- Open Access
Colorimetric detection of both total genomic and loci-specific DNA methylation from limited DNA inputs
© Wee et al. 2015
Received: 12 May 2015
Accepted: 25 June 2015
Published: 11 July 2015
Aberrant DNA methylation marks are potential disease biomarkers, and detecting both total genomic and gene-specific DNA methylation can aid in clinical decisions. While a plethora of methods exist in research, simpler, more convenient alternatives are needed to enhance both routine diagnostics and research.
Herein, we describe colorimetric assays using methyl-binding domain (MBD) proteins for rapid and convenient evaluation of total genomic and gene-specific methylation from 50 ng or less DNA input in under 2 h. As little as 5 % methylation differences can be detected and are enhanced by a novel MBD protocol for improved specificity. Our assays could differentiate naïve from de-methylating drug-treated cells and detect the presence of a methylated prostate cancer biomarker in the urine. Finally, the assay was evolved onto disposable screen-printed electrodes for convenient detection of gene-specific methylation in urine.
Rapid MBD-based colorimetric and electrochemical approaches to detect DNA methylation from limited samples were successfully demonstrated and applied to clinical samples. We envision that the ease, low sample requirements and speed of these assays could have both clinical and research-wide applications.
Aberrant epigenetic changes in DNA are potential disease biomarkers [1–3]. A form of epigenetic change is the methylation of the cytosine (5mC) in cytosine/guanine dinucleotides (CpG), particularly in CpG islands (CGI) of regulatory regions that function to regulate cellular processes [1–3]. Azanucleoside drugs such as 5-aza-2′-deoxycytidine (5-Aza) have been used therapeutically with some success to reactivate silenced genes in epigenetic diseases [4–6]. In addition, genome-wide hypomethylation is also associated with tumorigenesis [2, 3] and hence may be useful as an early screening strategy for cancer. While hypomethylation is associated with tumorigenesis, regulatory sequences at specific loci, such as that of tumour suppressor genes, are hypermethylated and detection of which are potentially useful in stratifying patient cohorts and informing clinical decisions [1–3, 7]. Most approaches, however, detect DNA methylation via bisulfite conversion [8, 9] of DNA followed by some form of sequencing [10–13]. To avoid the problems associated with bisulfite conversion, affinity capture approaches, such as methyl-binding domain (MBD) proteins or antibodies raised against 5mC, have been adapted to Next Generation Sequencing  platforms or other optical detection methods for both genome-wide [15, 16] and gene-specific  applications. Useful, simpler, more convenient methods to detect both genome-wide and gene-specific methylation are still lacking and would be useful for both routine diagnostics and research.
MBD enrichment approaches are useful and convenient because they avoid the limitations of bisulfite conversion while being very highly specific for 5mC on native double-stranded DNA but not hydroxymethylated (5hmC) or unmethylated DNA . Unfortunately, MBD enrichment approaches are limited by their difficulty in quantifying methylation levels and typically quantitative PCR (or sequencing) is used to measure enrichment levels as a proxy estimate of differential methylation . Additionally, the stringency of MBD enrichment reduces with limiting DNA inputs and various strategies including high-salt buffers  and alternative MBD enzymes  have been devised. Nonetheless, one is still able to infer, with very high stringency, the degree/density of methylation based on the buffer conditions required to recover enriched DNA [14, 19]. In short, the methylation outcomes derived from MBD approaches are generally binary, i.e., yes/no calls and therefore, ideal for identifying highly differentially methylated regions (HDMRs).
