DNA Methylation Analysis Triggered by Bulge Specific Immuno

Aug 10, 2012 - (1) Cytosine methylation at CpG islands has received particular attention because it is thought to be involved in controlling genetic e...
0 downloads 0 Views 1MB Size
Download PDF
Article pubs.acs.org/ac

DNA Methylation Analysis Triggered by Bulge Specific ImmunoRecognition Ryoji Kurita* and Osamu Niwa National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba Central 6, 1-1-1 Higashi, Tsukuba, Ibaraki, Japan 305-8566 S Supporting Information *

ABSTRACT: We report the sequence-selective discrimination of the cytosine methylation status in DNA with anti methylcytosine antibody for the first time. This was realized by employing an affinity measurement involving the target methylcytosine in a bulge region and anti methylcytosine antibody, following hybridization with a bulge-inducing DNA to ensure that only the target methylcytosine is located in the bulge. The affinity of the antibody for methylcytosine in the bulge was 79% of that in a single strand of DNA; however, the affinity for nontarget methylcytosine in a double strand of DNA decreased greatly. This is because the antibody cannot bind with an inwardly turned methylcytosine in the duplex region owing to the large antibody size. In contrast, the methylcytosine in the bulge is recognized by the antibody because it is available to rotate freely owing to the single bond between deoxyribose and phosphate in a DNA chain. By employing the difference between the affinity in the bulge and that in the duplex, we could determine selectively whether or not the target cytosine was methylated in an O6-methylguanine DNA methyltransferase (MGMT) promoter sequence with a single base level. The proposed bulge-specific assay technique can be combined with a widely used absorbance measurement method that employs the color change in tetramethyl benzidine induced by horseradish peroxidase-labeled secondary antibody. The sequence-selective discrimination of the methylation status could also be obtained with various types of interfering genomic DNA contamination without any conventional bisulfite treatment, polymerase chain reaction, (PCR) or electrophoresis.

T

assessed before a detection assay is undertaken. Moreover, the bisulfite treatment makes the analyte DNA thymine-rich since unmethylated cytosine is converted to thymine, and this complicates the design of specific probes for PCR amplification.8 A restriction enzyme, which selectively catalyzes the scission of a specific sequence containing methylcytosine, can also be used to determine methylation in DNA.9,10 However, restriction digests may be biased and limited depending on the available restriction enzyme. New methylcytosine assays have been reported that do not require bisulfite treatment, a restriction enzyme, PCR, or a sequencer.11−17 For example, methods have been reported for labeling methylcytosine with methyl-CpG binding proteins,12,13 osmium complex,14,15 and vanadium complex.17 Unfortunately, thymine is also labeled via OsO4 in addition to methylcytosine. Therefore, it is difficult to distinguish methylcytosine from thymine in a methylcytosine assay. Mixtures of V2O5 or NaIO4 and LiBr were used in an anaerobic condition to differentiate methylcytosine from both cytosine and thymine, followed by a hot piperidine treatment and electrophoretic analysis.17 A

he methylation of the 5′ carbon of cytosine in DNA is an epigenetic modification that regulates gene expression and plays crucial roles in embryonic development.1 Cytosine methylation at CpG islands has received particular attention because it is thought to be involved in controlling genetic expression, including that in cancer,2 genomic imprinting,3 cellular differentiation, and Alzheimer’s disease.4 5-Methylcytosine is now recognized as the fifth DNA base containing heritable information. Therefore, highly sensitive, accurate, and quantitative information concerning cytosine methylation in DNA would be valuable with respect to genetic disease diagnosis. Two major cytosine methylation assay methods have been reported: bisulfite treatment followed by PCR and sequencing and DNA restriction digests. A bisulfite-based determination method is widely used to distinguish between cytosine and methylcytosine.5−7 Treatment with bisulfite converts cytosine to uracil, while methylcytosine remains unaffected. Therefore, information about methylcytosine in DNA can be obtained at the single base level by determining the differences between the sequences of bisulfite-treated and untreated samples. However DNA degradation occurs during bisulfite treatment owing to oxidative damage, namely, depurination under the required acidic and thermal conditions. Therefore, to avoid misleading results, the quality of the bisulfite-treated DNA must be © 2012 American Chemical Society

Received: June 19, 2012 Accepted: August 10, 2012 Published: August 10, 2012 7533

dx.doi.org/10.1021/ac301702y | Anal. Chem. 2012, 84, 7533−7538

Analytical Chemistry

Article

Figure 1. Schematic of sequence-selective methylation analysis with an antibody in (a) a target region and (b) a nontarget region. mC indicates methylcytosine. The analyte DNA is hybridized with bulge-inducing DNA to place the target (methyl-)cytosine in a bulge region. (a) Methylcytosine in the bulge is recognized by the antibody owing to the rotation of the methylcytosine. (b) Methylcytosine is not recognized in a hybridized region owing to the large antibody size.

