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Detection of DNA methylation of G-quadruplex and i-motif-forming sequences by measuring the initial elongation efficiency of PCR Wataru Yoshida, Hitomi Yoshioka, Daniyah Habiballah Bay, Keisuke Iida, Kazunori Ikebukuro, Kazuo Nagasawa, and Isao Karube Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b00982 • Publication Date (Web): 28 Jun 2016 Downloaded from http://pubs.acs.org on July 3, 2016

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Analytical Chemistry

Detection of DNA methylation of G-quadruplex and i-motif-forming sequences by measuring the initial elongation efficiency of PCR Wataru Yoshida,†,* Hitomi Yoshioka,† Daniyah Habiballah Bay,†,‡ Keisuke Iida,§ Kazunori Ikebukuro,ǁ Kazuo Nagasawa,ǁ and Isao Karube† †

School of Bioscience and Biotechnology, Tokyo University of Technology, 1404-1 Katakuramachi, Hachioji, Tokyo, 1920982, Japan ‡ Biology Department, Umm Al-Qura University, 715, Makkah, 21955, Kingdom of Saudi Arabia § Graduate School of Science and Engineering, Saitama University, c/o Saitama Cancer Center, Ina, Kitaadachi-gun, Saitama, 362-0806, Japan ǁ Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology, 2-24-16 Naka-cho, Koganei, Tokyo, 184-8588, Japan ABSTRACT: DNA methylation has been proposed as one of the promising biomarkers for cancer diagnosis. In this study, we developed a DNA methylation detection system utilizing G-quadruplex and i-motif-forming sequences that requires neither sodium bisulfite treatment nor methylated DNA ligands. We hypothesized that G-quadruplex and i-motif structures would be stabilized by DNA methylation and arrest DNA polymerase activity during quantitative polymerase chain reaction (qPCR). The PCR products from VEGF, RET G-quadruplex, and i-motif-forming sequences were used as templates and analyzed by qPCR. Our results indicated that the initial elongation efficiency of PCR decreased with increasing DNA methylation levels in the G-quadruplex and i-motifforming sequences. Moreover, we demonstrated that the initial elongation efficiency of PCR decreased with increased DNA methylation of the VEGF region on genomic DNA. These results indicated that DNA methylation of the G-quadruplex and i-motifforming sequences on genomic DNA can be detected by qPCR.

DNA methylation is a common epigenetic modification that is important for transcriptional regulation of gene expression.1 In mammals, DNA methylation usually occurs in CpG islands, which are found in 72% of gene promoters.2,3 Hypomethylation is associated with overexpression of oncogenes in cancer cells. Conversely, the hypermethylation of CpG islands results in transcriptional silencing of tumor suppressor genes and ultimately in tumorigenesis.4–6 These characteristic epigenetic changes cast a spotlight on DNA methylation as a potential biomarker for cancer diagnosis.7–9 Sodium bisulfite-based assays are the most widely used methods for detecting DNA methylation. However, these methods require long hours and use harsh conditions such as low pH and high temperature, which lead to loss of some starting material.10–12 Several methods utilizing methylated DNA ligands to detect DNA methylation without sodium bisulfite treatment have been reported; however there are difficulties in preparing the specific ligand molecules and these methods require an additional step to bind the ligands to methylated DNA.13–15 To conveniently detect DNA methylation, a method that utilizes neither sodium bisulfite treatment nor methylated DNA ligands is required. We focused on G-quadruplex (G4) and i-motif structures to detect DNA methylation. G4 is a secondary DNA structure that is composed of two or more stacking G-tetrads. A Gtetrad is a planar array of four guanine bases connected by a Hoogsteen hydrogen bond and stabilized by a monovalent