Colorimetric readouts are also popular in molecular diagnostics because they can be evaluated with the naked eye and have the option for (semi)quantification. One popular colorimetric system is the horse radish peroxidase HRP/H2O2 system coupled to a chromogen e.g., 3,3′,5,5′-tetramethylbenzidine (TMB) substrate to generate a coloured by-product to signal the presence of a biomolecule, (HDMRs in this case). Herein, we describe MBD-based colorimetric assays for rapid, naked-eye evaluation of either overall genome-wide or gene-specific methylation detection. In addition, we also describe a novel method for rapid yet highly specific MBD enrichment from low nanogram amounts of DNA input for loci-specific applications that was subsequently applied to urine DNA derived from prostate cancer patients. Finally, since TMB is electrochemically active , we adapted our assay onto commercially available screen-printed electrodes as a potential alternative electrochemical approach for detecting DNA methylation. To the best of our knowledge, these are the first demonstrations of colorimetric and electrochemical evaluations of DNA methylation via MBD and may have wide applications in both research and diagnostics.
Results and discussion
The MBD/HRP assays
To enable gene-specific applications (Fig. 1b), digested DNA was first subjected to a novel protocol for rapid yet highly specific MBD enrichment from limiting samples (details in a later section). This was then followed with an isothermal amplification of the desired region with biotin-deoxynucleotides (dNTPs) to generate polymers of biotin/DNA which then served as substrates for the HRP/TMB colorimetric reaction and therefore indicating the presence of a HDMR.
Detecting genome-wide methylation
While we demonstrate a wide dynamic range of methylation in this study, the method may be tuned to narrower ranges by adjusting the buffer conditions to detect meaningful levels of methylation over normal levels. However, given the current limits in detection and reproducibility, the assay would more likely be suited to detect differential methylation changes of 10 % or more in a clinical setting.
Total DNA methylation is traditionally detected by HPLC  and mass spectrometry (MS)  methods. While our MBD assay uses less starting material, it is difficult to directly compare the methylation-based analytical performances. This is because both HPLC and MS approaches consider all cytosines in the genome whereas our assay is an enrichment approach that only considers a subset of cytosines and therefore a different measure of methylation. Nonetheless, compared to the published data in a previous MBD/flow cytometry study , the current approach had similar analytical performance but only required a fraction of the processing time (2 versus 24 h). Finally, in comparison to the published data in a recent study via a proximity oxygen channelling chemistry platform , both approaches had similar performance in detecting methylated DNA. However, the oxygen channelling platform requires very specialized equipment for detection whereas the colorimetric strategy described herein does not.
Improved MBD protocol for highly specific enrichment from limited DNA input
A potential limitation of MBD enrichment strategies is non-specific capture of unmethylated DNA. This problem is especially pronounced with low DNA input because of increased background from non-specifically captured unmethylated DNA and is further confounded when coupled to an exponential DNA amplification for gene-specific applications. Various strategies to alleviate this problem include multiple methylation-sensitive restriction digestion , exotic MBD complexes , high-salt-binding buffers and incubation at low temperatures . While these approaches improved specificity, their long overnight incubation may not be feasible for applications requiring fast turnovers e.g., routine diagnostics.
Nonetheless, the results of these studies suggested that when MBD was in excess, as in the case of low sample input, MBD non-selectively bound both methylated and unmethylated DNA. Hence, we hypothesized that increasing the DNA to MBD ratio could restore the selectivity of MBD for methylated DNA. One possible way to increase the DNA/MBD ratio is to introduce exogenous carrier/blocking DNA, such as salmon sperm DNA, to sequester free MBD.
Detecting GSTP1 methylation with the MBD/HRP assay
Confident of the stringency displayed by our MBD enrichment protocol, we next turned our attention to designing a methylation assay against GSTP1, a clinically important HDMR biomarker of prostate cancer [26, 27]. Currently, the methylation status of GSTP1 is the only FDA-approved methylation biomarker undergoing clinical trials  and is usually detected by head loop PCR . While potentially useful, the approach has various limitations such as the need for bisulfite conversion and complicated primer design. Simpler assays are hence needed to facilitate widespread adoption. The MBD/HRP approach described herein may fulfil this need.