Table 1. Variations in Cytosine Methylation in a MGMT Gene Sequencea

a

Underlined cytosine(s) is methylated.

cytosine antibody. Our idea involves measuring the affinity between target cytosine (or methylcytosine) in a bulge region and the antimethylcytosine antibody following hybridization with a bulge-inducing probe DNA so that the target is located in the bulge as shown in Figure 1a. This idea is based on the fact that there is a single bond between deoxyribose and phosphate in DNA that allows it to rotate freely. This means that only the target cytosine (or methylcytosine) in the bulge turns outward. In contrast, methylcytosine in a nontarget region paired with guanine turns inward; therefore, it cannot be recognized by the antibody as shown in Figure 1b. We succeeded in determining whether or not cytosine is methylated with a single base level in an MGMT promoter.

sequence-selective cleavage assay technique with a metal complex at a DNA bulge has been reported;18 however, this approach requires hot piperidine treatment and electrophoresis. The drawbacks of the chemical modification methods in regards to methylcytosine are the relatively long detection time and the need for a high concentration (around micromolar) DNA sample. We proposed a label-free electrochemical technique for detecting methylcytosine by utilizing the oxidation potential difference between cytosine and methylcytosine.16 However, this method also requires a high (approximately micromolar) DNA sample concentration; moreover there is no sequence selectivity. Anti methylcytosine antibody has been used for the immunoprecipitation or concentration of methyl-CpG regions in a DNA sample.19−21 Recently, immunochemical methods for detecting methylcytosine with anti methylcytosine antibody have been reported for assessing the methylation level that employ capillary electrophoresis,22 magnetic particles,23 microspheres,24 a nitrocellulose membrane,25 and a DNA microarray.26 We have also reported an electrochemiluminescencebased immunoassay technique for measuring the amount of methylcytosine in a single strand of DNA.27 These results suggest that the immunoassay has the potential to detect methylcytosine with a system as simple as immunochromatography. However, previously reported immunochemical methods for detecting methylcytosine, including our recent work, have no sequence selectivity and so we can quantify only the total amount of methylcytosine in the analyte DNA. Therefore, there is a real need to be able to detect single methylcytosine and its position in the DNA sequence. In this article, we report the first sequence-selective determination of methylcytosine in DNA with an antimethyl-

■

EXPERIMENTAL SECTION Reagents. Mouse monoclonal anti methylcytosine antibody (clone code 33D3) was purchased from Aviva Systems Biology. Rabbit polyclonal mouse IgG labeled with horseradish peroxidase purchased from Abcam was used as a secondary antibody. 3,3′,5,5′-Tetramethyl benzidine was purchased from Nacalai. Phosphate buffered saline (PBS) and bovine serum albumin were purchased from Sigma (St. Louis, MO). Various DNA sequences containing methylcytosine were obtained from bacteriophage DNA (48 502 base pairs, MW of approximately 32 300 kDa, Takara Bio) to investigate sequence specificity. DNA (200 μg) was incubated with AluI (Takara Bio) for 4 h at 37 °C to obtain 144 sequences, and then all CpG sites were methylated with 40 units of CpG methyltransferase (New England Laboratories) in the presence of 1.5 mM S-adenosylmethionine as a methyl donor at 37 °C overnight in a buffer supplied by a manufacturer (50 mM NaCl, 10 mM Tris-HCl, 10 mM MgCl2, 1 mM dithiothreitol, pH 7.9). 7534

