High-fidelity fractionation of ssDNA fragments differing in size by one-base on a spiral-channel electrophoretic chip
High-fidelity fractionation of ssDNA fragments differing in size by one-base on a spiral-channel electrophoretic chip
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DOI：
10.1002/elps.200900455
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发表时间：
2009-12-01
期刊：
影响因子：
2.9
通讯作者：
Misawa, Hiroaki
中科院分区：
文献类型：
--
作者：
Sun, Kai;Suzuki, Nobuko;Misawa, Hiroaki
为了从高覆盖度基因表达谱 (HiCEP) 分析生成的选择性 PCR 产物中分离出感兴趣的片段，非常需要能够区分和分离大小相差一个碱基（碱基对）的片段的高分辨率。我们在此报告一种用于自动高保真分馏的新型 4 英寸直径螺旋通道芯片装置。 180、181和182个碱基的重叠DNA片段被成功分离，大小仅相差一个碱基。对收集的片段进行 PCR 扩增，然后通过大小检查分析、DNA 测序和同源搜索进行评估。高分辨率分级分离的实现得益于以下因素的综合贡献：(i) 使用 30 cm 长的螺旋通道进行高分辨率分离，(ii) 避免 CE 期间未选择片段污染的封闭技术，以及 (iii) 精确的微尺度目标提取。通过优化仅与目标片段相对应的提取位置和提取时间，由未选择的片段引起的污染已大大降低至可忽略的水平。该技术可适应广泛的应用，例如蛋白质或细胞收集，其中对高纯度的要求比回收的分级材料的量更重要。
For the fractionation of fragments of interest from selective PCR products generated by high coverage gene expression profiling (HiCEP) analysis, high-resolution with the ability to discriminate and fractionate fragments differing by one base (base pair) in size is highly required. We report here on a new 4-inch diameter spiral-channel chip device for automatic high-fidelity fractionation. Overlapping DNA fragments of 180, 181 and 182 bases, with only one-base difference in size, were successfully fractionated. The collected fragments were PCR amplified, and then evaluated by size checking analysis, DNA sequencing, and homolog search. The high-resolution fractionation has been achieved because of the combined contributions of (i) the high-resolution separation using a 30 cm long spiral channel, (ii) a blocking technique to avoid contamination from unselected fragments during CE, and (iii) precise micro-scale target extraction. Contaminations due to unselected fractions have been greatly decreased to a negligible level by optimization of the extraction position and extraction time corresponding to the targeted segment only. This technique can be adapted to a wide range of applications, such as protein or cell collections where requirements for the high purity are more important than the amount of recovered fractionated material.