The assay was further extended to the ESR1 gene promoter which is also aberrantly methylated in cancers . To this end, we assayed Jurkat cells before and after 5-Aza treatment with M-WGA and U-WGA as specificity controls (Additional file 1: Figure S5). As expected, ESR1 was de-methylated in the 5-Aza-treated cells compared to naïve Jurkat cells. The de-methylation of both GSTP1 and ESR1 in 5-Aza-treated cells were consistent with the genome-wide hypomethylation observed in Fig. 3.
Based on the data shown here, a MBD/HRP-based approach may be useful for both total genomic and gene level methylation. However, a known limitation of all MBD-based approaches is the inability to provide methylation information at individual CpGs. In addition, MBD is also not able to provide 5hmC information . While methods exist for 5hmC, there is no simple single-step method besides HPLC and/or MS able to simultaneously interrogate both 5mC and 5hmC. Nonetheless, this MBD/HRP approach could be adapted to an analogous assay for 5hmC using affinity reagents currently available for 5hmC .
Adapting to an electrochemical assay using disposable screen-printed electrodes
In conclusion, we have developed simple and rapid approaches to detect total genomic DNA methylation and at specific gene sequences. The assays were first developed on a colorimetric readout for naked-eye evaluation and subsequently onto electrochemical approach for convenient diagnostic and research applications. We also described a novel MBD protocol with improved stringency by exploiting the competitive binding of non-human carrier DNA to sequester excess MBD. The high sensitivity, ease and low sample requirements of our approach may be useful for routine diagnostics and a wide range of applications unlike traditional methods.
Availability of supporting data
The data sets supporting the results of this article are included within the article and its additional files.
To demonstrate the feasibility of the assay, a 119-bp DNA fragment model system over the GSPTP1 gene was PCR amplified using standard procedures using the Kapa2G Robust PCR kit (KapaBiosystems, USA) with forward and reverse primers (AACCCCCTTATCCCTCCGTCGTGTGGCTTTTAC and AAACAGGTTCCTCCGAAGATTTCACACAACACT, respectively (Integrated DNA Technologies, Australia). Amplicons were then purified using QIAquick Gel extraction kit (Qiagen, Australia). To generate methylated sequences, amplicons were treated with SssI methyltransferase (New England Biolabs, USA) overnight as recommended by the manufacturer and purified using the Agencourt AMPure XP kit (Beckman Coulter, Australia). An aliquot of the SssI-treated DNA was used to evaluate the methylation conversion efficiency by digestion with the methylation-sensitive HpaII restriction enzyme (New England Biolabs, USA). Only reactions with no detectable digestion via gel electrophoresis were used in downstream experiments. Finally, the amplicons were treated with 5 μM biotin-14-dUTP (Thermo Fisher, Australia) and terminal deoxynucleotidyl transferase (TdT, New England Biolabs, USA) as recommended by the manufacturer.
WGA DNA was generated using the REPLI-g UltraFast Mini kit (Qiagen, Australia) and purified using the DNeasy Blood and Tissue kit (Qiagen, Australia). An aliquot of WGA DNA was then treated with SssI methyltransferase overnight and purified to generate highly methylated genomic DNA (M-WGA). An aliquot of the SssI-treated DNA was used to evaluate the methylation conversion efficiency by digestion with the methylation-sensitive HpaII restriction enzyme (New England Biolabs, USA). Only reactions with no detectable digestion via gel electrophoresis were used in downstream experiments. Genomic DNA from Jurkat cells representing before and after 5-aza-2′-deoxycytidine treatment were purchased from New England Biolabs (NEB).
HeLa and DuCap cells were purchased from ATCC and cultured according to the manufacturer’s instructions. gDNA was extracted using the DNeasy Blood and Tissue kit. Thirty millilitres urine samples from prostate cancer patients were collected with the relevant ethics approval from The University of Queensland Institutional Human Research Ethics Committee (Approval No. 201400012) and processed within 5 h with the ZR Urine DNA Isolation Kit (Zymo Research, USA).
