Jung Yun Kyung, Kim Jungkyu, Mathies Richard A
†Department of Chemistry, University of California, Berkeley, California 94720, United States.
‡School of Natural Science, Ulsan National Institute of Science and Technology, Ulsan 689-798, Republic of Korea.
Anal Chem. 2015 Mar 17;87(6):3165-70. doi: 10.1021/ac5048696. Epub 2015 Feb 23.
A PDMS-based microfluidic linear hydrogel array is developed for multiplexed single nucleotide polymorphism (SNP) detection. A sequence of three-dimensional (3D) hydrogel plugs containing the desired DNA probes is prepared by UV polymerization within a PDMS microchannel system. The fluorescently labeled target DNA is then electrophoresed through the sequence of hydrogel plugs for hybridization. Continued electrophoresis provides an electrophoretic wash that removes nonspecific binders. The capture gel array is imaged after washing at various temperatures (temperature gradient electrophoresis) to further distinguish perfect matches from mismatches. The ability of this microdevice to perform multiplex SNP genotyping is demonstrated by analyzing a mixture of model E. coli bacterial targets. This microfluidic hydrogel array is ∼1000 times more sensitive than planar microarrays due to the 3D gel capture, the hybridization time is much shorter due to electrophoretic control of the transport properties, and the stringent wash with temperature gradient electrophoresis enables analysis of single nucleotide mismatches with high specificity.
开发了一种基于聚二甲基硅氧烷（PDMS）的微流控线性水凝胶阵列，用于多重单核苷酸多态性（SNP）检测。通过在PDMS微通道系统内进行紫外光聚合，制备了一系列包含所需DNA探针的三维（3D）水凝胶塞。然后，将荧光标记的目标DNA通过水凝胶塞序列进行电泳以进行杂交。持续的电泳提供了一种电泳洗涤，可去除非特异性结合物。在不同温度下洗涤后（温度梯度电泳）对捕获凝胶阵列进行成像，以进一步区分完全匹配和错配。通过分析模型大肠杆菌细菌靶标的混合物，证明了这种微型设备进行多重SNP基因分型的能力。由于3D凝胶捕获，这种微流控水凝胶阵列比平面微阵列敏感约1000倍，由于对传输特性的电泳控制，杂交时间要短得多，并且温度梯度电泳的严格洗涤能够以高特异性分析单核苷酸错配。
