Accurate Detection of Methylated Cytosine in Complex Methylation

Jun 17, 2013 - Entrepreneurs get in on the ground floor with CBD from hemp. Grass Valley, Ore., is about two hours east of Portland, on the dry side o...
0 downloads 0 Views 543KB Size
Download PDF
Technical Note pubs.acs.org/ac

Accurate Detection of Methylated Cytosine in Complex Methylation Landscapes Ramkumar Palanisamy,†,‡ Ashley R. Connolly,*,†,‡ and Matt Trau*,†,§ †

Centre for Biomarker Research and Development, Australian Institute for Bioengineering and Nanotechnology, The University of Queensland, Brisbane, QLD 4072, Australia § School of Chemistry and Molecular Biosciences, The University of Queensland, Brisbane, QLD 4072, Australia S Supporting Information *

ABSTRACT: Monitoring DNA methylation can be a useful biomarker for disease diagnosis and prognosis. However, monitoring the methylation status of a specific cytosine biomarker is often confounded by heterogeneous peripheral DNA methylation. To address this issue, molecular inversion probes were designed with inosine strategically positioned to complement suspected DNA methylation sites. This enabled the methylation status of a specific cytosine to be accurately measured with a high level of specificity, irrespective of adjacent epigenetic modifications.

C

amplified to detectable levels using common DNA sequences on each probe. The specificity of LDA can be increased by coupling the two probes with a DNA backbone, so they function as a molecular inversion probe (MIP).15,16 The backbone contains a DNA barcode unique to the methylation site, and common primer binding sites to facilitate multiplex analysis. Despite the accuracy, quantifiability and multiplexability of MIPs, prior knowledge of the local “methylation landscape” remains essential for accurate analysis of methylated epialleles. Since this may vary between cells, tissues, and patients, there is a demand for methods that can accurately detect the methylation status of a cytosine in a complex methylation landscape. To address this problem, universal nucleotides were strategically incorporated into a MIP. Universal inosine nucleotides were strategically positioned to complement suspected cytosine methylation sites in DNA. This enabled the methylation status of a specific cytosine to be interrogated, irrespective of the adjacent cytosine methylation pattern. MIPs containing inosine were found to be useful for specific, sensitive, and accurate analysis of cytosine methylation. This approach has the potential to enable multiplex analysis of cytosine methylation in a complex methylation landscape, which may help to detect, diagnose, and better treat cancer.

ytosine methylation is an epigenetic modification in which a methyl group is added to the C5 position of cytosine in genomic DNA.1 This modification is essential for normal cellular function, but aberrant DNA methylation may alter gene expression and cause cancer.2−4 Recent advances in genome wide methylation analysis have revealed thousands of potential methylation biomarkers in a variety of different types of cancer.5,6 Accurate detection and quantification of some of these biomarkers may help to diagnose cancer and dictate treatment in applications of personalized medicine. A popular method to analyze cytosine methylation involves chemically treating a DNA extract with sodium bisulfite.7 Unmethylated cytosine is converted to uracil, while methylated cytosine remains unchanged. This nucleotide conversion can be detected by amplifying the suspected methylated region to detectable levels using the polymerase chain reaction (PCR).8,9 However, fragmentation of DNA during bisulfite treatment can often retard PCR amplification.10 Furthermore, a thorough understanding of the local “methylation landscape” is essential to avoid preferential amplification of a single methylated epiallele, which can confound analysis. The amount of tissue available for diagnosis can also limit the number of diagnostic procedures performed, particularly during DNA analysis, where the number of biomarkers analyzed is often limited by the multiplexability of PCR.11 As a result, ligation-dependent amplification (LDA) assays have emerged as an alternative method for analyzing DNA methylation.12−14 This approach enables many different suspected DNA methylation sites to be analyzed in a single multiplex assay.15 Two DNA probes designed to “frame” a suspected cytosine methylation site in bisulfite-treated DNA undergo ligation only if they complement the nucleotide at the suspected methylation site. The ligated probes can then be © 2013 American Chemical Society