dx.doi.org/10.1021/ac301702y | Anal. Chem. 2012, 84, 7533−7538

Analytical Chemistry

Article

Table 2. Bulge-Inducer DNA Sequences to Form a Bulge

Finally, the DNA sample was heated at 95 °C for 10 min and then quickly cooled in an ice bath to obtain single stranded DNA fragments. Blocking reagent was purchased from Pierce (catalog no. 37516, Rockford, IL). Two buffer solutions were prepared for the methylcytosine assay. One was PBS containing 0.05 (v/v) % Tween 20 as a washing buffer, and the other was PBS containing 0.05 (v/v)% Tween 20 and 0.1 (w/v) % bovine serum albumin (Sigma, St. Louis, MO) as an incubation buffer. Designs of Analyte and Bulge-Inducing DNA. The analyte DNA sequence is 5′-TTG CGC GGC GTC CGT CCT GTT GAC TTC-3′, which is part of the MGMT promoter gene. The positions of cytosine methylation(s) were changed as summarized in Table 1. Five kinds of 5′ amino-modified bulgeinducing DNA were synthesized for hybridization with the analyte DNA. The sequences were changed to form a 1, 3, 5, or 7 base bulge as summarized in Table 2. Inducer-1 is a sequence that fully matches the analyte DNA, so it hybridizes with the analyte DNA to form a complementary duplex without a bulge. The analyte and bulge-inducing DNA oligonucleotides were synthesized and purified with high performance liquid chromatography by Hokkaido System Science. Bulge-Specific Discrimination of Cytosine Methylation. A volume of 100 μL of 100 nM (10 pmol) bulge-inducing DNA was immobilized on a microtiter plate with an active ester surface (Sumitomo Bakelite, BS-61602) by incubation at 80 °C for 2 h. Excess bulge-inducing DNA was removed by rinsing the plate twice with 300 μL of distilled water, and 100 μL of analyte DNA was then added and incubated for 2 h at 37 °C for hybridization. The analyte DNA concentrations were varied from 0 to 100 nM. Unreacted analyte DNA was removed by rinsing the plate with washing buffer, and then the microtiter plate was blocked with blocking buffer for 30 min at room temperature. Next, 50 μL of 500 ng/mL antimethylcytosine antibody in incubation buffer was added to each well and incubated at 37 °C for 1 h. The concentration was optimized by adding 0.01−2 μg/mL antibody as shown in Figure S-1 in the Supporting Information. Each well was rinsed with washing buffer, and then 50 μL of 500 ng/mL horseradish peroxidaselabeled secondary antibody was added and incubated for 30 min at 37 °C. Each well was rinsed and incubated with 50 μL of 3,3′,5,5′-tetramethyl benzidine for 10 min at room temperature. Finally, the absorbance of each well was read at 450 nm with a microplate reader (Biorad, model 680) after the enzymatic reaction had been stopped with 50 μL of 2 N HCl.

antibody that bound to the DNA duplex was measured. Figure 2a shows schematics of the DNA duplex forms between

Figure 2. (a) Schematics of duplex forms between Analyte-1 and Inducer-1−5. Duplex-A forms a complementary full-match duplex. Duplex-B, C, D, and E have 7, 5, 3, and 1 base bulges in each duplex. The ★ indicates the position of methylcytosine. (b) Calibration curves for Analyte-1 when the bulge size is varied. Methylcytosine is located in a 7-base (◊, Duplex-B), 5-base (□, Duplex-C), 3-base (△, DuplexD), and 1-base (○, Duplex-E) bulge, respectively. In Duplex-A ( × ), methylcytosine is paired with guanine.

Analyte-1 and Inducer-1−5. In Duplex-A, Analyte-1 is completely hybridized with Inducer-1 without a bulge. However, in Duplex-B, C, D and E, each duplex has a different sized bulge. Figure 2b shows the results of our methylcytosine assay. The absorbance increased as the Analyte-1 concentrations increased in Duplex-B, C, D, and E. This is reasonable because the amount of hybridized and captured Analyte-1 that contains methylcytosine increases as the Analyte-1 concentration increases. In contrast, no increase in the absorbance was observed as Analyte-1 increased in full-match Duplex-A. These