metal ion.16–19 Several studies have reported that guanine-rich sites located in gene promoter regions and telomeres have the propensity to form G4 structures.17–22 Several small molecules that specifically bind to G4 structures have been developed because the G4 forming region has been recognized as a drug target.23–25 Stabilization of G4 structures formed in promoter regions may play a role in regulating gene expression.18 A recent study reported that a BCL-2 G4 structure was stabilized by methylation of the CpG in the BCL-2 G4 and that stabilization inhibited the extension reaction of DNA polymerase.26 The complementary C-rich sequences of G4 form an i-motif structure: a four-stranded secondary DNA structure composed of two parallel-stranded DNA duplexes that is stabilized by acidic conditions.27,28 A few studies have shown that i-motif structures may regulate transcription in a similar manner to G4. In another study, methylation of the i-motif structures increased the affinity of base-pairing energies and proton,29 suggesting the ability of stabilizing i-motif structures. Therefore, we hypothesized that the initial elongation efficiency of polymerase chain reaction (PCR) would decrease when the target G4 and i-motif regions are methylated; thus, DNA methylation could be detected by quantitative PCR (qPCR). In this study, we aimed to develop a method for detecting DNA methylation using qPCR without either sodium bisulfite treatment or methylated DNA ligands (Figure 1).

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Figure 1. Schematic representation of DNA methylation detection in G4 and i-motif-forming sequences using qPCR.

EXPERIMENTAL SECTION Preparation of methylated PCR products. Human genomic DNA was purified from HUVEC or HeLa cells using DNeasy blood and tissue kit (Qiagen). Genomic DNA (5.0 µg) was used as a template in the amplification of G4 and i-motif regions in VEGF, RET, and c-MYC genes by PCR in 500 µL solution. PCR primers were designed by Primer 3.30 The sequences of the primers are shown in Table S1. PCR was performed using Ex Taq HS (Takara) with a buffer [25 mM TAPS (N-Tris(hydroxymethyl)methyl-3aminopropanesulfonic Acid) (pH 9.3), 2 mM MgCl2, 0.1 mM DTT, 5% DMSO]. The thermocycling conditions were as follows: 95°C for 5 min, followed by 35 cycles of 95°C for 30 s, 59°C for 30 s, and 72°C for 30 s. After purification of the PCR products, 40 U of CpG methyltransferase (NEB) was used to methylate 2 µg of each PCR product, followed by phenol chloroform purification. The concentration of the methylated DNA was measured using a spectrophotometer (BioSpectrometer; Eppendorf). To confirm DNA methylation, 50 ng of methylated DNA was treated with 3 U of the methylation-sensitive restriction enzyme HpaII at 37°C for 1 h and then analyzed by a 15% polyacrylamide gel in 1× TBE buffer. Mutant VEGF and RET DNA were prepared using overlap PCR. In overlap PCR, 3 fmol of two oligonucleotides, VEGF_MT_F and VEGF_MT_R for mutant VEGF, and RET_MT_F and RET_MT_R for mutant RET, were used as template DNAs (Table S1). In mutant VEGF amplification, overlap PCR was performed using Ex Taq HS (Takara) with the supplied buffer and the primers used above. The thermocycling conditions were as follows: 95°C for 5 min, followed by 20 cycles of 95°C for 30 s, 59°C for 30 s, and 72°C for 30 s. In mutant RET amplification, overlap PCR was performed by Ex Taq HS (Takara) with a buffer [25 mM TAPS (pH 9.3), 2 mM MgCl2, 0.1 mM DTT, 5% DMSO]. The thermocycling conditions were as follows: 95°C for 5 min, followed by 25 cycles of 95°C for 30 s, 59°C for 30 s and 72°C for 30 s. The PCR products were methylated as described above. qPCR analysis for PCR products. Unmethylated and methylated PCR products were mixed to prepare template

