Highly Selective Detection of 5-Methylcytosine in Genomic DNA

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Highly selective detection of 5-methylcytosine in genomic DNA based on asymmetric PCR and specific DNA damaging reagents Yafen Wang, Chaoxing Liu, Tianlu Wang, Tingting Hong, Haomiao Su, Shuyi Yu, Hongwei Song, Songmei Liu, Xin Zhou, Wuxiang Mao, and Xiang Zhou Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.5b04939 • Publication Date (Web): 09 Feb 2016 Downloaded from http://pubs.acs.org on February 19, 2016

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Analytical Chemistry

Highly selective detection of 5-methylcytosine in genomic DNA based on asymmetric PCR and specific DNA damaging reagents Yafen Wang†, Chaoxing Liu†, Tianlu Wang†, Tingting Hong†, Haomiao Su†, Shuyi Yu†, Hongwei Song†, Songmei Liu⊥, Xin Zhou⊥, Wuxiang Mao*,§ & Xiang Zhou*, † † College of Chemistry and Molecular Sciences, Institute of Advanced Studies, Wuhan University, Wuhan, Hubei, 430072, P. R. China § School of Chemistry and Environmental Engineering, Wuhan Institute of Technology, Wuhan, Hubei, 430073, P. R. China ⊥ Zhongnan Hospital, Wuhan University, Wuhan, Hubei, 430071, P. R. China

Supporting Information Placeholder

ABSTRACT: DNA methylation is a significant epigenetic modification of the genome that is involved in regulating many cellular processes. An increasing number of human diseases have been discovered to be associated with aberrant DNA methylation, and aberrant DNA methylation has been deemed to be a potential biomarker for diseases such as cancers. A safe, nontoxic and sensitive method for accurate detection of 5-methylcytosine in genomic DNA is extremely useful for early diagnosis and therapy of cancers. In this paper, we established a novel system to detect 5-methylcytosine, which is based on bisulfite treatment, asymmetric PCR and specific DNA damaging reagents. Our method could be used for identifying the loci of 5mC in genomic DNA and detecting the DNA methylation levels in tissues as well.

With the discovery of new techniques for whole-genome sequencing, several modification nucleobases have been identified since the first noncanonical nucleobase was discovered several decades ago.1-3 Methylation of the 5-position of cytosine (C) at CpG sites is an essential epigenetic modification, which plays an important role in the regulation of gene,2 genomic imprinting,4 X chromosome inactivation and various diseases, especially in cancers.5-7 Furthermore, in active demethylation process, 5methylcytosine (5mC) could be oxidized to 5hydroxymethylcytosine (5hmC) and then to 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) by TET enzyme.8,9 Hypomethylation in the genome will further disturb the stability of some genes, which will induce overexpression of oncogenes and various diseases,10,11 while hypermethylation within the promoter regions of tumour suppressor genes is associated with inappropriate gene silencing.12-14 Therefore, levels of DNA methylation could be considered as a biomarker of cancers for early diagnosis.12,15 Thus, the detection of 5mC is an urgent and significant work. However, the subtle difference between 5mC and C makes it difficult to distinguish between them. To date, an ample variety of methods to detect 5mC at CpG islands at specific gene sites in tumour suppressor genes have been established. The classic approaches take advantage of bisulfite treatment.16,17 It is known that bisulfite can selectively deaminate cytosine into uracil but has no effect on 5mC. Methylation– specific PCR (MSP) 16 uses specific primers, which is a classic method to discriminate between C and 5mC. Other available methods such as restriction enzymes,18-20 single-molecule techniques,21,22 affinity enrichment,23 capillary electrophoresis (CE),24 nanopore 25 have also been developed to detect the 5mC of

