The Experts below are selected from a list of 249 Experts worldwide ranked by ideXlab platform

Chun-yang Zhang - One of the best experts on this subject based on the ideXlab platform.

  • Integration of single-molecule detection with endonuclease IV-assisted signal amplification for sensitive DNA Methylation Assay.
    Chemical communications (Cambridge England), 2021
    Co-Authors: Yan Zhang, Xiaoran Zou, Jian-ge Qiu, Chun-yang Zhang
    Abstract:

    We demonstrate the development of a new fluorescent biosensor for sensitive DNA Methylation Assay by integrating single-molecule detection with endo IV-assisted signal amplification. This biosensor possesses the characteristics of good selectivity and high sensitivity with a detection limit of 7.3 × 10-17 M. It can distinguish as low as 0.01% Methylation level, and can analyze genomic DNA Methylation even in a single cancer cell.

  • Recent advances in biosensors for in vitro detection and in vivo imaging of DNA Methylation.
    Biosensors & bioelectronics, 2020
    Co-Authors: Qian Zhang, Chun-yang Zhang
    Abstract:

    DNA Methylation is the predominant epigenetic modification that participates in many fundamental cellular processes through posttranscriptional regulation of gene expression. Aberrant DNA Methylation is closely associated with a variety of human diseases including cancers. Therefore, accurate and sensitive detection of DNA Methylation may greatly facilitate the epigenetic biological researches and disease diagnosis. In recent years, a series of novel biosensors have been developed for highly sensitive detection of DNA Methylation, but an overview of recent advances in biosensors for in vitro detection and especially live-cell imaging of DNA Methylation is absent. In this review, we summarize the emerging biosensors for in vitro and in vivo DNA Methylation Assays in the past five years (2015-2020). Based on the signal types, the biosensors for in vitro DNA Methylation Assay are classified into five categories including fluorescent, electrochemical, colorimetric, surface enhanced Raman spectroscopy, mass spectrometry, and surface plasmon resonance biosensors, while the biosensors for in vivo DNA Methylation Assay mainly rely on fluorescent imaging. We review the strategies, features and applications of these biosensors, and provide a new insight into the challenges and future directions in this area.

  • Sensitive detection of methylated DNA using the short linear quencher-fluorophore probe and two-stage isothermal amplification Assay.
    Biosensors & bioelectronics, 2013
    Co-Authors: Guichi Zhu, Kun Yang, Chun-yang Zhang
    Abstract:

    Abstract DNA Methylation is an important epigenetic modification of genomes and is associated with various human diseases. Here, we develop a sensitive approach for DNA Methylation Assay using the short linear quencher–fluorophore DNA probe (QF probe) and two-stage isothermal amplification. With bisulfite treatment, the methylated DNA target is able to hybridize with the template to initiate the strand displacement amplification (SDA), generating abundant triggers which can further hybridize with the QF probes to form the DNA duplexes. The subsequent recognition of DNA duplexes and the cleavage of QF probes by the nicking enzyme can initiate the nicking enzyme signal amplification (NESA), inducing a significant fluorescence enhancement. While in the absence of methylated DNA, neither SDA nor NESA reaction can be initiated and no fluorescence enhancement is observed. This method exhibits high sensitivity with a detection limit of 0.78 pM, and can distinguish as low as 0.1% Methylation level from the mixture of methylated and unmethylated DNA. Notably, the introduction of SDA into NESA can improve the detection sensitivity by up to 2 orders of magnitude as compared with the NESA Assay, and it can even discriminate single-base mismatched methylated DNA.