For total genomic methylation studies, 50 ng of gDNA (both WGA and cell line derived) was enzymatically digested with the endonucleases DpnII and MseI (7.5 units each, NEB) at 37 °C in a 20-μL reaction supplemented with the NEB Buffer 3.1 system to generate <1000 bp DNA fragments with 5′ overhangs. After 30 min, the reaction was supplemented to a final volume of 25 μL with 5 units of Klenow fragment (3′→5′ exo-) DNA polymerase (NEB, USA) and 5 μM of biotin-14-dUTP, dATP, dGTP and dCTP and incubated at 37 °C for another 30 min to fill in the overhangs and biotinylate the fragmented DNA. The reactions were then heat inactivated at 75 °C for 20 min.
For gene-specific applications, 50 ng of gDNA was digested with MseI and MluCI (NEB, USA) instead at 37 °C for 30 mins in a 20-μL reaction supplemented with the NEB CutSmart Buffer system.
Total genomic methylation assays
For total genomic methylation studies, digested gDNA reactions were first diluted tenfold in water. One microlitre (i.e., 200 pg of DNA) was then used in the MBD/HRP assay to estimate levels of methylation. MBD-modified magnetic beads (NEB, USA) were prepared and used as recommended by the manufacturer. To enrich for methylated DNA, the provided 1× MBD buffer was supplemented with 600 mM NaCl. After a 15-min incubation with DNA targets, the MBD beads were isolated with a magnet and the supernatant was removed. The MBD beads were then resuspended in 20 μL of 1/1000 HRP solution (BD Biosciences, Australia) in 1× MBD buffer for 10 mins. The MBD beads were then washed four times with 1× MBD buffer. Finally, 100 μL of 1-Step™ TMB substrate solution (Thermo Scientific, Australia) and colorimetric changes were monitored at 650 nm over 30 min on the EnSpire® plate reader (Perkin Elmer, Australia). Absorbance values at 20 min were used in subsequent data analysis.
For gene-specific applications, 19 μL of digested DNA was used for MBD enrichment in a modified protocol. Briefly, one tenth of the recommended MBD/bead mix was used in a 100-μL reaction. MBD buffer (1×), MBD buffer (1.25×) or MBD buffer (1.25×) supplemented with 50 ng of salmon sperm DNA (Sigma Aldrich, Australia) was used for a 15-min MBD enrichment step at 4 °C. After three 5-min washes with 1× MBD buffer, the captured DNA was eluted in 5 μL 2.5 M NaCl solution. To purify the enriched DNA for downstream amplification, 10 μL of Agencourt AMPure XP bead solution was added to the same tube and purification proceeded as recommended by the manufacturer. The purified DNA was finally eluted in 20 μL water. The remaining 1 μL of digested DNA was diluted tenfold in water and used as input controls in subsequent amplifications.
To detect gene-specific methylation, the isothermal recombinase polymerase amplification  (RPA, TwistDx, UK) was employed to amplify the GSTP1 locus with primers described above in a modified RPA protocol. Briefly, 1 μL of gDNA from the above step was used for each 12.5 μL RPA reaction supplemented with 10 μM biotin-14-dUTP and 7 mM MgOAc at 37 °C for 15 min. After amplification, 12.5 μL of Agencourt AMPure XP bead solution was used to remove excess biotin and purify amplicons. The purified amplicons were eluted in 15 μL of water where 3 μL was subjected to gel electrophoresis to verify amplification.
GSTP1 primers were as described earlier. ESR1 forward and reverse primers were GTTCGTCCTGGGACTGCACTTGCTCCCGTC and AGATGCTTTGGTGTGGAGGGTCATGGTCATGGT, respectively.
To detect amplicons colorimetrically, 1 μL of purified amplicons was reacted with 20 μL SA-HRP (1/2000 dilution in 1× PBS) and SA magnetic beads (1/20 dilution in 1× PBS, NEB) for 5 mins. After collecting DNA-bound beads with a magnet and three 1× PBS washes, 50 μL of TMB substrate solution was allowed to react with the captured HRP and absorbance readings were taken after a 15-min incubation.