■

RESULTS Assay Design. Methylation of genomic DNA at certain cytosine guanine dinucleotides (CpG sites) may serve as a biomarker of cancer.17 However, CpG methylation can often be Received: November 6, 2012 Accepted: June 17, 2013 Published: June 17, 2013 6575

dx.doi.org/10.1021/ac3031948 | Anal. Chem. 2013, 85, 6575−6579

Analytical Chemistry

Technical Note

In a standard assay, both MIPs were mixed with the synthetic target. Following hybridization and ligation (Figure 1A), the circularized MIP was amplified with a fluorescently labeled primer (Figure 1B) to generate a single stranded fluorescent amplicon (Figure 1C). The fluorescent amplicon was hybridized to optically encoded microbeads containing a complementary DNA barcode immobilized on the surface (Figure 1D). This strategy enabled amplicons to be rapidly analyzed and quantified using flow cytometry (Figure 1E). Inosine Hybridization Efficiency. Inosine has been identified as a “universal” nucleoside, because it is capable of forming hydrogen bonds with cytosine, uracil, and adenine.20,21 As a result, it has been used in variety of applications to increase the efficiency with which oligonucleotides hydrogen-bond to regions of nucleoside degeneracy.22,23 Inosine has also been shown to be functionally active in ligation and polymerization reactions and is a suitable substrate to template DNA amplification.24,25 However, the use of inosine in MIPs as a method to increase the flexibility of methylation analysis has not yet been considered. This concept was evaluated in a model assay using a synthetic target (UU) that contained seven uracils, representing seven unmethylated CpG sites (see Table S1 in the Supporting Information). Uracil at the fourth CpG site was interrogated using iMIPA, which contained adenine at the 3′ end. iMIPA also contained inosine in the hybridization domains at positions complementary to the remaining six unmethylated CpG sites. A similar assay was performed using a second MIP, in which all inosines were replaced with adenine (MIPA). This enabled a direct comparison of the stability of the uracil hydrogen bond with inosine and adenine. Following ligation and amplification, the amount of fluorescence on the surface of optically encoded microbeads provided a measure of the progress of each reaction. The MIP that contained adenine in the hybridization domains (MIPA) produced a fluorescent signal that was 7.5fold higher than that measured using iMIPA, which contained inosine in the hybridization domains (see Figure S1 in the Supporting Information). This suggests adenine forms a morestable hydrogen bond with uracil. However, inosine does hybridize to uracil, but with an 86% reduction in efficiency. Nevertheless, strategically incorporating inosines into the hybridization domains of a MIP is an effective strategy to promote the formation of hydrogen bonds with either cytosine or uracil in bisulfite-treated DNA. This approach has the potential to increase the flexibility of methylation analysis. Temperature and Inosine Hybridization. Elevated temperatures compromise the stability of hydrogen bonds and base stacking interactions in duplex DNA.26 This is particularly evident as the number of nucleotides in the duplex is reduced. The DNA hybridization domains within a MIP are designed to form a 20 bp duplex adjacent to a suspected methylation site. MIPs used in the assay also contain inosine, which can compromise the stability of the DNA duplex.27 This may have an impact on the yield of the ligation reaction, which was evaluated at different temperatures to ensure DNA methylation could be detected with high sensitivity. This was evaluated in a model assay using a synthetic target (UU) that contained seven uracils, representing seven unmethylated CpG sites (see Table S1 in the Supporting Information). Uracil at the fourth CpG site was interrogated using iMIPA, which contained adenine at the 3′ end. iMIPA also contained inosine in the hybridization domains at positions

difficult to detect because adjacent methylation sites often bias the amplification of a single epiallele, which can confound analysis.18 Nevertheless, the methylation status of a specific CpG site in a complex methylation landscape can be accurately measured using multiple MIPs designed to detect all possible epialleles in a single multiplexed assay.19 It is also conceivable that the methylation status of a specific cytosine in a complex methylation landscape can be measured using only a single MIP. This involves incorporating “universal” inosine nucleotides into the MIP domains that hybridize to the DNA target (hybridization domains). This strategy would enable the universal nucleotides to hydrogen bond to either cytosine or uracil at a site of methylation ambiguity (see Figure 1).20