■

RESULTS AND DISCUSSION Immuno-Recognition of Methylcytosine in Bulges. First, DNA containing methylcytosine was hybridized with fullmatch DNA or 1−7 mismatch DNA to form a bulge in the DNA duplex, and then the amount of antimethylcytosine 7535

dx.doi.org/10.1021/ac301702y | Anal. Chem. 2012, 84, 7533−7538

Analytical Chemistry

Article

Figure 2b, and the signal for 2 nM Duplex-E was larger than 3 times the standard deviation of the background. The detection limit of our proposed method is about 2 or 3 orders of magnitude better than that of chemical modification methods in regards to methylcytosine combined with electrophoretic analysis14,17 or direct electrochemical detection.16 Selectivity and Sequence Specificity for Methylcytosine Assay in a Bulge. Selectivity for methylcytosine in a bulge against nontarget methylcytosine in the same duplex was investigated as follows. Figure 4a shows schematics of a DNA

results indicate that the methylcytosine in the bulge was selectively detected by an immune reaction. This was due to the hard binding of an antibody with inward turned methylcytosine in the hybridized DNA region owing to the large antibody size (approximately 150 kDa). Since the antibody diameter (10 nm) is much larger than that of DNA duplex (2 nm), the antibody cannot recognize the inward turned methylcytosine in the duplex. However, the methylcytosine in the bulge can rotate freely since deoxyribose and phosphate groups in a DNA chain are connected with a single bond, and there is no other interaction such as a hydrogen bond. A sufficient increase in absorbance was obtained only at a single-base bulge, and no significant increase was observed as the bulge size was increased to seven bases. Detection in a single-base bulge is advantageous for methylation analysis at a single-base level. Furthermore, a duplex is thermally stable because there is only a single-base mismatch, and a shorter bulge-inducing DNA would be applicable. In addition, it was simple to design bulge-inducing DNA since it consists solely of the elimination of guanine paired with the target methylcytosine from a sequence fully matched with the analyte DNA. Scatchard plots were obtained to compare the dissociation constant values (Kd) of the antibody and methylcytosine when the methylcytosine was in a single-stranded DNA or in a singlebase bulge in a double stranded DNA (the same as Duplex-E shown in Figure 2). Each Kd value in Figure 3 was estimated

Figure 4. (a) Schematics of duplex forms between Inducer-5 and Analyte-1−4. The ★ indicates methylcytosine, and the ☆ indicates cytosine. (b) The symbols indicate calibration curves for Analyte-1 (○, Duplex-E), Analyte-2 ( ×, Duplex-F), Analyte-3 (△, Duplex-G), and Analyte-4 (□, Duplex-H).

duplex formed between Inducer-5 and Analytes-1−4. The sequences of Analytes-1−4 are the same; however, the methylation region differs as shown in Table 1. In Analyte-1, only the target cytosine is methylated and the other cytosine bases are not. In Analyte-2, the target cytosine is not methylated and one of the nontarget cytosine bases is methylated. In Analyte-3, the target and one of the nontarget cytosine bases are methylated. In Analyte-4 no cytosine was methylated. Figure 4b shows measurement results for Analytes-1−4 with Inducer-5. The absorbance increased as the concentrations of Analyte-1 and Analyte-3 increased; however, no increase was observed for Analyte-2 and Analyte-4. This is because the cytosine in the bulges in Duplex-E (Analyte-1) and Duplex-G (Analyte-3) is methylated; however, the cytosine in the bulges in Duplex-F (Analyte-2) and Duplex-H (Analyte-4) is not methylated. The important point is that no absorption increase was observed in Duplex-F (Analyte-2), which contains one methylcytosine in the nontarget region. This is because the antibody cannot bind with an inner methylcytosine in a hybridized DNA as described in Figure 2. Moreover, no significant difference was observed between Duplex-E and Duplex-G. These results clearly demonstrate that we can distinguish whether or not the target cytosine is methylated with high selectivity without any effect from the status of other

Figure 3. Results of a Scatchard plot assay for methylcytosine in a single strand of DNA (○) and in a bulge region (□). The bulge form is the same as that of Duplex-E in Figure 2a.