To enable an electrochemical readout, 50 μL of 0.5 M H2SO4 was used to stop the HRP/TMB reaction after 15 mins and to convert TMB to its electrochemically active form . Fifty microlitres of the resulting TMB solution was added onto screen-printed electrodes (CH Instruments, USA) and electrochemical response was detected using ampometry at 150 mV over 30s on a potentiostat (CH Instruments, USA). Current response at 10 s was used for subsequent analysis.
We gratefully acknowledge funding received by our laboratories from the National Breast Cancer Foundation of Australia (CG-08-07 and CG-12-07). These grants have significantly contributed to the environment to stimulate the research described here.
- Klose RJ, Bird AP. Genomic DNA methylation: the mark and its mediators. Trends Biochem Sci. 2006;31(2):89–97.PubMedView ArticleGoogle Scholar
- Friso S, Udali S, Guarini P, Pellegrini C, Pattini P, Moruzzi S, et al. Global DNA hypomethylation in peripheral blood mononuclear cells as a biomarker of cancer risk. Cancer Epidemiol Biomarkers Prev. 2013;22(3):348–55. doi:10.1158/1055-9965.EPI-12-0859.PubMed CentralPubMedView ArticleGoogle Scholar
- Ehrlich M. DNA hypomethylation in cancer cells. Epigenomics. 2009;1(2):239–59. doi:10.2217/epi.09.33.PubMed CentralPubMedView ArticleGoogle Scholar
- Jones PA, Taylor SM. Cellular differentiation, cytidine analogs and DNA methylation. Cell. 1980;20(1):85–93.PubMedView ArticleGoogle Scholar
- Yoo CB, Jones PA. Epigenetic therapy of cancer: past, present and future. Nat Rev Drug Discov. 2006;5(1):37–50. doi:10.1038/nrd1930.PubMedView ArticleGoogle Scholar
- Egger G, Liang G, Aparicio A, Jones PA. Epigenetics in human disease and prospects for epigenetic therapy. Nature. 2004;429(6990):457–63. doi:10.1038/nature02625.PubMedView ArticleGoogle Scholar
- Wee EJ, Peters K, Nair SS, Hulf T, Stein S, Wagner S, et al. Mapping the regulatory sequences controlling 93 breast cancer-associated miRNA genes leads to the identification of two functional promoters of the Hsa-mir-200b cluster, methylation of which is associated with metastasis or hormone receptor status in advanced breast cancer. Oncogene. 2012;31(38):4182–95. doi:10.1038/onc.2011.584.PubMed CentralPubMedView ArticleGoogle Scholar
- Frommer M, McDonald LE, Millar DS, Collis CM, Watt F, Grigg GW, et al. A genomic sequencing protocol that yields a positive display of 5-methylcytosine residues in individual DNA strands. Proc Natl Acad Sci U S A. 1992;89(5):1827–31.PubMed CentralPubMedView ArticleGoogle Scholar
- Clark SJ, Harrison J, Paul CL, Frommer M. High sensitivity mapping of methylated cytosines. Nucleic Acids Res. 1994;22(15):2990–7.PubMed CentralPubMedView ArticleGoogle Scholar
- Taylor KH, Kramer RS, Davis JW, Guo J, Duff DJ, Xu D, et al. Ultradeep bisulfite sequencing analysis of DNA methylation patterns in multiple gene promoters by 454 sequencing. Cancer Res. 2007;67(18):8511–8.PubMedView ArticleGoogle Scholar
- Gu H, Smith ZD, Bock C, Boyle P, Gnirke A, Meissner A. Preparation of reduced representation bisulfite sequencing libraries for genome-scale DNA methylation profiling. Nat Protoc. 2011;6(4):468–81.PubMedView ArticleGoogle Scholar