DNA.26 Meanwhile, a host of chemical strategies have emerged, which is able to selectively convert either 5mC or C in DNA. The representative method is chemical modification of Maxam−Gilbert,27 which uses hydrazine to selectively degrade cytosine in DNA sequences. Meanwhile, many other reagents have been found to differentiate between 5mC and C. For instance, the combination of V2O5 and LiBr is a good choice to tackle the challenge of the detection of 5mC.28 There is no doubt that for further application to detection of 5mC in genomic DNA, these methods have their own superiority and disadvantages. Although many chemical methods have already been established, such as OsO4 has different reaction activity between 5mC and C owning to the different nucleophilicities of the double bonds in 5mC and C,29,30 thymidines could be oxidized by OsO4 and the toxicity of OsO4 may induce some problems. Hence, a simple, nontoxic and sensitive method for accurate detection of 5mC in genomic DNA is urgently needed. In our previous work, the N-halogeno-N-sodiobenzenesulfonamide (compound 1) reagent could be synthesized and applied to distinguish indirectly between 5mC and C in DNA sequences,31 but it is limited to the detection of 5mC in DNA sequences directly. Also, our group found that potassium tungstate and hydrogen peroxide (K2WO4/H2O2) can specifically recognize guanines in non-duplex regions of nucleic acids.32 In this paper, we introduced the bisulfite treatment into our previous work to detect 5mC directly. By means of asymmetric PCR and the specific DNA damaging reagents, the method we establish could detect 5mC in genomic DNA.

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EXPERIMENT SECTION Materials. The DNA oligonucleotides with no hexachlorofluorescein (HEX) labelled and the oligomers labelled with HEX were purchased from Sangon Biotechnology Co., Ltd. (Shanghai, China). The final polyacrylamide gel electrophoresis product was scanned with Pharos FX Molecular imager (Bio-Rad, USA) operated in the fluorescence mode. DNA concentration was quantified by NanoDrop 2000c (Thermo Scientific, USA). Potassium Tungstate and Hydrogen Peroxide were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Bisulfite Conversion Kit was purchased from New England BioLabs (Ipswich, MA). Hotstart Taq polymerase was obtained from Takara (Dalian, China). General AllGen Kit was purchased from CWBIO. All water used in this study was ultrapure water (18.2 MΩ/cm). Extracted Genomic DNA from cell. HepG-2 and MCF-7 were cultured in RPMI-1640 supplemented with 10% fetal bovine serum (FBS) and 5% penicillin-streptomycin. Genomic DNA were extracted and purified by CWBIO Genomic DNA kit according to the manufacturer’s instruction. Bisulfite treatment. Bisulfite conversion was performed according to the previous literature.16 5 µL DNA (10 µM), 45 µL ultrapure water and 5.5 µL NaOH (3M) was mixed and incubated at 42 °C for 30 min for DNA denaturation. Then 30 µL freshly prepared hydroquinone (10 mM) was added to obtain a pale yellow solution, subsequent 520 µL freshly prepared sodium bisulfite (3.6 M, pH = 5.0) was added to the reaction tube and up-down reversal gently for mixing. The mixture was covered by 200 µL liquid paraffin before incubated at 50 °C for 16 h. The mixture were desalted by Sep-Pak® Vac C18 1cc and vacuum dried. The final DNA was dissolved in 30 µL ultrapure water. Bisulfite treatment of genomic DNA is treated by bisulfite conversion kit according to the manufacturer’s instruction. Primer design and PCR amplification. In order to achieve a successfully PCR product, no CpG sites exists in the primers for asymmetric PCR. The PCR mixture contain 5 µL reaction buffer (10 ×), 4 µL dNTPs (2.5 mM), 2.5 U Hotstart Taq polymerase, 10 µL HEX labelled forward primer (10 µM), 1 µL reverse primer (1 µM) (for another asymmetric PCR, forward primer: HEXlabelled-reverse primer = 1:100) and bisulfite-treated DNA (50 ng for genomic DNA and 10 ng for 76-mer template DNA) in a final volume of 50 µL. The asymmetric PCR amplification were carried out under the following procedure: 95 °C for 15 min, and then 40 cycles of PCR at 94 °C for 30 s, 48 °C for 30 s and 72 °C for 30 s, followed by final extension for 3 min at 72 °C. After that, prechilled ethanol (1 mL, 100%) and 10 µL CH3COONaCH3COOH buffer (3 M, pH = 5.2) were added into this mixture for precipitation. This mixture was frozen at −80 °C for 2 h and then centrifuged at 4 °C (12,000 g) for 20 min. After removal of supernatant, the DNA precipitate was vacuum dried, then redissolved in ddH2O. Bisulfite-treated ODNs reacted with compound 1. 6 µL bisulfite-treated ODN was added into the mixture of 2 µL Tris-HCl buffer (1 M, pH = 5.0), 2 µL compound 1 (20 mM) and 10 µL acetonitrile. After vortex and centrifugation, the mixture was incubated at 50 °C for 10 min. Then 1 mL prechilled 100% ethanol and 10 µL CH3COONa-CH3COOH buffer (3 M, pH = 5.2) were added into the reaction tube and vortexing. The mixture was frozen at −80 °C for 2 h, and centrifuged at 4 °C (12,000 g) for 20 minutes. After removal of supernatant, the DNA precipitate was dried by Speedvac. Then the DNA was redissolved in 100 µL (10%) piperidine and incubated at 90 °C for 30 min. After the piperidine treatment, the DNA was precipitated again as the step mentioned above and then dried by Speedvac.

