In-Gel Technology for PCR Genotyping and Pathogen Detection
In-Gel Technology for PCR Genotyping and Pathogen Detection
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DOI：
10.1021/ac1013157
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发表时间：
2010-10-01
影响因子：
7.4
通讯作者：
Acker, Jason P.
中科院分区：
文献类型：
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作者：
Atrazhev, Alexey;Manage, Dammika P.;Acker, Jason P.
这项工作描述了使用聚丙烯酰胺凝胶和PCR试剂在模具中光聚合，以创建一个阵列的半固体职位，作为反应容器的并行PCR扩增的外部添加的模板。DNA扩增发生在圆柱形的自立式9 × 9凝胶柱阵列中，每个凝胶柱的体积小于1 μ L。在聚合之前加入嵌入染料的凝胶的光聚合允许采集实时PCR数据和熔解曲线分析数据，而不需要任何类型的PCR后染色程序。当模板DNA在凝胶内聚合时或当外源性模板添加到预制凝胶柱顶上时，PCR同样有效且可重复。以BK病毒尿症患者的尿液或纯化DNA为模板进行PCR扩增。每个凝胶后阵列可以使用多个引物组，没有可检测的交叉污染。在单个凝胶柱中通过PCR一致地检测到少至34个BK病毒模板。在人基因组DNA（gDNA）中扩增HPA 1和FGFR 2基因需要少至2-5 ng的gDNA模板/凝胶柱。该设备原型包括用于PCR热循环的Peltier元件和用于捕获荧光以用于产物检测的CCD相机。我们的技术适合于集成在护理点微型设备中。
This work describes the use of polyacrylamide gel and PCR reagents photopolymerized in a mold to create an array of semisolid posts that serve as reaction vessels for parallel PCR amplification of an externally added template. DNA amplification occurred in a cylindrical, self-standing 9 x 9 array of gel posts each less than 1 mu L in volume. Photopolymerization of the gel with an intercalating dye added prior to polymerization permitted acquisition of real-time PCR data and melting curve analysis data without the need for any type of post-PCR staining procedures. PCR was equally efficient and reproducible when template DNA was polymerized within the gel or when exogenous template was added atop precast gel posts. PCR amplification occurred with template from purified DNA or from raw urine of patients with BK viruria. Multiple primer sets can be utilized per gel post array with no detectable cross contamination. As few as 34 BK virus templates were consistently detected by PCR in an individual gel post. Amplification of HPA1 and FGFR2 genes in human genomic DNA (gDNA) required as little as 2-5 ng of gDNA template/gel post. The device prototype includes a Peltier element for PCR thermal cycling and a CCD camera to capture fluorescence for product detection. Our technology is amenable to integration in point of care microdevices.