  • sensitive and label free DNA Methylation detection by ligation mediated hyperbranched rolling circle amplification
    Analytical Chemistry, 2012
    Co-Authors: Anping Cao, Chun-yang Zhang
    Abstract:

    Sensitive and specific detection of DNA Methylation in CpG sites of genomic DNA is imperative for rapid epigenetic evaluation and early cancer diagnosis. Here, we employ for the first time the thermostable ligation for methylated DNA discrimination and hyperbranched rolling circle amplification (HRCA) for signal enhancement, without the need for restriction enzymes, PCR amplification, or fluorescence-labeled probes. After bisulfite treatment of methylated DNA, the Methylation-specific linear padlock probe can be circularized only in the presence of methylated DNA and serves subsequently as a template for HRCA, whose products are easily detected using SYBR Green I and a standard fluorometer. While in the presence of unmethylated DNA, the linear padlock probe cannot be circularized because of the defectively matched substrate, and no HRCA occurs. This ligation-mediated HRCA-based method exhibits excellent specificity and high sensitivity with a detection limit of 0.8 fM and a detection range of 4 orders of magnitude, and it can even distinguish as low as 0.01% Methylation level from the mixture, which is superior to most currently used methods for DNA Methylation Assay. This method can be further applied to analyze genomic DNA in human lung cancer cells.

  • Single base extension reaction-based surface enhanced Raman spectroscopy for DNA Methylation Assay.
    Biosensors & bioelectronics, 2011
    Co-Authors: Chun-yang Zhang
    Abstract:

    DNA Methylation is a key diagnostic maker for genetic disease, cancer progression and pharmcogenomics. So far various techniques have been developed for DNA Methylation Assay, but most of them are laborious and time-consuming. Here we develop a simple and highly sensitive DNA Methylation Assay based on single base extension reaction and surface enhanced Raman spectroscopy (SERS). In the presence of methylated DNA, gold nanoparticle-modified capture probe can couple with a cyanine 5-deoxyribonucleoside triphosphate (cy5-dGTP) through single base extension reaction, and generates a high SERS signal after further addition of gold nanoparticles to increase the local electromagnetic field. While in the presence of unmethylated DNA, gold nanoparticle-modified capture probe cannot couple with cy5-dGTP due to the presence of a mismatch base, and no SERS signal is observed. This single base extension reaction-based SERS can determine methylated DNA with a detection limit of 3 pM, and can even distinguish as low as 1% Methylation level in tumor suppressor gene CDKN2/p16/MTS1 (p16) from the mixtures. Notably, the sensitivity of this Assay has improved by 5 orders of magnitude as compared to reported gold nanoparticle-based colorimetric Assay, and by 2 orders of magnitude as compared to microarray-based Methylation-sensitive single nucleotide primer extension Assay (Ms-SNuPE). This method might be further applied to detect the Methylation status in tumor-linked genes for cancer diagnosis.

Eve O’reilly - One of the best experts on this subject based on the ideXlab platform.

  • A urine-based DNA Methylation Assay, ProCUrE, to identify clinically significant prostate cancer
    Clinical epigenetics, 2018
    Co-Authors: Fang Zhao, Ekaterina Olkhov-mitsel, Shivani Kamdar, Renu Jeyapala, Julia Garcia, Rachel Hurst, Marcelino Yazbek Hanna, Robert D. Mills, Alexandra V. Tuzova, Eve O’reilly
    Abstract:

    Prevention of unnecessary biopsies and overtreatment of indolent disease remains a challenge in the management of prostate cancer. Novel non-invasive tests that can identify clinically significant (intermediate-risk and high-risk) diseases are needed to improve risk stratification and monitoring of prostate cancer patients. Here, we investigated a panel of six DNA Methylation biomarkers in urine samples collected post-digital rectal exam from patients undergoing prostate biopsy, for their utility to guide decision making for diagnostic biopsy and early detection of aggressive prostate cancer. We recruited 408 patients in risk categories ranging from benign to low-, intermediate-, and high-risk prostate cancer from three international cohorts. Patients were separated into 2/3 training and 1/3 validation cohorts. Methylation biomarkers were analyzed in post-digital rectal exam urinary sediment DNA by quantitative MethyLight Assay and investigated for their association with any or aggressive prostate cancers. We developed a Prostate Cancer Urinary Epigenetic (ProCUrE) Assay based on an optimal two-gene (HOXD3 and GSTP1) LASSO model, derived from Methylation values in the training cohort, and assessed ProCUrE’s diagnostic and prognostic ability for prostate cancer in both the training and validation cohorts. ProCUrE demonstrated improved prostate cancer diagnosis and identification of patients with clinically significant disease in both the training and validation cohorts. Using three different risk stratification criteria (Gleason score, D’Amico criteria, and CAPRA score), we found that the positive predictive value for ProCUrE was higher (59.4–78%) than prostate specific antigen (PSA) (38.2–72.1%) for all risk category comparisons. ProCUrE also demonstrated additive value to PSA in identifying GS ≥ 7 PCa compared to PSA alone (DeLong’s test p = 0.039), as well as additive value to the PCPT risk calculator for identifying any PCa and GS ≥ 7 PCa (DeLong’s test p = 0.011 and 0.022, respectively). ProCUrE is a promising non-invasive urinary Methylation Assay for the early detection and prognostication of prostate cancer. ProCUrE has the potential to supplement PSA testing to identify patients with clinically significant prostate cancer.