PCR product reacts with compound 1 and hot pipedrine. For the reaction with compound 1, 6 µL PCR product was added into the mixture of 2 µL Tris-HCl buffer (1 M, pH = 5.0), 2 µL compound 1 (20 mM) and 10 µL acetonitrile. After vortex and centrifugation, the mixture was incubated at 50 °C for 15 min. Then 1 mL prechilled 100% ethanol and 10 µL CH3COONaCH3COOH buffer (3 M, pH = 5.2) were added into the reaction tube and vortex. The mixture was frozen at −80 °C for 2 h, and centrifuged at 4 °C (12,000 g) for 20 min. After the hot piperidine treatment, the DNA was precipitated as the steps mentioned above and then dried by Speedvac. PCR product reacts with /H2O2 and hot piperdine. For the reaction with K2WO4/H2O2, 10 µL PCR product was added into the mixture of 30 µL Na2HPO4-NaH2PO4 buffer (10 mM, pH = 7.0), 5 µL K2WO4 (100 mM) and 5 µL H2O2 (1 M). After vortex and centrifugation, the mixture was incubated at 37 °C for 2 h. Then 1 mL prechilled 100% ethanol and 10 µL CH3COONaCH3COOH buffer (3 M, pH = 5.2) were added into the reaction tube and mixing. The mixture was frozen at −80 °C for 2 h, and centrifuged at 4 °C (12,000 g) for 20 min. After the hot piperidine treatment, the DNA was precipitated as the procedures mentioned above and then was dried by Speedvac. RESULTS AND DISCUSSION Principle of bisulfite treatment and compound 1 to detect 5mC. The principle we design for detection of 5mC is illustrated in figure 1a. Initially, HEX-labelled DNA is treated by bisulfite

Figure 1. (a) Schematic illustration of detection the loci of 5mC in nucleic acid sequences treated by bisulfite and compound 1. (b) Schematic illustration of detection the loci of 5mC in genomic

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DNA by asymmetric PCR, the product of PCR is treated by compound 1 and hot piperidine. conversion, which could convert unmethylated cytosine to uracil (U) while leaving 5mC intact. Because N-sodio-N-bromo-mnitrobenzenesulfonamide (compound 1) can selectively react with C and 5mC in DNA,31 after bisulfite treatment, only 5mC at the initial sites could react with compound 1, and would be further cleaved after hot piperidine treatment. While, for the C at the initial site, no reaction and cleavage can be observed because compound 1 does not react with U. Thus, the number and loci of 5mC in initial DNA can be observed through the new bands in PAGE.

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detect the loci and number of 5mC in DNA sequence by a convenient and precise way. Principle of asymmetric PCR-based and compound 1 treatment assay. The relationship between tumours and DNA methylation has been attracted tremendous attention by researchers. Encouraged by the successful detection of 5mC in long sequences, we attempt to detect 5mC in genomic DNA. Due to the fact that only 5mC remains to read as C after bisulfite treatment and PCR amplification, 5mC can be distinguished from C through chemical sequencing using our strategy combined with the

Evaluation of the feasibility of bisulfite treatment and compound 1 to detect 5mC assay. Whether compound 1 could react with 5mC but not U is vital to the second step in scheme 1a. So, we firstly chose ODNs that contain both U and 5mC sites in the sequences to verify the feasibility of the strategy described in figure 1a. Only 5mC in the DNAs could be detected in PAGE (Figure 2a , lane 2-4). Furthermore, negligible reaction at the

