Highly sensitive detection of DNA hypermethylation in melanoma cancer cells.
Highly sensitive detection of DNA hypermethylation in melanoma cancer cells.
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DOI：
10.1016/j.bios.2018.10.018
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发表时间：
2019-01-15
影响因子：
12.6
通讯作者：
Wang SX
中科院分区：
文献类型：
--
作者：
Nesvet J;Rizzi G;Wang SX
抑癌基因启动子区 CpG 岛的异常高甲基化是早期癌症检测的一个有前途的生物标志物。这种甲基化状态反映在原发肿瘤脱落的 ctDNA 的甲基化模式中；然而，为了实现液体活检ctDNA甲基化检测在早期癌症诊断中的全面临床应用，必须提高现有技术的灵敏度和多重性。此外，该测定必须便宜且易于在临床环境中进行。我们报告了甲基化特异性 PCR (MSP) 与巨磁阻 (GMR) 生物传感器熔解曲线分析的集成，以大大提高我们的 DNA 杂交检测甲基化检测的灵敏度。我们的 GMR 传感器采用合成 DNA 探针进行功能化，该探针针对 MSP 扩增子中的甲基化或非甲基化 CpG 位点，并测量两个探针之间的解链温度 (Tm) 差异 (ΔTm)，从而使溶液中甲基化 DNA 的分析检测限低至 0.1%。此外，甲基化：非甲基化混合物连续稀释的 ΔTm 线性回归可以量化甲基化百分比，这可能具有诊断和预后用途。最后，我们对两个不同的基因进行了多重 MSP，并展示了我们的 GMR 测定法解析该混合物的能力，尽管它们的扩增子在标准 EvaGreen 熔解分析中的 Tm 重叠。我们的检测方法的多重检测能力及其增强的灵敏度，无需深度测序，代表了实现血浆中甲基化 ctDNA 检测方法的重要一步，用于临床环境中的早期癌症检测。
Aberrant hypermethylation of CpG islands in the promoter region of tumor suppressor genes is a promising biomarker for early cancer detection. This methylation status is reflected in the methylation pattern of ctDNA shed from the primary tumor; however, to realize the full clinical utility of ctDNA methylation detection via liquid biopsy for early cancer diagnosis, improvements in the sensitivity and multiplexability of existing technologies must be improved. Additionally, the assay must be cheap and easy to perform in a clinical setting. We report the integration of methylation specific PCR (MSP) to melt curve analysis on giant magnetoresistive (GMR) biosensors to greatly enhance the sensitivity of our DNA hybridization assay for methylation detection. Our GMR sensor is functionalized with synthetic DNA probes that target methylated or unmethylated CpG sites in the MSP amplicon, and measures the difference in melting temperature (Tm) between the two probes (ΔTm), giving an analytical limit of detection down to 0.1% methylated DNA in solution. Additionally, linear regression of ΔTm’s for serial dilutions of methylated:unmethylated mixtures allows for quantification of methylation percentage, which could have diagnostic and prognostic utility. Lastly, we performed multiplexed MSP on two different genes, and show the ability of our GMR assay to resolve this mixture, despite their amplicons’ overlapping Tm’s in standard EvaGreen melt analysis. The multiplexing ability of our assay and its enhanced sensitivity, without necessitating deep sequencing, represent important steps toward realizing an assay for the detection of methylated ctDNA in plasma for early cancer detection in a clinical setting.
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影响因子：
6.1
作者：
Earhart CM;Hughes CE;Gaster RS;Ooi CC;Wilson RJ;Zhou LY;Humke EW;Xu L;Wong DJ;Willingham SB;Schwartz EJ;Weissman IL;Jeffrey SS;Neal JW;Rohatgi R;Wakelee HA;Wang SX
通讯作者：
Wang SX
影响因子：
12.6
作者：
Hall DA;Gaster RS;Lin T;Osterfeld SJ;Han S;Murmann B;Wang SX
通讯作者：
Wang SX
影响因子：
7.4
作者：
Lee, Jung-Rok;Choi, Joohong;Wang, Shan X.
通讯作者：
Wang, Shan X.
影响因子：
--
作者：
Mehrotra M;Singh RR;Loghavi S;Duose DY;Barkoh BA;Behrens C;Patel KP;Routbort MJ;Kopetz S;Broaddus RR;Medeiros LJ;Wistuba II;Luthra R
通讯作者：
Luthra R
影响因子：
11.5
作者：
Ooki, Akira;Maleki, Zahra;Hoque, Mohammad Obaidul
通讯作者：
Hoque, Mohammad Obaidul