Peter W Laird - One of the best experts on this subject based on the ideXlab platform.

  • Abstract 1620: Effects of γ-glutamyl hydrolase and folylpolyglutamyl synthase modulation on gene-specific promoter CpG island Methylation
    Cancer Research, 2011
    Co-Authors: Toshinori Hinoue, Daniel J. Weisenberger, Kyoung-jin Sohn, Peter W Laird
    Abstract:

    Background: Folate mediates the transfer of one-carbon units for most biological Methylation reactions including DNA Methylation, which is catalyzed by DNA methyltransferase (DNMT). Genomic DNA hypoMethylation and gene-specific promoter CpG island hyperMethylation are important epigenetic mechanisms of carcinogenesis. DNA Methylation and DNMT also are potential therapeutic targets and may modify the effect of specific chemotherapeutic agents. Intracellular folate homeostasis is maintained by folylpolyglutamate synthase (FPGS) and γ-glutamyl hydrolase (GGH) for folate-dependent one-carbon transfer reactions and antifolate-induced cytotoxic effects. We have previously shown that GGH/FPGS modulation significantly affects global DNA Methylation and DNMT activity in human HCT116 colon and MDA-MB-435 breast cancer cells in a predictably manner. We investigated whether GGH/FPGS modulation would also affect gene-specific promoter CpG island Methylation. Methods: We generated an in vitro model of GGH overexpression/inhibition in HCT116 and MDA-MB-435 cells by transfecting the cells with the sense GGH cDNA or GGH-targeted siRNA, respectively. An in vitro model of FPGS overexpression/inhibition in HCT116 was generated by transfecting the cells with the sense or antisense FPGS cDNA, respectively. An in vitro model of FPGS overexpression/inhibition in MDA-MB-435 cells was generated by transfecting cells with the sense FPGS cDNA or FPGS-targeted siRNA, respectively. Illumina Infinium DNA Methylation Assay was used to interrogate 27,578 individual CpG loci in 14,495 different gene promoters. Results: MDA-MB-435 cells showed more CpG Methylation alterations in response to GGH and FPGS modulation than HCT116 cells. Sixty-one hyper- and 54 hypomethylated genes were common between HCT116 and MDA-MB-435 cell lines in GGH overexpression while 117 hyper- and 129 hypomethylated genes were common between these cell lines in GGH inhibition. In both cell lines, 64 hyper- and 52 hypomethylated genes were common in FPGS overexpression whereas 31 hyper- and 38 hypomethylated genes were common in FPGS inhibition. Preliminary analysis demonstrated that genes associated with efflux and influx of (anti)folate (ABCC1, ABCC2, ABCC3, ABCG2, SLC19A1), de novo purine synthesis, glutathione transferase (GSTM1, GSTP1, GSTT1), growth regulation (IGF2, IGFBP3), mismatch repair (MLH1), tumor suppressor (PTEN) and transcription (RUNX3) were hyper- or hypomethylated in both cell lines. Conclusions: Our data indicate that GGH/FPGS modulation can affect gene-specific promoter CpG island Methylation as well as genomic DNA Methylation, which might influence chemosensitivity of colon and breast cancer cells to 5FU and antifolates. Studies are underway to investigate the functional ramifications of the altered CpG Methylation in response to GGH/FPGS modulation in these cell lines. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1620. doi:10.1158/1538-7445.AM2011-1620