Figure 1. (A) A molecular inversion probe (MIP) was designed to detect unmethylated cytosine in bisulfite treated DNA. iMIPA contained a 3′-adenine designed to detect uracil at CpG site 4 in a synthetic DNA target (UU). iMIPA also contained inosine in the DNA hybridization domain, which enabled it to hybridize to the target, irrespective of the methylation status of flanking CpG sites. A second iMIPG contained a 3′-guanine to facilitate the analysis of methylated cytosine in the target and served as a mismatched nucleotide control. MIPs were hybridized to the target (A), ligated, and the circularized products were amplified to detectable levels using a fluorescent primer in a sensitive asymmetric PCR assay (B). The extent of ligation and amplification was measured by hybridizing the fluorescent amplicon (C) to optically encoded microbeads (D), which enabled the proportion of uracil (or cytosine) in the target to be rapidly measured using flow cytometry (E).

This concept was evaluated in a model assay using a synthetic 43 base oligonucleotide target containing 7 CpG sites (see Table S1 in the Supporting Information). A MIP with adenine at the 3′ end (the interrogation base) was designed to detect unmethylated cytosine (uracil) at the fourth CpG site in a DNA target. The MIP (iMIPA) also contained six inosines, strategically positioned to complement the remaining six CpG sites in the DNA target. The DNA backbone contained two universal primer binding sites and a unique DNA barcode to facilitate amplification and detection of the ligated product (Figure 1). A second MIP, also containing six inosines (iMIPG) was synthesized with guanine at the 3′ end to facilitate the detection of methylated cytosine in the same DNA target. This MIP served as a “mismatched nucleotide” control in the assay and contained a different DNA barcode, which enabled the deconvolution of both MIPs during multiplex analysis. 6576

dx.doi.org/10.1021/ac3031948 | Anal. Chem. 2013, 85, 6575−6579

Analytical Chemistry

Technical Note

complementary to the remaining six unmethylated CpG sites (uracil) in the target. The ligation reaction was performed at 65 °C, and the amount of fluorescence on the surface of optically encoded microbeads revealed the extent of the reaction (see Figure S2A in the Supporting Information). When a replicate reaction was performed at 45 °C, the extent of ligation was found to increase by 1.8-fold. This substantial increase in ligation was further perpetuated by increasing the ligation time from 2 min to 60 min (see Figure S2B in the Supporting Information), which resulted in an additional 3.0-fold increase in the amount of DNA measured. The considerable increase in the amount of ligated product at a lower temperature was attributed to the formation of morestable inosine hydrogen bonds in the DNA duplex. The lower temperature presumably reduced the rate of ligation, but morecircularized MIP formed as the reaction was allowed to progress for a longer period of time. The DNA ligase remained functional over a large temperature range, despite the presence of non-natural inosine nucleotides in the DNA substrate. Assays using MIPs containing inosine in the hybridization domain maintain a high level of sensitivity and specificity when performed at 45 °C. Inosine Binding Specificity. The methylation status of a specific cytosine can be interrogated using MIPs that contain inosine, since it can form stable hydrogen bonds with either cytosine or uracil in bisulfite-treated DNA. However, inosine is also capable of hydrogen bonding to additional nucleosides, which may promote nonspecific hybridization and possibly limit the specificity and accuracy of the assay.21,28 Therefore, the specificity of the assay was evaluated in a synthetic system using two MIPs that had the same hybridization domain, containing multiple inosines. However, each MIP differed at its interrogation base. An assay was performed using iMIPA, which contained a 3′-adenine to facilitate the detection of unmethylated cytosine at the fourth CpG site of a synthetic target, which contained multiple uracils (target UU; see Table S1 in the Supporting Information). The amount of nonspecific hybridization and ligation to the same target (UU) was also assayed in a replicate reaction using iMIPG, which contained a 3′-guanine to detect methylated cytosine. Substantially more product was produced (32.5-fold) when iMIPA was used to detect the unmethylated DNA target (Figure 2). Only a small amount of product was produced when iMIPG was used in a replicate assay with the same target, despite both MIPs containing inosine and differing by only a single nucleotide at the interrogation site. The specificity of the assay was also evaluated using a morecomplex target that represented heterogeneously methylated DNA. A target containing an unmethylated cytosine (uracil) at the fourth CpG site, flanked by multiple methylated cytosines (CU) was analyzed using a MIP designed to detect the central uracil (iMIPA). The extent of nonspecific hybridization and ligation was also assayed in a replicate reaction using a MIP designed to detect methylated cytosine (iMIPG) at the same position in target CU. As expected, substantially more product was produced (32.5-fold) when the iMIPA was used in the assay, yet a small amount of product was also produced when iMIPG was used with the same target. This level of discrimination demonstrates that strategic incorporation of inosines into MIPs at sites of ambiguous methylation is an effective strategy for accurately interrogating the methylation status of a specific cytosine, irrespective of the methylation status of adjacent CpG dinucleotides.