DNA at a methylation frequency of 0%, 20%, 40%, 60%, 80%, and 100%. The 20 µL of qPCR solution contained 0.5 µM primer pairs, 1.0 × 107 copies of the template DNA (0%, 20%, 40%, 60%, 80%, or 100% methylated DNA), and SYBR Premix Ex Taq II (Tli RNaseH Plus; Takara) was prepared and then performed qPCR using 7900HT fast real-time PCR system (ABI). The thermocycling conditions were as follows: 95°C for 5 min, followed by 40 cycles of 95°C for 30 s, 59°C for 30 s, and 72°C for 30 s. The fluorescence intensity of SYBR green I = 1.0 was used to calculate the Ct value. The mean Ct value, standard deviation (SD), and the increase in value from that observed at 0% methylation DNA were calculated. Methylated and unmethylated DNA were diluted to prepare 1.0 × 104 to 1.0 × 109 copies of the template DNA, and qPCR was performed as described above to analyze the dependency of the template DNA copy number. For titration analysis of the RET G4 and i-motif-forming sequence, RET DNA and mutant RET DNA were mixed to prepare template DNA at G4 and i-motif-forming sequence contents of 0%, 20%, 40%, 60%, 80%, and 100%. Mixtures of the methylated RET DNA and methylated mutant RET DNA were also prepared; qPCR was subsequently performed at 1.0 × 107 copies of the template DNA, as described above. For analysis of the qPCR reaction solution, 20 µL of qPCR solution containing 0.5 U of Ex Taq HS (Takara), 0.5 µM primer pairs, 1 mM dNTPs (250 µM each), 1.0 × 107 copies of the unmethylated or methylated VEGF DNA, and 20,000-fold diluted SYBR green I (Lonza) was prepared in a buffer [25 mM TAPS (pH 9.3), 0.1 mM DTT, and 2 mM MgCl2] and then subjected to qPCR using the 7900HT fast real-time PCR system. The thermocycling conditions were as follows: 95°C for 5 min, followed by 40 cycles of 95°C for 30 s, 59°C for 30 s, and 72°C for 30 s. The fluorescence intensity of SYBR Green I = 0.3 was used to calculate the Ct value. The mean Ct value and standard deviation (SD) were calculated. To analyze the effect of PCR additives, 50 mM KCl; 5% dimethyl sulfoxide (DMSO); 1 M betaine hydrochloride; and 1%, 0.1%, or 0.01% Triton X-100 were added to the qPCR solution. Using as a buffer [25 mM TAPS (pH 9.3), 0.1 mM DTT,


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Figure 2. qPCR analysis of methylated G4 and i-motif-forming sequences using the SYBR premix. qPCR results of VEGF, RET, and cMYC G4 and i-motif DNA (A) and VEGF and RET mutant DNAs (B). The increase in the Ct value from the Ct value of each unmethylated DNA is shown (mean ± sd, N = 4).

2 mM MgCl2, and 0.1% Triton X-100], qPCR was performed with 0%, 20%, 40%, 60%, 80%, and 100% methylated VEGF, RET, and c-MYC DNAs. Methylated and unmethylated DNAs were diluted to prepare 1.0 × 102 to 1.0 × 109 copies of the template DNA, and qPCR was performed to analyze its dependence on the template DNA copy number. After the reaction, the PCR products were analyzed on 2% agarose gel. To analyze the dependence of the reaction on the type of DNA polymerase in the buffer, qPCR was performed with 1.0 × 107 copies of VEGF DNA using 0.5 U of Taq DNA polymerase (NEB), 0.5 U of Blend Taq DNA polymerase (Toyobo), and 0.4 U of KOD-Plus-Neo (Toyobo) instead of Ex Taq HS DNA polymerase. Preparation of methylated genomic DNA. HeLa genomic DNA (100 µg) was methylated by 100 U of CpG methyltransferase, followed by phenol chloroform purification. The concentration of the methylated DNA was measured using a spectrophotometer (BioSpectrometer; Eppendorf). To confirm the DNA methylation level of the VEGF G4 and i-motif-forming region, bisulfite treatment of genomic DNA (1 µg) was performed using the EpiTect Bisulfite Kit (Qiagen); PCR was performed using Ex taq HS (Takara). The PCR products were cloned using the pGEM-T Vector Systems (Promega) and transformed into E. coli DH5α (Toyobo). Individual clones were sequenced using a 3730xl DNA analyzer (Thermo Fisher Scientific). PCR primers are shown in Table S1. qPCR analysis for genomic DNAs. HeLa genomic DNA, methylated HeLa genomic DNA, and a mixture of both were used as the qPCR templates. The 20 µL of qPCR solution containing 0.5 U of Ex Taq HS (Takara), 0.5 µM primer pairs, 1 mM dNTPs (250 µM each), 100 ng (2.8 × 104 copies) of genomic DNA, and 20,000-fold diluted SYBR green I (Lonza) in the buffer [25 mM TAPS (pH 9.3), 0.1 mM DTT, 2 mM MgCl2, 0.1% Triton-X100] was prepared; then, qPCR was performed. The following were the thermocycling conditions: 95°C for 5 min, followed by 40 cycles of 95°C for 30 s, 59°C for 30 s, and 72°C for 30 s. Fluorescence intensity of SYBR Green I = 0.3 was used to calculate the Ct value. The mean and Ct value and standard deviation (SD) were calculated.