from the inverse of the slopes in the Scatchard plots. Kd in single-stranded DNA is 1.09 nM and Kd in a bulge is 1.38 nM. A decrease of about 21% was observed for methylcytosine in the bulge. This would be the decrease in the collision probability between the antibody and the methylcytosine caused by the steric hindrance while methylcytosine turns inward. Methylcytosine in a bulge can be recognized by the antibody only when it turns outward. Therefore, some reduction in antibody binding is reasonable because the antibody cannot bind to methylcytosine when it turns inward. Even so, a high affinity of 79% was maintained, and singlemethylation in DNA can be detected with a detection limit of a few nanomolar (subpicomole) as shown in Figure 2b. The relative standard deviation was 8.5% when measuring 2 nM Analyte-1 with Inducer-5 (Duplex-E). There was no crossover of error bars between Duplex-E and Duplex-A at 2 nM in 7536

dx.doi.org/10.1021/ac301702y | Anal. Chem. 2012, 84, 7533−7538

Analytical Chemistry

Article

bulge specific immuno-recognition. The single methylation status of target cytosine was determined without any effect from the methylation status in the nontarget region. This is because the high affinity of the antibody for methylcytosine in a single-base bulge region is maintained, whereas the affinity for nontarget methylcytosine in a hybridized region decreases greatly owing to the size of the bulky antibody compared with the diameter of the DNA duplex. No serious error was observed in the presence of various interfering sequences, and the proposed assay technique was confirmed to have satisfactory sequence specificity.

cytosine methylation in the nontarget region. Recently, some very important CpG sites have been found that significantly affect gene regulation with only a single methylation.28−30 The focus of methylation studies has now shifted from the methylation frequency to the role of each methylation with a single CpG level. The proposed method is unsuitable for global methylation analysis; however, the site-specific methylation status can be obtained more quickly than with conventional bisulfite-based assays. We investigated the sequence specificity of our bulge specific assay against various sequences with Inducer-5 and genomic DNA as follows. Two interfering samples were prepared, one consisted of unmethylated λDNA fragments treated with AluI and the other comprised methylated λDNA fragments treated with AluI and CpG methyltransferase. The enzyme-treated λDNA sample contained a wide variety of sequences since the λDNA sequence had 143 AluI scission sites and 3112 CpG sites.31 Figure 5 shows the results for Analyte-1 and Analyte-4

■

ASSOCIATED CONTENT

S Supporting Information *

Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.

■

AUTHOR INFORMATION

Corresponding Author

*Phone: +81-29-861-6158. Fax: +81-29-861-6177. E-mail: r. [email protected]. Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS Part of this study was supported by NEDO Japan, and the Grant-in-Aid for Young Scientists (B), Grant No. 23710152. We also thank Ms. Arai for technical assistance with the measurements and Mr. Meacock for revising the language of the manuscript.

■

REFERENCES

(1) Yoder, J. A.; Walsh, C. P.; Bestor, T. H. Trends Genet. 1997, 13, 335−340. (2) Jones, P. A. Cancer Res. 1996, 56, 2463−2467. (3) Monk, M. Dev. Genet. 1995, 17, 188−197. (4) Ledoux, S.; Nalbantoglu, J.; Cashman, N. R. Mol. Brain Res. 1994, 24, 140−144. (5) Clark, S. J.; Harrison, J.; Paul, C. L.; Frommer, M. Nucleic Acids Res. 1994, 22, 2990−2997. (6) Docherty, S. J.; Davis, O. S. P.; Haworth, C. M. A.; Plomin, R.; Mill, J. Methods 2010, 52, 255−258. (7) Herman, J. G.; Graff, J. R.; Myohanen, S.; Nelkin, B. D.; Baylin, S. B. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 9821−9826. (8) DNA Methylation - From Genomics to Technology; Tatarinova, T., Kerton, O., Eds. InTech: Rijeka, Croatia, 2012. (9) Rush, L. J.; Plass, C. Anal. Biochem. 2002, 307, 191−201. (10) Xiong, Z. G.; Laird, P. W. Nucleic Acids Res. 1997, 25, 2532− 2534. (11) Münzel, M.; Lercher, L.; Müller, M.; Carell, T. Nucleic Acids Res. 2010, 38, e192. (12) Yu, Y.; Blair, S.; Gillespie, D.; Jensen, R.; Myszka, D.; Badran, A. H.; Ghosh, I.; Chagovetz, A. Anal. Chem. 2010, 82, 5012−5019. (13) Acevedo, L. G.; Sanz, A.; Jelinek, M. A. Epigenomics 2011, 3, 93−101. (14) Tanaka, K.; Tainaka, K.; Kamei, T.; Okamoto, A. J. Am. Chem. Soc. 2007, 129, 5612−5620. (15) Tanaka, K.; Tainaka, K.; Umemoto, T.; Nomura, A.; Okamoto, A. J. Am. Chem. Soc. 2007, 129, 14511−14517. (16) Kato, D.; Sekioka, N.; Ueda, A.; Kurita, R.; Hirono, S.; Suzuki, K.; Niwa, O. J. Am. Chem. Soc. 2008, 130, 3716−3717. (17) Bareyt, S.; Carell, T. Angew. Chem., Int. Ed. 2008, 47, 181−184. (18) Okamoto, A.; Tainaka, K.; Kamei, T. Org. Biomol. Chem. 2006, 4, 1638−1640.

