Low-density arrays were assembled into microfluidic channels hot-embossed in poly(methyl methacrylate) (PMMA) to allow the detection of low-abundant mutations in gene fragments (K-ras) that carry point mutations with high diagnostic value for colorectal cancers. Following spotting, the chip was assembled with a cover plate and the array accessed using microfluidics in order to enhance the kinetics associated with hybridization. The array was configured with zip code sequences (24-mers) that were complementary to sequences present on the target. The hybridization targets were generated using an allele-specific ligase detection reaction (LDR), in which two primers (discriminating primer that carriers the complement base to the mutation being interrogated and a common primer) that flank the point mutation and were ligated joined together) only when the particular mutation was present in the genomic DNA. The discriminating primer contained on its 5'-end the zip code complement (directs the LDR product to the appropriate site of the array), and the common primer carried on its 3' end a fluorescent dye (near-IR dye IRD-800). The coupling chemistry (5'-amine-containing oligonucleotide tethered to PMMA surface) was optimized to maximize the loading level of the zip code oligonucleotide, improve hybridization sensitivity (detection of low-abundant mutant DNAs in high copy numbers of normal sequences), and increase the stability of the linkage chemistry to permit re-interrogation of the array. It was found that microfluidic addressing of the array reduced the hybridization time from 3 h for a conventional array to less than 1 min. In addition, the coupling chemistry allowed reuse of the array > 12 times before noticing significant loss of hybridization signal. The array was used to detect a point mutation in a K-ras oncogene at a level of 1 mutant DNA in 10,000 wild-type sequences.

中文翻译：

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将微阵列组装在由聚（甲基丙烯酸甲酯）制成的微流控芯片中，用于检测低丰度的DNA突变。

将低密度阵列组装到在聚甲基丙烯酸甲酯（PMMA）中热压花的微流控通道中，以检测基因片段（K-ras）中的低丰度突变，这些基因片段具有对大肠癌具有高诊断价值的点突变。点样后，将芯片与盖板组装在一起，并使用微流控技术访问阵列，以增强与杂交相关的动力学。该阵列配置有邮政编码序列（24个单体），该序列与靶标上存在的序列互补。杂交目标是使用等位基因特异性连接酶检测反应（LDR）产生的，仅当基因组DNA中存在特定突变时，其中两个引物（被询问突变的携带补体碱基的区别性引物和一个共同的引物）位于点突变旁并连接在一起。区分引物在其5'端包含邮政编码补码（将LDR产物导向阵列的适当位点），而普通引物在其3'端带有荧光染料（近红外染料IRD-800） 。优化了偶联化学（拴在PMMA表面的含5'-胺的寡核苷酸）以最大化邮政编码寡核苷酸的负载水平，提高杂交灵敏度（检测高拷贝数的正常序列中的低丰度突变DNA），并增加了化学键的稳定性，从而可以对阵列进行重新询问。发现阵列的微流体寻址将杂交时间从常规阵列的3小时减少到少于1分钟。另外，在注意到杂交信号的显着损失之前，偶联化学允许阵列重复使用＞ 12次。该阵列用于检测K-ras癌基因中的点突变，其水平为10,000个野生型序列中的1个突变体DNA。