RESULTS AND DISCUSSION Preparation of methylated PCR products as template DNA. The G4 and i-motif-forming sequences of VEGF31 and RET32 were used as the test sequences. As a control, we used c-MYC G4 and i-motif-forming sequences33 because they lack a CpG sequence. These G4 and i-motif regions were amplified from human genomic DNA using PCR. The sequences of 192bp VEGF, 188-bp RET, and 191-bp c-MYC PCR products are shown in Figure S1. The PCR products were methylated by CpG methyl transferase. To confirm the DNA methylation state, the PCR products were treated with the methylationsensitive restriction enzyme HapII and analyzed using native polyacrylamide gel electrophoresis (PAGE) (Figure S2). Mutant VEGF and RET DNAs were constructed using overlap PCR. In the mutant DNA, guanine bases, which form the Gtetrads, were substituted with thymine bases, except in CpG sequences (Figure S1). The mutant DNAs were methylated, and the DNA methylation state was confirmed by the methods described above (Figure S2). The unmethylated and methylated DNAs were mixed to prepare 0%, 20%, 40%, 60%, 80%, and 100% methylated DNA samples and were used as template DNA for qPCR. qPCR analysis of methylated G4 and i-motif DNA. In qPCR analysis, 1.0 × 107 copies of template DNA was used in a reaction volume of 20 µL. The SYBR Premix Ex Taq II was utilized as a qPCR reagent. The initial elongation efficiency of PCR was evaluated by measuring increase in the cycle threshold (Ct) values (∆Ct) from the unmethylated DNA template. The ∆Ct values increased (reduced initial elongation efficiency) with increasing methylation levels of G4 and i-motif VEGF and RET DNA templates (Figure 2A, Figure S3A, 3SC). However, the ∆Ct value had no correlation with methylation levels of c-MYC G4 and i-motif DNA templates containing no CpG sequences in the G4 and i-motif-forming sequences (Figure 2A, Figure S3E). These results indicated that the initial elongation efficiency of PCR decreased with increasing DNA methylation levels of VEGF and RET DNAs containing CpG sequences in the G4 and i-motif-forming sequences. Next, mutant VEGF and RET DNAs, which could not form G4 and



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Figure 3. qPCR analysis of 1.0 × 104 to 1.0 × 109 copies of VEGF G4 and i-motif DNA (A) and RET G4 and i-motif DNA (B) using the SYBR premix (mean ± SD, N = 4).

