Integrated two-step gene synthesis in a microfluidic device
Integrated two-step gene synthesis in a microfluidic device
复制标题
DOI：
10.1039/b807688j
复制
发表时间：
2009-01-01
期刊：
影响因子：
6.1
通讯作者：
Ying, Jackie Y.
中科院分区：
文献类型：
--
作者：
Huang, Mo Chao;Ye, Hongye;Ying, Jackie Y.
在此，我们提出了一种集成的微流体装置，能够进行两步基因合成，以组装成具有所需编码序列的基因的寡核苷酸池。该装置包括两个聚合酶链反应（PCR），温度控制的水凝胶阀，电磁微混合器，穿梭微混合器，体积计，和基于磁珠的固相PCR纯化，使用快速成型方法制造而无需光刻工艺。所制造的装置与小型化热循环仪组合以进行基因合成。首先通过聚合酶链组装（PCA）将寡核苷酸组装成基因，并通过第二PCR扩增全长基因。通过固相PCR纯化从PCR反应混合物中进一步分离合成的基因。我们已经成功地使用该装置合成了一个绿色荧光蛋白片段（GFPuv）（760 bp），并获得了与在商业热循环仪内的PCR管中进行的实验相当的合成产率和错误率。通过DNA测序确定的所得错误率为1/250 bp。据我们所知，这是第一个展示集成两步基因合成的微流体装置。
Herein we present an integrated microfluidic device capable of performing two-step gene synthesis to assemble a pool of oligonucleotides into genes with the desired coding sequence. The device comprised of two polymerase chain reactions (PCRs), temperature-controlled hydrogel valves, electromagnetic micromixer, shuttle micromixer, volume meters, and magnetic beads based solid-phase PCR purification, fabricated using a fast prototyping method without lithography process. The fabricated device is combined with a miniaturized thermal cycler to perform gene synthesis. Oligonucleotides were first assembled into genes by polymerase chain assembly(PCA), and the full-length gene was amplified by a second PCR. The synthesized gene was further separated from the PCR reaction mixture by the solid-phase PCR purification. We have successfully used this device to synthesize a green fluorescent protein fragment (GFPuv) (760 bp), and obtained comparable synthesis yield and error rate with experiments conducted in a PCR tube within a commercial thermal cycler. The resulting error rate determined by DNA sequencing was 1 per 250 bp. To our knowledge, this is the first microfluidic device demonstrating integrated two-step gene synthesis.