false-positive signals is carefully and well done in the experiment,our method will be highly sensitive for methylation assay. To achieve this goal,choice of ligase and temperature of ligation/digestion should be optimized. For detecting a large number of samples,when the CpG test sites can be recognized by the methylation-sensitive restriction endonucleases,this method will show obvious advantages of low labor intensity,high-throughput and high sensitivity. 4. Conclusion

Combining the ‘‘simultaneously-ligate-and-digest’’ strategy and HRCA-3-D microarray,a method was developed to detect methylation qualitatively. This rapid and easy method has the advantages of low labor intensity and high-throughput. To the best of our knowledge,the padlock probe in this method is used for the first time in methylation assays. This method may hopefully be used for rapid screening of samples in early diagnosis.


This work was supported by the Scientific Research Foundation of Mianyang Normal University (No. QD2012A10) and Educational Commission of Sichuang Province,China (No. 13ZB0275).

Appendix A. Supplementary data

Supplementary data associated with this article can be found,in the online version,at

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