School of Applied and Engineering Physics, Cornell University , Ithaca, New York 14853, United States.
Anal Chem. 2018 Feb 20;90(4):2601-2608. doi: 10.1021/acs.analchem.7b04120. Epub 2018 Feb 1.
We present a microfluidic device for specifically capturing cancer cells and isolating their genomic DNA (gDNA) for specific amplification and sequence analysis. To capture cancer cells within the device, nucleic acid aptamers that specifically bind to cancer cells were immobilized within a channel containing micropillars designed to increase capture efficiency. The captured cells were lysed in situ, and their gDNA was isolated by physical entanglement within a second smaller-dimensioned micropillar array. This type of isolation allows the gDNA to be retained and purified within the channel and enables amplification and analysis to be performed on the gDNA without the loss of the original template. We developed a technique for selectively amplifying genes from whole gDNA using multiple displacement amplification. The amplified gene samples were sequenced, and the resulting sequence information was compared against the known wild-type gene to identify any mutations. We have tested cervical and ovarian cancer cells for mutations in the TP53 gene using this technology. This approach offers a way to monitor multiple genetic mutations in the same small population of cells, which is beneficial given the wide diversity in cancer cells, and therefore it requires very few cells to be extracted from a patient sample.
我们提出了一种微流控装置，用于特异性捕获癌细胞并分离其基因组 DNA（gDNA），以进行特定的扩增和序列分析。为了在装置内捕获癌细胞，将特异性结合癌细胞的核酸适体固定在含有微柱的通道内，这些微柱设计用于提高捕获效率。捕获的细胞在原位裂解，其 gDNA 被物理纠缠在第二个较小尺寸的微柱阵列中而被分离。这种类型的分离允许 gDNA 保留在通道内并被纯化，并允许在不丢失原始模板的情况下对 gDNA 进行扩增和分析。我们开发了一种使用多次置换扩增从全 gDNA 中选择性扩增基因的技术。对扩增的基因样本进行测序，并将得到的序列信息与已知的野生型基因进行比较，以识别任何突变。我们已经使用该技术测试了宫颈癌和卵巢癌细胞中 TP53 基因的突变。这种方法提供了一种在同一小群细胞中监测多种基因突变的方法，鉴于癌细胞的广泛多样性，这是有益的，因此从患者样本中提取很少的细胞即可。
