Tam Brooke E, Hao Yining, Sikes Hadley D
Dept. of Chemical Engineering, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA, 02139.
Biotechnol Prog. 2018 Nov;34(6):1589-1595. doi: 10.1002/btpr.2644. Epub 2018 Jul 27.
Gene-specific promoter methylation is involved in gene silencing and is an important cancer biomarker. Cancer-specific methylation patterns have been observed and clinically validated for numerous gene promoters, but the knowledge gleaned from this large body of work is currently under-utilized in the clinic. Methylation-specific PCR is currently the gold standard method for clinical methylation assessment, but several research groups have proposed hybridization-based techniques which could be simpler to implement and provide more accurate results. However, the sensitivity of this easier alternative must be improved dramatically in order to compete with methylation-specific PCR. Efficient sample capture is a key step in maximizing sensitivity, so here we investigate the key parameters involved in (i) maximizing the capture of gene-specific target DNA molecules at the surfaces of functionalized, magnetic microparticles and (ii) recognizing DNA methylation using an engineered methyl-CpG-binding domain (MBD) protein. The magnetic bead density, the probe concentration, and the MBD concentration were very important for maximizing detection, and other variables such as the hybridization time also impacted the target capture efficiency but had a smaller effect on the overall methylation assay. The effect of genomic DNA on the capture of the target sequence was also investigated, and model methylated vs. unmethylated target sequences could be distinguished in the presence of 1 ng/μL genomic DNA. The findings we report related to the underlying binding events involved in hybridization-based epigenotyping can be leveraged in combination with the many signal amplification and detection approaches that are currently being developed. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:1589-1595, 2018.
基因特异性启动子甲基化与基因沉默有关，是一种重要的癌症生物标志物。已观察到多种基因启动子具有癌症特异性甲基化模式，并在临床上得到验证，但目前临床对从大量此类研究中获得的知识利用不足。甲基化特异性PCR是目前临床甲基化评估的金标准方法，但几个研究小组提出了基于杂交的技术，这些技术可能更易于实施且能提供更准确的结果。然而，为了与甲基化特异性PCR竞争，这种更简便方法的灵敏度必须大幅提高。高效的样本捕获是实现灵敏度最大化的关键步骤，因此我们在此研究了涉及以下方面的关键参数：（i）在功能化磁性微粒表面最大化捕获基因特异性靶DNA分子；（ii）使用工程化甲基化CpG结合域（MBD）蛋白识别DNA甲基化。磁珠密度、探针浓度和MBD浓度对于最大化检测非常重要，其他变量如杂交时间也会影响靶标捕获效率，但对整体甲基化检测的影响较小。还研究了基因组DNA对靶序列捕获的影响，在存在1 ng/μL基因组DNA的情况下，可以区分甲基化与未甲基化的模型靶序列。我们报告的与基于杂交的表观基因分型中潜在结合事件相关的研究结果，可与目前正在开发的许多信号放大和检测方法结合利用。© 2018美国化学工程师学会生物技术进展，34:1589 - 1595，2018年。