  • cobra a sensitive and quantitative DNA Methylation Assay
    Nucleic Acids Research, 1997
    Co-Authors: Zhenggang Xiong, Peter W Laird
    Abstract:

    We report here on a quantitative technique called COBRA to determine DNA Methylation levels at specific gene loci in small amounts of genomic DNA. Restriction enzyme digestion is used to reveal Methylation-dependent sequence differences in PCR products of sodium bisulfite-treated DNA as described previously. We show that Methylation levels in the original DNA sample are represented by the relative amounts of digested and undigested PCR product in a linearly quantitative fashion across a wide spectrum of DNA Methylation levels. In addition, we show that this technique can be reliably applied to DNA obtained from microdissected paraffin-embedded tissue samples. COBRA thus combines the powerful features of ease of use, quantitative accuracy, and compatibility with paraffin sections.

Gwo-bin Lee - One of the best experts on this subject based on the ideXlab platform.

  • A DNA Methylation Assay for detection of ovarian cancer cells using a HpaII/MspI digestion-based PCR Assay in an integrated microfluidic system
    Microfluidics and Nanofluidics, 2013
    Co-Authors: Chih-hung Wang, Keng Fu Hsu, Cheng Yang Chou, Hsien-chih Lai, Tong-miin Liou, Gwo-bin Lee
    Abstract:

    Early and accurate diagnosis of cancer plays a very important role in favorable clinical outcomes. DNA Methylation of tumor suppressor genes has been recognized as a diagnostic biomarker for early carcinogenesis. The presence of 5-methylcytosine in the CpG islands in the promoter region of a tumor suppressor gene is an important indicator of DNA Methylation. However, the standard detection Assay utilizing a bisulfite treatment and Hpa II/ Msp I endonuclease digestion is a tedious and lengthy process and requires a relatively large amount of DNA for testing. In this study, the methylated DNAs of various tumor suppressor genes, HAAO, HOXA9 and SFRP5, were chosen as candidates for detection of ovarian cancer cells. The entire experimental process for the DNA Methylation Assay, including target DNA isolation, Hpa II/ Msp I endonuclease digestion, and nucleic acid amplification has been realized in an integrated microfluidic system. The limit of detection using this developed system has been experimentally determined to be 10^2 cells/reaction. The entire process from sample loading to analysis of the results only took 3 h which is much faster than the existing protocols. Different sources of biosamples, such as cells, ascites and serums, could be detected with the methylated DNA, indicating that this developed microfluidic system could be adapted for clinical use. Thus, this developed microsystem may be a promising platform for the rapid and early diagnosis of cancers.

  • a DNA Methylation Assay for detection of ovarian cancer cells using a hpaii mspi digestion based pcr Assay in an integrated microfluidic system
    Microfluidics and Nanofluidics, 2013
    Co-Authors: Chih-hung Wang, Keng Fu Hsu, Cheng Yang Chou, Hsien-chih Lai, Tong-miin Liou, Gwo-bin Lee
    Abstract:

    Early and accurate diagnosis of cancer plays a very important role in favorable clinical outcomes. DNA Methylation of tumor suppressor genes has been recognized as a diagnostic biomarker for early carcinogenesis. The presence of 5-methylcytosine in the CpG islands in the promoter region of a tumor suppressor gene is an important indicator of DNA Methylation. However, the standard detection Assay utilizing a bisulfite treatment and HpaII/ MspI endonuclease digestion is a tedious and lengthy process and requires a relatively large amount of DNA for testing. In this study, the methylated DNAs of various tumor suppressor genes, HAAO, HOXA9 and SFRP5, were chosen as candidates for detection of ovarian cancer cells. The entire experimental process for the DNA Methylation Assay, including target DNA isolation, HpaII/MspI endo- nuclease digestion, and nucleic acid amplification has been realized in an integrated microfluidic system. The limit of detection using this developed system has been experi- mentally determined to be 10 2 cells/reaction. The entire process from sample loading to analysis of the results only took 3 h which is much faster than the existing protocols. Different sources of biosamples, such as cells, ascites and serums, could be detected with the methylated DNA, indicating that this developed microfluidic system could be adapted for clinical use. Thus, this developed microsystem may be a promising platform for the rapid and early diagnosis of cancers.