- Hirst M, Marra MA. Next generation sequencing based approaches to epigenomics. Brief Funct Genomics. 2010;9(5–6):455–65.PubMed CentralPubMedView ArticleGoogle Scholar
- Zerilli F, Bonanno C, Shehi E, Amicarelli G, Adlerstein D, Makrigiorgos GM. Methylation-specific loop-mediated isothermal amplification for detecting hypermethylated DNA in simplex and multiplex formats. Clin Chem. 2010;56(8):1287–96.PubMedView ArticleGoogle Scholar
- Nair SS, Coolen MW, Stirzaker C, Song JZ, Statham AL, Strbenac D, et al. Comparison of methyl-DNA immunoprecipitation (MeDIP) and methyl-CpG binding domain (MBD) protein capture for genome-wide DNA methylation analysis reveal CpG sequence coverage bias. Epigenetics Off J DNA Methylation Soc. 2011;6(1):34–44. doi:10.4161/epi.6.1.13313.View ArticleGoogle Scholar
- Wee EJ, Trau M. Measuring whole genome methylation via oxygen channelling chemistry. Chem Commun. 2014;50(74):10894–6. doi:10.1039/c4cc04623d.View ArticleGoogle Scholar
- Corrie SR, Sova P, Feng Q, Blair T, Kiviat NB, Trau M. Bisulfite-free analysis of 5MeC-binding proteins and locus-specific methylation density using a microparticle-based flow cytometry assay. Analyst. 2011;136(4):688–91. doi:10.1039/c0an00790k.PubMedView ArticleGoogle Scholar
- Lee TY, Shin Y, Park MK. A simple, low-cost, and rapid device for a DNA methylation-specific amplification/detection system using a flexible plastic and silicon complex. Lab Chip. 2014;14(21):4220–9. doi:10.1039/c4lc00804a.PubMedView ArticleGoogle Scholar
- Khrapunov S, Warren C, Cheng H, Berko ER, Greally JM, Brenowitz M. Unusual characteristics of the DNA binding domain of epigenetic regulatory protein MeCP2 determine its binding specificity. Biochemistry. 2014;53(21):3379–91. doi:10.1021/bi500424z.PubMed CentralPubMedView ArticleGoogle Scholar
- Warton K, Lin V, Navin T, Armstrong NJ, Kaplan W, Ying K, et al. Methylation-capture and Next-Generation Sequencing of free circulating DNA from human plasma. BMC Genomics. 2014;15:476. doi:10.1186/1471-2164-15-476.PubMed CentralPubMedView ArticleGoogle Scholar
- Oliver VF, Wan J, Agarwal S, Zack DJ, Qian J, Merbs SL. A novel methyl-binding domain protein enrichment method for identifying genome-wide tissue-specific DNA methylation from nanogram DNA samples. Epigenetics Chromatin. 2013;6(1):17. doi:10.1186/1756-8935-6-17.PubMed CentralPubMedView ArticleGoogle Scholar
- Fanjul-Bolado P, Gonzalez-Garcia MB, Costa-Garcia A. Amperometric detection in TMB/HRP-based assays. Anal Bioanal Chem. 2005;382(2):297–302. doi:10.1007/s00216-005-3084-9.PubMedView ArticleGoogle Scholar
- Fraga MF, Esteller M. DNA methylation: a profile of methods and applications. BioTechniques. 2002;33(3):632. 4, 6–49.PubMedGoogle Scholar
- Singer J, Schnute Jr WC, Shively JE, Todd CW, Riggs AD. Sensitive detection of 5-methylcytosine and quantitation of the 5-methylcytosine/cytosine ratio in DNA by gas chromatography--mass spectrometry using multiple specific ion monitoring. Anal Biochem. 1979;94(2):297–301.PubMedView ArticleGoogle Scholar