Figure 2. (a) Polyacrylamide gel electrophoresis analysis of cleaved products of ODN 1-3 after relevant treatments. Lane 1: HEX-4-mer marker; Lane 2-4: ODN 1 to ODN 3 were treated with compound 1 and hot piperdine; Lane 5: HEX-11-mer marker. (b) Polyacrylamide gel electrophoresis analysis of cleaved products of ODN 2’ after relevant treatments. Lane 1: HEX-4-mer marker; Lane 2: ODN 2 was treated with compound 1 and hot piperdine; Lane 3: Bisulfite-treated ODN 2’ was treated with compound 1 and hot piperdine; Lane 4: HEX-20-mer marker. vulnerable U sites of the DNA was observed, which is presumably due to the strongly electron-withdrawing ability of carbonyl group in U. To verify the feasibility of compound 1 in the discrimination of 5mC from C after bisulfite treatment, DNA sequence (ODN 2’) was selected, in which U is completely replaced by C compared with ODN 2. After bisulfite treatment, ODN 2’ was subjected to the same treatment compared with ODN 2 and only the 5mC site can be cleaved after the treatment of bisufite and Compound 1, which coincide with the result of ODN 2 (Figure 2b, lane 2 and lane 3). Thereby, these data manifest that the method could selectively detect the accurate loci and number of 5mC in DNA strand. Inspired by the sensitivity and accuracy of these results, we further apply this method to selectively detect 5mC in short sequences of VHL tumour-suppressor gene with different methylation levels (ODN 4-6). Denaturing PAGE analyses of bisulfite-treated ODN 4-6 containing different amount of 5mC were depicted in figure 3, which clearly indicated the number and accurate loci of the 5mC in the tested DNA sequences without producing any additional cleavage at the sites of C. The sensitivity and accuracy of this method was tested later by applying this method to longer sequence containing seven 5mC in 75 nt DNA (Figure S1). Taken together, this method mentioned above possesses the potential to

Figure 3. Polyacrylamide gelelectrophoresis analysis of cleaved product of ODN 4-6 after relevant treatments. (a) lane 1-2: Maxam−Gilbert A+G and G sequencing lanes for OND 4; Lane 3: Bisulfite-treated ODN 4 is treated with compound 1 and hot piperdine. (b) Lane 1-2: Maxam−Gilbert A+G and G sequencing lanes for OND 5; Lane 3: Bisulfite-treated ODN 5 is treated with compound 1 and hot piperdine. (c) Lane 1-2: Maxam−Gilbert A+G and G sequencing lanes for OND 6; Lane 3: Bisulfite-treated ODN 5 is treated with compound 1 and hot piperdine. asymmetric PCR. The principle is illustrated in figure 1b. Genomic DNA was extracted from cells or tissues, followed by bisulfite treatment, which was used as the templates for the following asymmetric PCR. During PCR amplification, U is replicated as thymine, while 5mC is maintained as cytosine,33 thus asymmetric PCR was introduced to obtain an interested single-stranded DNA (ssDNA). The loci of C in the interested ssDNA of asymmetric PCR product corresponding to 5mC of the targeted genomic DNA could be detected by compound 1 and hot piperdine using the protocol described above. Evaluation of asymmetric PCR-based and compound 1 assay. At first, synthesised DNA which contain different 5mC sites in 76 nt DNA sequences are chosen to test the feasibility of figure 1b. Figure 4a shows the electrophoresis result of ODN 8a which

Figure 4. Polyacrylamide gel electrophoresis analysis of ODN 8a and ODN 8b based on compound 1 and asymmetric PCR. (a) Lane 1: HEX-41-mer marker; Lane 2: The PCR product of ODN 8a without treatment; Lane 3: The PCR product of ODN 8a was treated with compound 1 and hot piperdine. (b) Lane 1: Maxam−Gilbert C sequencing lanes for ODN 9 to indicate the sites of 5mC in ODN 8b; Lane 2: The PCR product of ODN 8b without