Figure 2. Uracil at the fourth CpG site in target (UU) was interrogated using iMIPA that contained adenine at the 3′ end. This MIP also contained inosine in the hybridization domains at positions complementary to CpG sites in the target. Nonspecific ligation was measured by analyzing the same target using iMIPG, which also contained inosines in the hybridization domains but had a 3′-guanine. The binding affinity of inosine was measured by performing a similar reaction using a synthetic target that also contained uracil in the interrogation site, but was flanked by cytosines (target CU). This represented an unmethylated CpG site surrounded by methylated DNA. The MIPs were ligated, amplified, and the amount of fluorescence on the surface of optically encoded microbeads revealed the extent of the reaction. The fluorescence values represent the mean ± standard deviation of triplicate assays.

Analysis of Genomic DNA. Incorporating inosines into a MIP at sites of ambiguous methylation is an effective strategy to detect methylated cytosine in a synthetic DNA target. However, the specificity of the assay is likely to vary with the complexity of the target. Therefore, the specificity of the assay was evaluated in a biological system using genomic DNA extracted from cells. Bisulfite-treated genomic DNA extracted from T47D cells was assayed with a MIP designed to detect methylated cytosine at position +1709 in the EN1 gene (iMIPG). The hybridization domains of iMIPG also contained inosines at positions complementary to six adjacent CpG sites in the region of interest. Replicate assays containing bisulfite-treated DNA and iMIPG were ligated, amplified, and analyzed. This revealed cytosine at position +1709 was methylated in T47D cells (Figure 3), which was confirmed by DNA sequence analysis (see Figure S3A in the Supporting Information). When a similar assay was performed on amplified genomic DNA, no signal was produced, because of the absence of methylated cytosine at position +1709 of the EN1 gene. This was attributed to inadequate replication of methyl-derivatized cytosine during genomic amplification. As a result, cytosine was demethylated and remained undetected by iMIPG. Demethylation at this position was also confirmed by sequence analysis (see Figure S3B in the Supporting Information), which demonstrates that a specific cytosine in a genomic DNA extract can be analyzed with a high level of specificity. Although a unique DNA sequence can be detected with high specificity, bisulfite-treated DNA has reduced nucleotide complexity and high sequence degeneracy. Analysis is therefore limited to unique DNA sequences in bisulfite-treated DNA. This was evident during the analysis of unmethylated cytosine 6577