- Lisanti S, Omar WA, Tomaszewski B, De Prins S, Jacobs G, Koppen G, et al. Comparison of methods for quantification of global DNA methylation in human cells and tissues. PLoS One. 2013;8(11):e79044. doi:10.1371/journal.pone.0079044.PubMed CentralPubMedView ArticleGoogle Scholar
- Yegnasubramanian S, Lin X, Haffner MC, DeMarzo AM, Nelson WG. Combination of methylated-DNA precipitation and methylation-sensitive restriction enzymes (COMPARE-MS) for the rapid, sensitive and quantitative detection of DNA methylation. Nucleic Acids Res. 2006;34(3):e19. doi:10.1093/nar/gnj022.PubMed CentralPubMedView ArticleGoogle Scholar
- Lee WH, Morton RA, Epstein JI, Brooks JD, Campbell PA, Bova GS, et al. Cytidine methylation of regulatory sequences near the pi-class glutathione S-transferase gene accompanies human prostatic carcinogenesis. Proc Natl Acad Sci U S A. 1994;91(24):11733–7.PubMed CentralPubMedView ArticleGoogle Scholar
- Millar DS, Ow KK, Paul CL, Russell PJ, Molloy PL, Clark SJ. Detailed methylation analysis of the glutathione S-transferase pi (GSTP1) gene in prostate cancer. Oncogene. 1999;18(6):1313–24. doi:10.1038/sj.onc.1202415.PubMedView ArticleGoogle Scholar
- Mahon KL, Qu W, Devaney J, Paul C, Castillo L, Wykes RJ, et al. Methylated glutathione S-transferase 1 (mGSTP1) is a potential plasma free DNA epigenetic marker of prognosis and response to chemotherapy in castrate-resistant prostate cancer. Br J Cancer. 2014;111(9):1802–9. doi:10.1038/bjc.2014.463.PubMedView ArticleGoogle Scholar
- Rand KN, Ho T, Qu W, Mitchell SM, White R, Clark SJ, et al. Headloop suppression PCR and its application to selective amplification of methylated DNA sequences. Nucleic Acids Res. 2005;33(14):e127. doi:10.1093/nar/gni120.PubMed CentralPubMedView ArticleGoogle Scholar
- Li LC, Carroll PR, Dahiya R. Epigenetic changes in prostate cancer: implication for diagnosis and treatment. J Natl Cancer Inst. 2005;97(2):103–15. doi:10.1093/jnci/dji010.PubMedView ArticleGoogle Scholar
- Thomson JP, Hunter JM, Nestor CE, Dunican DS, Terranova R, Moggs JG, et al. Comparative analysis of affinity-based 5-hydroxymethylation enrichment techniques. Nucleic Acids Res. 2013;41(22):e206. doi:10.1093/nar/gkt1080.PubMed CentralPubMedView ArticleGoogle Scholar
- Soleymani L, Fang Z, Sargent EH, Kelley SO. Programming the detection limits of biosensors through controlled nanostructuring. Nat Nanotechnol. 2009;4(12):844–8. doi:10.1038/nnano.2009.276.PubMedView ArticleGoogle Scholar
- Wee EJ, Rauf S, Koo KM, Shiddiky MJ, Trau M. mu-eLCR: a microfabricated device for electrochemical detection of DNA base changes in breast cancer cell lines. Lab Chip. 2013;13(22):4385–91. doi:10.1039/c3lc50528f.PubMedView ArticleGoogle Scholar
- Wee EJ, Rauf S, Shiddiky MJ, Dobrovic A, Trau M. DNA ligase-based strategy for quantifying heterogeneous DNA methylation without sequencing. Clin Chem. 2015;61(1):163–71. doi:10.1373/clinchem.2014.227546.PubMedView ArticleGoogle Scholar
- Piepenburg O, Williams CH, Stemple DL, Armes NA. DNA detection using recombination proteins. PLoS Biol. 2006;4(7):e204. doi:10.1371/journal.pbio.0040204.PubMed CentralPubMedView ArticleGoogle Scholar
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