i-motif structures, were tested to determine the initial elongation efficiency of PCR. The methylation level showed no correlation with the ∆Ct value of the mutant DNA templates (Figure 2B, Figure S3B, S3D). Although the VEGF and RET mutant DNAs are GC-rich sequences: the GC content is 66% and 74%, respectively, the initial elongation efficiency of unmethylated and methylated VEGF and RET mutant DNAs did not differ. This result indicated that methylated GC-rich sequences containing no G4 and i-motif-forming sequences did not affect PCR efficiency and that the presence of methylated G4 and i-motif-forming sequences in template DNA reduced the initial elongation efficiency of PCR. Next, titration analysis of the RET G4 and i-motif-forming sequence was performed. RET DNA and mutant RET DNA were mixed to prepare template DNA at G4 and i-motifforming sequence content of 0%, 20%, 40%, 60%, 80%, and 100%. A mixture of the methylated RET DNA and methylated mutant RET DNA was also prepared and then analyzed by qPCR. As a result, the Ct values increased with increasing G4 and i-motif content level, and the inhibitory effect was enhanced by DNA methylation (Figure S4). These results indicated that the G4 structure would form during the PCR and that the DNA methylation of the G4 and i-motif-forming sequence enhanced the reduction of the initial elongation efficiency of PCR. To analyze the dependency of template DNA copy number, qPCR was performed with 1.0 × 104 to 1.0 × 109 copies of the template DNAs. A difference in the amplification efficiency of unmethylated and methylated DNA templates was detected at 1.0 × 106 to 1.0 × 109 copies of VEGF DNA (Figure 3A), and at 1.0 × 105 to 1.0 × 109 copies of RET DNA (Figure 3B). These results indicated that the detection limit of this DNA methylation detection system using the SYBR premix was 1.0 × 106 copies and 1.0 × 105 copies of VEGF and RET DNAs, respectively. In this DNA methylation sensing system, ∆Ct values depend on the increased stability of G4 and i-motif by DNA methylation, while the detection limit depends on the amplification efficiency of unmethylated DNA. Although 1.0 × 105 copies of the unmethylated VEGF DNA were not amplified by PCR, 1.0 × 105 copies of the unmethylated RET DNA were

amplified. Therefore, the detection limit of methylated RET DNA is lower than that of VEGF DNA. Analysis of qPCR reaction solution. SYBR Premix Ex Taq II was used as the qPCR reagent; however, the components of the premix are not known. According to the instructions of Ex Taq HS, 25 mM TAPS (pH 9.3) with 0.1 mM DTT, 2 mM MgCl2, and 50 mM KCl was utilized as the reaction buffer. To analyze the effect of the buffer components on the qPCR, qPCR for VEGF DNA was performed in a buffer containing 0.5 U of Ex Taq HS, 0.5 µM primer pairs, 1 mM dNTPs, and 1.0 × 107 copies of VEGF DNA and SYBR Green I. Under these conditions, the PCR product was not detected. The qPCR was performed without KCl because K+ stabilizes G4 structures; however, the PCR amplification efficiency was lower than that of the SYBR premix (Figure S5A). These results indicated the requirement of PCR additives that destabilize the secondary structure of the template DNA. Therefore, 1 M betaine; 5% DMSO; and 0.01%, 0.1%, or 1% Triton X-100 were added to the reaction buffer without KCl. The addition of 1 M betaine or 0.01% Triton X-100 did not affect the PCR amplification efficiency (Figure S5B, S5C). When 5% DMSO or 1% Triton X-100 was added, the PCR amplification efficiency increased, but the Ct value was not affected by DNA methylation (Figure S5D, S5E). Conversely, the PCR amplification efficiency was improved by the addition of 0.1% Triton X-100, and the Ct values increased with increasing methylation levels of VEGF DNA (Figure 4, Figure S5F, S6). The increase of Ct values with DNA methylation was also detected using RET DNA (Figure 4, Figure S5G, S6). On the other hand, the ∆Ct value had no correlation with the methylation levels of c-MYC DNA (Figure 4, Figure S5H, S6). qPCR was performed with 1.0 × 102 to 1.0 × 109 copies of template DNA to analyze the detection limits for VEGF and RET DNA in the optimized buffer. Differences in the initial elongation efficiencies of unmethylated and methylated DNA templates were detected at 1.0 × 103 to 1.0 × 109 copies of VEGF DNA (Figure 5A, S7) and at 1.0 × 105 to 1.0 × 109 copies of RET DNA (Figure 5B, S7). These results indicated that the detection limit of methylated VEGF DNA was improved using the optimized buffer as compared with the SYBR