dx.doi.org/10.1021/ac3031948 | Anal. Chem. 2013, 85, 6575−6579

Analytical Chemistry

Technical Note

approach obviates the requirement for multiple oligonucleotides to detect a methylated cytosine accurately. Optimal analysis was performed at a reduced temperature (45 °C) to increase the stability of the inosine−uracil hydrogen bond. Under these conditions, the efficiency of the DNA ligase was reduced but the enzyme retained its specificity, despite a substantial deviation from its thermal optimum of 70 °C.39 As the ligation reaction progressed, the yield increased up to 5 fold within 60 min. This was attributed to the increased stability of the inosine−uracil duplex, despite the slower ligation kinetics at 45 °C. However, the inosine−uracil duplex is likely to be progressively destabilized with the incorporation of additional inosines in the DNA hybridization domain of a MIP, which may influence both the yield and specificity of the assay. The methylation status of a cytosine in a complex methylation landscape was measured with high specificity, despite using “universal” inosine nucleotides in the DNA hybridization domain of a MIP. The high level of specificity was attributed to co-operative binding of the hybridization domains to the DNA target during the formation of the circularized DNA adduct. This approach was well-suited to maintaining a high level of specificity with the flexibility of incorporating “universal” inosine nucleotides in each hybridization domain. A small amount of nonspecific ligation (3%−7%) was found to be consistent with the promiscuity of DNA ligase in assays containing synthetic DNA.16,40 In practice, however, the specificity of ligation-dependent assays performed on morecomplex genomic extracts is expected to approach 0.1%.13 The specificity is also defined by the uniqueness of the DNA target sequence, which can often be made redundant following bisulfite treatment, which substantially reduces the nucleotide sequence complexity of DNA. Including inosines in the hybridization domains of a MIP enables the analysis of a methylated cytosine in a complex methylation landscape. Despite its flexibility, inosine hybridized to cytosine 35% more efficiently than uracil (Figure 2). Although this hybridization bias will have a minimal effect on the specificity of the assay, it is likely to influence the sensitivity, particularly during the analysis of a sample that contains a large excess of highly methylated DNA. Incorporating inosine into the DNA hybridization domains of MIPs is a viable strategy for accurately measuring the methylation status of a specific cytosine. This flexible approach enabled the MIPs to specifically detect cytosine methylation in bisulfite-treated genomic DNA (Figure 3). This approach may also be used to detect unmethylated cytosine by carefully designing MIP hybridization domains to detect a unique DNA sequence in bisulfite-treated DNA. In summary, we have demonstrated that the methylation status of a cytosine in a bisulfite-treated DNA extract can be determined with a high level of specificity and accuracy, by including inosines in the hybridization domain of a MIP at sites complementary to suspected cytosine methylation. This approach may help to clarify the role of methylated cytosine biomarkers in disease diagnosis and prognosis.

Figure 3. iMIPG contained a 3′-guanine designed to detect methylated cytosine at +1709 in the EN1 gene in genomic DNA extracted from T47D cells. This MIP also contained inosine in the hybridization domains at positions complementary to suspected neighboring CpG sites. The MIP was hybridized to bisulfite-treated genomic DNA, ligated, digested with exonuclease and the circularized product was amplified using PCR. In a similar assay, iMIPG was hybridized to whole-genome-amplified (WGA) bisulfite-treated genomic DNA, which was void of methylated cytosine. The extent of each reaction was analyzed using gel electrophoresis. The values represent the mean ± standard deviation of triplicate assays.

at the same position in genomic DNA. iMIPA was used to detect uracil at position +1709 in the EN1 gene, but it also contained degenerate DNA hybridization domains that did not accurately portray the methylation status of the specific cytosine in genomic DNA extracted from T47D cells (see Figure S4 in the Supporting Information).

■

DISCUSSION The implication of epigenetic modifications in various types of cancer has accelerated the development of technologies to measure cytosine methylation accurately.29−31 However, it is challenging to detect a methylated cytosine in a complex methylation landscape accurately, because the methylation status of adjacent cytosines can confound analysis. To facilitate more-accurate analysis, inosines were strategically incorporated into the hybridization domain of a MIP at sites complementary to suspected regions of cytosine methylation. This approach enabled the resultant cytosine or uracil in bisulfite-treated DNA to form a stable duplex with inosine, so the methylation status of a unique cytosine in a complex local methylation landscape could be measured with a high level of specificity. A variety of novel methods have been developed to analyze methylated DNA.32−35 Many involve primed amplification of an epiallele from bisulfite-treated DNA, which can often confound analysis, because unforeseen cytosine methylation will bias epiallele amplification.36 This can be overcome by designing multiple oligonucleotide primers to detect all possible epialleles.37 However, comprehensive representation of DNA spanning n suspected methylation sites requires 2n different oligonucleotides. As a result, amplification of epialleles rapidly becomes impractical as the number of suspected methylation sites in a primer binding domain increases, which is further compounded by the demands of assay optimization.38 Utilizing inosine in MIPs can facilitate simple and accurate analysis of a methylated cytosine in a single assay. This