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treatment; Lane 3: The PCR product of ODN 8b was treated with compound 1 and hot piperdine. contains one 5mC site in 76 nt DNA sequence. The asymmetric PCR product of ODN 8a was treated with compound 1 and hot piperdine, a new band was observed in PAGE (Figure 4a, lane 3), which was corresponding to site of 5mC in ODN 8a (Figure 4a, HEX-41-mer marker in lane 1). Two 5mC sites in 76 nt DNA sequence was also tested (ODN 8b), the result is described in figure 4b. These results suggested that the strategy could detect the accurate loci and number of 5mC in long sequences by virtue of asymmetric PCR. To investigate the relationship between tumours and transcriptional silencing induced by aberrant hypermethylation, E-cadherin (a tumour suppressor gene) is further characterized in two human cancer cell lines (MCF-7 and HepG2). As mentioned above, the template of PCR is from bisulfitetreated genomic DNA which was extracted form MCF-7 cells or HepG-2 cells. The segment we chose contained four 5mC at CpG sites in 102 bp length was tested, and the electrophoresis result was depicted in figure 5. No new bands was produced

Figure 5. Detection of E-cadherin suppressor gene in the human cancer cell lines (HepG-2 and MCF-7). Lane 1: Maxam−Gilbert C sequencing lanes of ODN 10; Lane 2: The PCR product of the bisulfite-treated genomic DNA extracted from MCF-7 cells; Lane 3: The same as lane 2 but treated with compound 1 and hot piperdine; Lane 4: The PCR product of the bisulfite-treated genomic DNA extracted from HepG-2 cells; Lane 5: The same as lane 4 but treated with compound 1 and hot piperdine, four cleavage products at the 5mC sites were observed. in MCF-7 (Figure 5, lane 3), however, four new bands were appeared in the tested segment of HepG-2 (Figure 5, lane 5). The results coincide with that CpG sites in the E-cadherin promoter are unmethylated in MCF-7 cells,34 whereas CpG sites in the Ecadherin promoter are hypermethylation in HepG-2 cells,33 manifesting the effective strategy for detecting 5mC in genomic DNA. Collectively, the strategy we proposed not only could detect synthesised DNA sequences but also could determine the number and accurate loci of 5mC in genomic DNA. Principle of asymmetric PCR-based and K2WO4/H2O2 treatment assay. Enlightened by the previous method we suggested, another strategy which was illustrated in figure 6 was prepared. Because of that only methylcytosine in the original DNA remains intact after bisulfite treatment and is matched to guanine (G) in the complementary DNA strand, the issue of methylation detection can be transformed into the detection of G in the complementary DNA strand. Thus, we have introduced asymmetric PCR to obtain the corresponding complementary DNA strand, the site of G represents the 5mC in the genomic DNA. Due to K2WO4/H2O2 can specifically recognize guanines in non-duplex regions of nucleic acids,32 recognition of G in the

product could identifiy the number and loci of 5mC in the tested genomic DNA. Evaluation of asymmetric PCR-based and K2WO4/H2O2 treatment assay. At the beginning, synthesised DNA sequences which contain different 5mC sites in 76 nt DNA sequences were chosen to test the feasibility of scheme 2. Figure 7a shows the electrophoresis result of ODN 8c which contains four 5mC sites in 76 nt DNA sequence. Lane 1 is Maxam−Gilbert G sequencing lanes for ODN 11 which could denote the loci of 5mC in ODN 8c. Lane 2 is the PCR product of ODN 8c. Lane 3 is the PCR product of ODN 8c treated by K2WO4/H2O2 and hot piperdine. Lane 4 is the PCR product of ODN 8c treated by dimethyl sulfate (DMS), which is a reagent for Maxam−Gilbert G sequencing. Obviously, four new bands were appeared in PAGE (Figure 7a, lane 3) which is consistent with the sites of 5mC in ODN 8c (Figure 7a, lane 1

Figure 6. Schematic illustration of detection the loci of 5mC in genomic DNA by asymmetric PCR, the product of PCR is treated with K2WO4/H2O2 and hot piperidine.