  • Enzyme digestion-based microfluidic system for DNA Methylation Assay
    2012 IEEE 25th International Conference on Micro Electro Mechanical Systems (MEMS), 2012
    Co-Authors: Chih-hung Wang, Keng Fu Hsu, Cheng Yang Chou, Gwo-bin Lee
    Abstract:

    Early and accurate diagnosis of cancer plays a very important role in cancer treatment. The DNA Methylation of tumor suppressor genes has been used as a diagnostic biomarker of early carcinogenesis. The 5-methylcytosine of CpG islands in the promoter region has been demonstrated as an evidence of DNA Methylation. In this study, the entire process for performing DNA Methylation Assay including capturing of target DNA, HpaII and MspI endonuclease digestion and nucleic acid amplification have been realized in an integrated microfluidic system. The limit of detection in the microfluidic system was experimentally found to be 102 cells/reaction. The entire process from sample loading to results observed only takes 3 hrs. Rapid diagnosis of ovarian cancer cells in the integrated microfluidic system has been demonstrated by using cell lines and clinical samples. The developed micro system may be promising for early diagnosis of cancers.

Fang Zhao - One of the best experts on this subject based on the ideXlab platform.

  • A urine-based DNA Methylation Assay, ProCUrE, to identify clinically significant prostate cancer
    Clinical epigenetics, 2018
    Co-Authors: Fang Zhao, Ekaterina Olkhov-mitsel, Shivani Kamdar, Renu Jeyapala, Julia Garcia, Rachel Hurst, Marcelino Yazbek Hanna, Robert D. Mills, Alexandra V. Tuzova, Eve O’reilly
    Abstract:

    Prevention of unnecessary biopsies and overtreatment of indolent disease remains a challenge in the management of prostate cancer. Novel non-invasive tests that can identify clinically significant (intermediate-risk and high-risk) diseases are needed to improve risk stratification and monitoring of prostate cancer patients. Here, we investigated a panel of six DNA Methylation biomarkers in urine samples collected post-digital rectal exam from patients undergoing prostate biopsy, for their utility to guide decision making for diagnostic biopsy and early detection of aggressive prostate cancer. We recruited 408 patients in risk categories ranging from benign to low-, intermediate-, and high-risk prostate cancer from three international cohorts. Patients were separated into 2/3 training and 1/3 validation cohorts. Methylation biomarkers were analyzed in post-digital rectal exam urinary sediment DNA by quantitative MethyLight Assay and investigated for their association with any or aggressive prostate cancers. We developed a Prostate Cancer Urinary Epigenetic (ProCUrE) Assay based on an optimal two-gene (HOXD3 and GSTP1) LASSO model, derived from Methylation values in the training cohort, and assessed ProCUrE’s diagnostic and prognostic ability for prostate cancer in both the training and validation cohorts. ProCUrE demonstrated improved prostate cancer diagnosis and identification of patients with clinically significant disease in both the training and validation cohorts. Using three different risk stratification criteria (Gleason score, D’Amico criteria, and CAPRA score), we found that the positive predictive value for ProCUrE was higher (59.4–78%) than prostate specific antigen (PSA) (38.2–72.1%) for all risk category comparisons. ProCUrE also demonstrated additive value to PSA in identifying GS ≥ 7 PCa compared to PSA alone (DeLong’s test p = 0.039), as well as additive value to the PCPT risk calculator for identifying any PCa and GS ≥ 7 PCa (DeLong’s test p = 0.011 and 0.022, respectively). ProCUrE is a promising non-invasive urinary Methylation Assay for the early detection and prognostication of prostate cancer. ProCUrE has the potential to supplement PSA testing to identify patients with clinically significant prostate cancer.