■

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information contains additional methods and experimental results. This material is available free of charge via the Internet at http://pubs.acs.org. 6578

dx.doi.org/10.1021/ac3031948 | Anal. Chem. 2013, 85, 6575−6579

Analytical Chemistry

■

Technical Note

(26) Yakovchuk, P.; Protozanova, E.; Frank-Kamenetskii, M. D. Nucleic Acids Res. 2006, 34, 564−574. (27) Martin, F. H.; Castro, M. M.; Aboul-ela, F.; Tinoco, I., Jr. Nucleic Acids Res. 1985, 13, 8927−8938. (28) Batzer, M. A.; Carlton, J. E.; Deininger, P. L. Nucleic Acids Res. 1991, 19, 5081. (29) Gitan, R. S.; Shi, H.; Chen, C. M.; Yan, P. S.; Huang, T. H. Genome Res. 2002, 12, 158−164. (30) Ehrich, M.; Nelson, M. R.; Stanssens, P.; Zabeau, M.; Liloglou, T.; Xinarianos, G.; Cantor, C. R.; Field, J. K.; van den Boom, D. Proc. Natl. Acad. Sci., U.S.A. 2005, 102, 15785−15790. (31) Wojdacz, T. K.; Dobrovic, A. Nucleic Acids Res. 2007, 35, e41. (32) Bird, A. P.; Taggart, M. H.; Smith, B. A. Cell 1979, 17, 889−901. (33) Gebhard, C.; Schwarzfischer, L.; Pham, T. H.; Schilling, E.; Klug, M.; Andreesen, R.; Rehli, M. Cancer Res. 2006, 66, 6118−6128. (34) 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. (35) Wittwer, C. T.; Reed, G. H.; Gundry, C. N.; Vandersteen, J. G.; Pryor, R. J. Clin. Chem. 2003, 49, 853−860. (36) Warnecke, P. M.; Stirzaker, C.; Melki, J. R.; Millar, D. S.; Paul, C. L.; Clark, S. J. Nucleic Acids Res. 1997, 25, 4422−4426. (37) Archey, W. B.; McEachern, K. A.; Robson, M.; Offit, K.; Vaziri, S. A.; Casey, G.; Borg, A.; Arrick, B. A. Oncogene 2002, 21, 7034−7041. (38) Polz, M. F.; Cavanaugh, C. M. Appl. Environ. Microbiol. 1998, 64, 3724−3730. (39) Jeon, S. J.; Ishikawa, K. FEBS Lett. 2003, 550, 69−73. (40) Zirvi, M.; Bergstrom, D. E.; Saurage, A. S.; Hammer, R. P.; Barany, F. Nucleic Acids Res. 1999, 27, e41.

AUTHOR INFORMATION

Corresponding Author

*Tel.: +61 7 33464172. Fax: +61 7 33463973. E-mail: [email protected] (A.R.C.), [email protected] (M.T.). Author Contributions ‡

Authors contributed equally to this work.

Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS We gratefully acknowledge funding and support from the National Breast Cancer Foundation (NBCF) of Australia through the National Collaborative Breast Cancer Research Grant (No. CG-08-07). We are very grateful to Mr. James Bates for preparing the microbeads used in these experiments.

