High-performance single cell genetic analysis using microfluidic emulsion generator arrays.
High-performance single cell genetic analysis using microfluidic emulsion generator arrays.
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DOI：
10.1021/ac902683t
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发表时间：
2010-04-15
影响因子：
7.4
通讯作者：
Mathies, Richard A.
中科院分区：
文献类型：
--
作者：
Zeng, Yong;Novak, Richard;Shuga, Joe;Smith, Martyn T.;Mathies, Richard A.
在单细胞水平进行高通量的基因和表型分析对于增进我们对细胞功能和功能失调的分子机制的理解至关重要。在此我们描述了一种高性能的单细胞基因分析（SCGA）技术，它将高通量微流控乳液生成与单细胞多重PCR相结合。开发了包含4、32和96个通道的微加工乳液发生器阵列（MEGA）装置，赋予了每小时产生多达3.4×10⁶纳升体积液滴的灵活能力。集成在MEGA芯片中的混合玻璃 - 聚二甲基硅氧烷隔膜微泵可实现均匀的液滴形成、可控的产生频率以及引物功能化微珠和细胞的有效运输和包封。开发了一种多重单细胞PCR方法来检测和定量野生型以及突变/致病细胞。在此方法中，用针对不同细胞类型特定基因的多个正向引物功能化的微珠用于液滴中的固相PCR。PCR之后，裂解液滴，收集微珠并通过多色流式细胞术快速分析。以大肠杆菌细胞作为模型，我们表明该技术能够在正常K12细胞的高背景下对致病性大肠杆菌O157细胞进行数字检测，检测限约为1:10⁵。这一结果表明多重SCGA是对复杂群体中基因变异进行高通量定量数字分析的一种有前景的工具。
High-throughput genetic and phenotypic analysis at the single cell level is critical to advance our understanding of the molecular mechanisms underlying cellular function and dysfunction. Here we describe a high-performance single cell genetic analysis (SCGA) technique that combines high-throughput microfluidic emulsion generation with single cell multiplex PCR. Microfabricated emulsion generator array (MEGA) devices containing 4, 32 and 96 channels are developed to confer a flexible capability of generating up to 3.4 × 106 nanoliter-volume droplets per hour. Hybrid glass-polydimethylsiloxane diaphragm micropumps integrated into the MEGA chips afford uniform droplet formation, controlled generation frequency, and effective transportation and encapsulation of primer functionalized microbeads and cells. A multiplex single cell PCR method is developed to detect and quantify both wild type and mutant/pathogenic cells. In this method, microbeads functionalized with multiple forward primers targeting specific genes from different cell types are used for solid-phase PCR in droplets. Following PCR, the droplets are lysed, the beads are pooled and rapidly analyzed by multi-color flow cytometry. Using E. coli bacterial cells as a model, we show that this technique enables digital detection of pathogenic E. coli O157 cells in a high background of normal K12 cells, with a detection limit on the order of 1:105. This result demonstrates that multiplex SCGA is a promising tool for high-throughput quantitative digital analysis of genetic variation in complex populations.
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影响因子：
168.9
作者：
Klein, CA;Blankenstein, TJF;Riethmüller, G
通讯作者：
Riethmüller, G
DOI：
10.1073/pnas.0507904102
发表时间：
2005-11-08
影响因子：
11.1
作者：
Diehl, F;Li, M;Vogelstein, B
通讯作者：
Vogelstein, B
影响因子：
14.9
作者：
Fuhrmann C;Schmidt-Kittler O;Stoecklein NH;Petat-Dutter K;Vay C;Bockler K;Reinhardt R;Ragg T;Klein CA
通讯作者：
Klein CA
DOI：
10.1038/nrc2604
发表时间：
2009-05
期刊：
Nature reviews. Cancer
影响因子：
--
作者：
通讯作者：
--
影响因子：
8.4
作者：
Cady, NC;Stelick, S;Batt, CA
通讯作者：
Batt, CA