Figure 7. Polyacrylamide gel electrophoresis analysis of ODN 8c and ODN 8d based on K2WO4/H2O2 and asymmetric PCR. (a) Lane 1: Maxam−Gilbert G sequencing for ODN 11 to indicate the sites of 5mC in ODN 8c; Lane 2: The PCR product of ODN 8c without treatment. Lane 3: The PCR product of ODN 8c was treated with K2WO4/H2O2 and hot piperdine. Lane 4: The PCR product of ODN 8c was treated with DMS under the conditions of Maxam−Gilbert G sequencing lanes. (b) Lane 1: Maxam−Gilbert G sequencing lanes of ODN 12 to indicate the sites of 5mC in ODN 8d; Lane 2: The PCR product of ODN 8d without treatment; Lane 3: The PCR product of ODN 8d was treated with K2WO4/H2O2 and hot piperidine.

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as marker). We also analysed six 5mC sites in 76 nt DNA sequence, the result was represented in figure 7b. Based on these results, we apply this method to genomic DNA. As expected, the strategy could also have excellent result in genomic DNA as shown in figure 8, lane 1 is Maxam−Gilbert G sequencing lanes for ODN 13 which could indicate the loci of 5mC in the analyzed segment; lane 2 and lane 4 are the PCR product from MCF-7 cells and HepG-2 cells respectively; lane 3 and lane 5 are the PCR product from MCF-7 cells and HepG-2 cells respectively and were treated with K2WO4/H2O2 and hot piperdine. Four new bands were appeared in HepG-2 while no new band was produced in MCF-7, which is consistent with previous work described above. From these results, our method for detection of the accurate loci and number of 5mC in genomic DNA has good results.

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CONCLUSIONS We have established a new convenient, non-toxic, low-cost system for the detection of 5mC in genomic DNA. The new system is based on bisulfite-mediated cytosine conversion, asymmetric PCR and specific DNA damaging reagents, which allow us to directly identify the number and accurate loci of the 5mC in genomic DNA. What’s more, this approach can be further applied to tissue analysis through detecting 5-methylcytosine in disease-related genes, which provides a potential tool for further application in future clinical diagnostics.

AUTHOR INFORMATION Corresponding Author *[email protected] *[email protected]

Notes The authors declare no competing financial interests.

ACKNOWLEDGMENT This work was supported by National Basic Research Program of China (973 Program) (2012CB720600, 2012CB720603, 2012CB720605); the National Science Foundation of China (No. 21432008 ， 91413109 ， 21372182).

REFERENCES (1) Robertson, K. D.; Wolffe, A. P. Na.t Rev. Genet. 2000, 1, 11-19.

Figure 8. Detection of E-cadherin suppressor gene in the human cancer cell lines HepG-2 and MCF-7. Lane 1: Maxam−Gilbert C sequencing lanes of ODN 13; Lane 2: The PCR product of the bisulfite-treated genomic DNA extracted from MCF-7 cells; Lane 3: The same as lane 2 but treated with K2WO4/H2O2 and hot piperdine; Lane 4: The PCR product of the bisulfite-treated genomic DNA extracted from HepG-2 cells; Lane 5: The same as lane 4 but treated with K2WO4/H2O2 and hot piperdine. CpG methylation detection in tissues. DNA methylation is a crucial epigenetic modification of the genome which is involved in regulating many cellular processes. A growing number of human diseases have been found to be linked with aberrant DNA methylation. To test the efficiency of the newly developed methods in the analysis of cancer tissues, we extracted DNA from liver tumour tissues and the corresponding precancerous tissues. A further analysis of 5mC expression level was performed. A comparatively higher expression level of 5mC in tumour tissues than precancerous tissues could be observed with the detection systems (Figure S3 and Figure S4). Based on our results, the newly developed platforms could be used for further applications in future clinical diagnostics. The comparision of two different schemes. In this paper, we have introduced two different schemes for the detection of 5mC in DNA sequences. Compared with the two methods we designed, both of them have advantages and disadvantages. The advantages of the first method (Scheme 1) is that the reaction is time saving which cost just several minutes and it can be used to detect 5mC in DNA sequences directly. However, the damaging reagents of N-halogeno-N-sodiobenzenesulfonamide need to be synthesized. Compared to the first method, the second method (Scheme 2) requires longer reaction time which need about 2 hours, but the DNA damaging reagent is readily available.

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Highly Selective Detection of 5-Methylcytosine in Genomic DNA

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