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Figure 4. qPCR analysis of VEGF, RET, and c-MYC G4 and imotif DNA using the optimized qPCR solution. The qPCR solution (20 µL) contained 0.5 U of Ex Taq HS, 0.5 µM primer pairs, 1 mM dNTPs, 1.0 × 107 copies of the 0%, 20%, 40%, 60%, 80%, and 100% methylated DNA, 20,000-fold diluted SYBR green I in a buffer [25 mM TAPS (pH 9.3), 0.1 mM DTT, 2 mM MgCl2, 0.1% Triton-X100]. The increase in the Ct value from the Ct value of each unmethylated DNA is shown (mean ± sd, N = 4).

Premix system. To investigate whether other DNA polymerases could be applied to the DNA methylation detection system, Taq DNA polymerase, Blend Taq DNA polymerase, and KOD-Plus-Neo were used instead of Ex Taq HS for qPCR with the optimized buffer. When KOD-Plus-Neo was used, no PCR product was detected. On the other hand, when Taq or Blend Taq DNA polymerase was used, the ∆Ct values increased with increasing methylation levels of VEGF DNA (Figure S8). These results indicated that these DNA polymerases could also be used for the DNA methylation detection system. Detection of DNA methylation of VEGF G4 and i-motifforming region on genomic DNA. qPCR was performed

using HeLa genomic DNA to investigate whether DNA methylation level of the VEGF region on human genomic DNA would be detected by qPCR. First, the methylation levels of the VEGF and c-MYC regions in HeLa genomic DNA were confirmed by bisulfite sequencing. The VEGF and c-MYC regions in HeLa genomic DNA were hypomethylated (Figure S9A, S9B). To prepare genomic DNA with hypermethylated VEGF and c-MYC regions, HeLa genomic DNA was methylated with CpG methyltransferase. Using the methylated HeLa genomic DNA, hypermethylation of the regions was confirmed (Figure S9C, S9D). In the VEGF G4 and i-motif-forming sequence, the methylation level was 85%. The methylated HeLa genomic DNA and HeLa genomic DNA were mixed in equal proportion to prepare genomic DNA of which the methylation level at the VEGF G4 and i-motif-forming region was 42.5%. For qPCR analysis, 100 ng (2.8 × 104 copies) of genomic DNA was used as the template. As a control, c-MYC G4 and i-motifforming region were used because the amplification efficiency of the c-MYC region was not dependent on DNA methylation. In the qPCR analysis for genomic DNA, the qPCR was performed in the optimized buffer [25 mM TAPS (pH 9.3), 0.1 mM DTT, 2 mM MgCl2, 0.1% Triton-X100]. As expected, when the c-MYC region was amplified, the Ct values were not changed (Figure 6, Figure S10A). Conversely, when the VEGF region was amplified, the Ct values increased with increasing methylation levels of the VEGF G4 and i-motif region (Figure 6, Figure S10B). These results confirmed the detection of methylation levels in VEGF G4 and i-motif-forming-region in genomic DNA. After qPCR, the PCR products were analyzed on agarose gel. Although the VEGF product of the correct size was detected, non-specific products were also detected (Figure S11), suggesting that the sensitivity would be improved using a fluorescently labeled VEGF-specific probe instead of SYBR Green I. Moreover, there are several PCR methods where the reaction volume is nL scale, such as the MicroTAS-based PCR system.34 By combining these small-scale PCR systems with the system described here, DNA methylation may be detected in fewer copies of genomic DNA with qPCR.