Figure 5. Sequence specificity against various sequences. The DNA concentration was always 10 nM, and the assay was performed with Inducer-5. Analyte-1 contains one target methylcytosine, and Analyte4 contains no methylcytosine.

in the presence and absence of the same concentration of methylated or unmethylated λDNA. No negative error was observed when Analyte-1 was measured in the presence of various single stranded DNA fragments. This means that Inducer-5 hybridizes effectively with Analyte-1 without being affected by the other DNA sequences and forms a duplex with a bulge selectively. Furthermore, no positive error was observed when Analyte-1 and Analyte-5 were measured in the presence of methylated λDNA fragments. These results clearly demonstrate that any nonspecific hybridization is negligible, and an intended bulge can be formed in the contamination of various sequences. With a significant difference, we could clearly distinguish the methylation status in the presence of interfering DNA fragments as shown in Figure 5.

■

CONCLUSIONS A sequence-selective DNA methylation assay technique with an antimethylcytosine antibody has been proposed that utilizes 7537

dx.doi.org/10.1021/ac301702y | Anal. Chem. 2012, 84, 7533−7538

Analytical Chemistry

Article

(19) Reynaud, C.; Bruno, C.; Boullanger, P.; Grange, J.; Barbesti, S.; Niveleau, A. Cancer Lett. 1992, 61, 255−262. (20) Darwish, I.; Emara, S.; Askal, H.; El-Rabbat, N.; Omura, K.; Hirose, K.; Akizawa, T.; Yoshioka, M. Anal. Chim. Acta 2000, 413, 79− 86. (21) Kremer, D.; Metzger, S.; Kolb-Bachofen, V. Anal. Biochem. 2012, 422, 74−78. (22) Wang, X. L.; Song, Y. L.; Song, M. Y.; Wang, Z. X.; Li, T.; Wang, H. L. Anal. Chem. 2009, 81, 7885−7891. (23) Wang, Z.; Wang, X.; Liu, S.; Yin, J.; Wang, H. Anal. Chem. 2010, 82, 9901−9908. (24) Ge, C.; Fang, Z.; Chen, J.; Liu, J.; Lu, X.; Zeng, L. Analyst 2012, 137, 2032−2035. (25) Deobagkar, D. D.; Panikar, C.; Rajpathak, S. N.; Shaiwale, N. S.; Mukherjee, S. Methods 2012, 56, 260−267. (26) Kelkar, A.; Deobagkar, D. Epigenetics 2009, 4, 311−316. (27) Kurita, R.; Arai, K.; Nakamoto, K.; Kato, D.; Niwa, O. Anal. Chem. 2012, 84, 1799−1803. (28) Nanduri, J.; Makarenko, V.; Reddy, V. D.; Yuan, G.; Pawar, A.; Wang, N.; Khan, S. A.; Zhang, X.; Kinsman, B.; Peng, Y.-J.; Kumar, G. K.; Fox, A. P.; Godley, L. A.; Semenza, G. L.; Prabhakar, N. R. Proc. Natl. Acad. Sci. U.S.A. 2012, 109, 2515−2520. (29) Furst, R. W.; Kliem, H.; Meyer, H. H. D.; Ulbrich, S. E. J. Steroid Biochem. Mol. Biol. 2012, 130, 96−104. (30) Nile, C. J.; Read, R. C.; Akil, M.; Duff, G. W.; Wilson, A. G. Arthritis Rheum. 2008, 58, 2686−2693. (31) http://blast.ddbj.nig.ac.jp/

7538

dx.doi.org/10.1021/ac301702y | Anal. Chem. 2012, 84, 7533−7538