■

REFERENCES

(1) Ehrlich, M.; Gama-Sosa, M. A.; Huang, L. H.; Midgett, R. M.; Kuo, K. C.; McCune, R. A.; Gehrke, C. Nucleic Acids Res. 1982, 10, 2709−2721. (2) Esteller, M. Oncogene 2002, 21, 5427−5440. (3) Shiota, K. Cytogenet Genome Res. 2004, 105, 325−334. (4) Robertson, K. D. Nat. Rev. Genet. 2005, 6, 597−610. (5) https://tcga-data.nci.nih.gov/tcga/. (6) http://bioinfo.hrbmu.edu.cn/diseasemeth. (7) Clark, S. J.; Harrison, J.; Paul, C. L.; Frommer, M. Nucleic Acids Res. 1994, 22, 2990−2997. (8) Candiloro, I. L.; Mikeska, T.; Dobrovic, A. Methods Mol. Biol. 2011, 791, 55−71. (9) Dallol, A.; Al-Ali, W.; Al-Shaibani, A.; Al-Mulla, F. In FormalinFixed Paraffin-Embedded Tissues: Methods and Protocols; Al-Mulla, F., Ed.; Methods in Molecular Biology, No. 724; Humana Press: New York, 2011; pp 191−204. (10) Raizis, A. M.; Schmitt, F.; Jost, J. P. Anal. Biochem. 1995, 226, 161−166. (11) Gunson, R. N.; Bennett, S.; Maclean, A.; Carman, W. F. J. Clin. Virol. 2008, 43, 372−375. (12) Dahl, C.; Guldberg, P. Nucleic Acids Res. 2007, 35, e144. (13) Hirani, R.; Connolly, A. R.; Putral, L.; Dobrovic, A.; Trau, M. Anal. Chem. 2011, 83, 8215−8221. (14) Hardenbol, P.; Baner, J.; Jain, M.; Nilsson, M.; Namsaraev, E. A.; Karlin-Neumann, G. A.; Fakhrai-Rad, H.; Ronaghi, M.; Willis, T. D.; Landegren, U.; Davis, R. W. Nat. Biotechnol. 2003, 21, 673−678. (15) Li, J. B.; Gao, Y.; Aach, J.; Zhang, K.; Kryukov, G. V.; Xie, B.; Ahlford, A.; Yoon, J. K.; Rosenbaum, A. M.; Zaranek, A. W.; LeProust, E.; Sunyaev, S. R.; Church, G. M. Genome Res. 2009, 19, 1606−1615. (16) Palanisamy, R.; Connolly, A. R.; Trau, M. Anal. Chem. 2011, 83, 2631−2637. (17) Belinsky, S. A. Nat. Rev. Cancer 2004, 4, 707−717. (18) Mikeska, T.; Candiloro, I. L.; Dobrovic, A. Epigenomics 2010, 2, 561−573. (19) Deng, J.; Shoemaker, R.; Xie, B.; Gore, A.; LeProust, E. M.; Antosiewicz-Bourget, J.; Egli, D.; Maherali, N.; Park, I. H.; Yu, J.; Daley, G. Q.; Eggan, K.; Hochedlinger, K.; Thomson, J.; Wang, W.; Gao, Y.; Zhang, K. Nat. Biotechnol. 2009, 27, 353−360. (20) Crick, F. H. C. J. Mol. Biol. 1966, 19, 548−555. (21) Janke, E. M.; Riechert-Krause, F.; Weisz, K. J. Phys. Chem. B 2011, 115, 8569−8574. (22) Knoth, K.; Roberds, S.; Poteet, C.; Tamkun, M. Nucleic Acids Res. 1988, 16, 10932. (23) Palva, A.; Vidgren, G.; Paulin, L. J. Microbiol Methods 1994, 19, 315−321. (24) Patil, R. V.; Dekker, E. E. Nucleic Acids Res. 1990, 18, 3080. (25) McKernan, K. M.; Blanchard, A.; Kotler, L.; Costa, G. Reagents, methods, and libraries for bead-based sequencing, U.S. Patent 8,329,404, Dec. 11, 2008. 6579

dx.doi.org/10.1021/ac3031948 | Anal. Chem. 2013, 85, 6575−6579