Figure 5. qPCR analysis of 1.0 × 102 to 1.0 × 109 copies of VEGF G4 and i-motif DNA (A) and RET G4 and i-motif DNA (B) using the optimized qPCR solution (mean ± SD, N = 4).



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DNA methylation on the initial elongation efficiency of PCR. Our results indicated a positive correlation between ∆Ct values and DNA methylation levels in VEGF and RET DNAs. Moreover, we demonstrated that the initial elongation efficiency of PCR decreased with increasing DNA methylation levels of the VEGF region on genomic DNA. These results indicated that DNA methylation of the G4 and i-motif-forming sequences on genomic DNA can be detected by qPCR.

ASSOCIATED CONTENT Supporting Information

The Supporting Information is available free of charge on the ACS Publications website.

Figure 6. Detection of the DNA methylation of VEGF G4 and imotif-forming region on genomic DNA by qPCR. (1) HeLa genomic DNA, (2) mixture of HeLa genomic DNA and methylated HeLa genomic DNA, and (3) methylated HeLa genomic DNA, were used as template. The qPCR solution (20 µL) contained 0.5 U of Ex Taq HS, 0.5 µM primer pairs, 1 mM dNTPs, 2.8× 104 copies of genomic DNA, 20,000 fold diluted SYBR green I in a buffer [25 mM TAPS (pH 9.3), 0.1 mM DTT, 2 mM MgCl2, 0.1% Triton-X100] (mean ± sd, N = 3).

Supporting Information including DNA sequences used in this study, sequence of PCR products, results of DNA methylation analysis by HapII, representative data of SYBR green fluorescence intensity on qPCR, titration analysis of the RET G4 and imotif-forming sequence, agarose gel analysis of qPCR products, qPCR analysis with Taq and Blend Taq DNA polymerases, bisulfite sequencing analysis of the VEGF and c-MYC G4, and i-motifforming regions on HeLa genomic DNAs. (Word)

AUTHOR INFORMATION Corresponding Author

*E-mail: [email protected]

The RET region was not amplified from HeLa genomic DNA by qPCR with the optimized buffer. On the other hand, the region could be amplified from HeLa genomic DNA by PCR with a buffer containing 25 mM TAPS (pH 9.3), 2 mM MgCl2, 0.1 mM DTT, and 5% DMSO, suggesting that optimization of qPCR buffer conditions would allow the detection of DNA methylation levels of the RET region on genomic DNA. A genome-wide DNA methylation analysis has shown that methylation typically occurs in CpG islands35 which are highly concentrated in promoter regions. In a previous study, G4forming sequences were identified in CpG islands using DNA microarray with a G4 ligand.36 Genome-wide analysis revealed that the human genome contains 716,310 G4 forming sequences.37 These results suggest that methylated G4 and i-motif structures might exist in various promoter regions that would be applicable to many assays. Therefore, we hypothesized that DNA methylation on various promoter regions could be detected by qPCR. Recently, Aschenbrenner et al. reported that DNA methylation could be detected by measuring PCR amplification efficiency by means of a 3’-mismatched primer with mutant DNA polymerase.38 In this method, a 3’-mismatched primer was extended more efficiently from a methylated cytosine than from an unmethylated cytosine on the template DNA, thereby suggesting that the initial elongation efficiency of PCR increased when the template DNA was methylated. In the DNA methylation detection system presented here, the initial elongation efficiency of PCR decreased with increasing template DNA methylation. Therefore, a combination of the DNA methylation detection system and 3’-mismatched primers with mutant DNA polymerase may provide accurate detection system.

CONCLUSIONS In this study, we used G4 and i-motif-forming sequences in VEGF, and RET as template DNA to investigate the effect of

Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

Notes The authors declare no competing ﬁnancial interest.

ACKNOWLEDGMENT This work was supported by Japan Society for the Promotion of Science (15K18278).

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