High-throughput single copy DNA amplification and cell analysis in engineered nanoliter droplets
High-throughput single copy DNA amplification and cell analysis in engineered nanoliter droplets
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DOI：
10.1021/ac800327d
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发表时间：
2008-05-15
影响因子：
7.4
通讯作者：
Mathies, Richard A.
中科院分区：
文献类型：
--
作者：
Kumaresan, Palani;Yang, Chaoyong James;Mathies, Richard A.
开发了一种高通量单拷贝遗传扩增（SCGA）工艺，该工艺利用微制造液滴发生器（mu DG）将单个DNA分子或细胞与引物功能化的微珠一起快速封装在均匀的PCR混合液滴中。纳升体积液滴独特地实现了适合远程测序和遗传分析的DNA目标的定量高产扩增。混合玻璃-聚二甲基硅氧烷（PDMS）微器件组件用于将微泵集成到mu DG中，该微泵提供均匀的液滴尺寸，可控的产生频率和有效的珠粒结合。批量PCR扩增后，将液滴裂解，回收微珠并通过流式细胞术快速分析。在单分子浓度下，DNA靶标的大小从380到1139 bp不等，使用SCGA进行定量扩增。从每个携带624 bp产物的100 amol的微珠中获得的远程测序结果表明，这些扩增子能够从单个微珠中实现原子摩尔级的Sanger测序，并提高焦磷酸测序的读取长度。对人淋巴细胞中甘油醛3磷酸脱氢酶（GAPDH）基因和细菌大肠杆菌K12细胞中g T B基因的成功单细胞分析表明，SCGA也将对单细胞进行高通量遗传分析。
A high-throughput single copy genetic amplification (SCGA) process is developed that utilizes a microfabricated droplet generator (mu DG) to rapidly encapsulate individual DNA molecules or cells together with primer functionalized microbeads in uniform PCR mix droplets. The nanoliter volume droplets uniquely enable quantitative high-yield amplification of DNA targets suitable for long-range sequencing and genetic analysis. A hybrid glass-polydimethylsiloxane (PDMS) microdevice assembly is used to integrate a micropump into the mu DG that provides uniform droplet size, controlled generation frequency, and effective bead incorporation. After bulk PCR amplification, the droplets are lysed and the beads are recovered and rapidly analyzed via flow cytometry. DNA targets ranging in size from 380 to 1139 bp at single molecule concentrations are quantitatively amplified using SCGA. Long-range sequencing results from beads each carrying similar to 100 amol of a 624 bp product demonstrate that these amplicons are competent for achieving attomole-scale Sanger sequencing from a single bead and for advancing pyrosequencing read-lengths. Successful single cell analysis of the glyceraldehyde 3 phosphate dehydrogenase (GAPDH) gene in human lymphocyte cells and of the g T B gene in bacterial Escherichia coli K12 cells establishes that SCGA will also be valuable for performing high-throughput genetic analysis on single cells.