Bare nanocapillary for DNA separation and genotyping analysis in gel-free solutions without application of external electric field
Bare nanocapillary for DNA separation and genotyping analysis in gel-free solutions without application of external electric field
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DOI：
10.1021/ac800549k
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发表时间：
2008-07-15
影响因子：
7.4
通讯作者：
Liu, Shaorong
中科院分区：
文献类型：
--
作者：
Wang, Xiayan;Wang, Shili;Liu, Shaorong
在这项工作中，我们演示了在压力驱动条件下使用纳米毛细管在无凝胶溶液中进行DNA分离和基因分型分析，而无需应用外电场。纳米毛细管类似于50 cm长、500 nm半径的裸熔融二氧化硅毛细管。DNA样品注入后，分析物以色谱分离的形式被洗脱出来。DNA分子的洗脱顺序严格遵循它们的大小，较长的DNA比较短的DNA被洗脱得更快。对于短的（几个碱基）和长的（数万个碱基对）DNA片段都获得了高分辨率。考察了洗脱液组成、洗脱压力等关键实验参数对分离效率和分辨率的影响。我们还将该技术应用于真实世界基因分型样本的DNA分离，以证明其在生物学应用中的可行性。从拟南芥植物基因组DNA粗制剂中扩增的PCR产物（未经任何纯化）直接注射到纳米毛细管中，PCR扩增的DNA片段被很好地分解，从而可以明确地识别杂合和纯合个体的样品。由于用于进行分离的毛细管是无涂层的，柱寿命实际上是无限的。在这些分析中消耗的唯一材料是洗脱液，因此，操作成本很低。
In this work, we demonstrate DNA separation and genotyping analysis in gel-free solutions using a nanocapillary under pressure-driven conditions without application of an external electric field. The nanocapillary is a similar to 50-cm-long and 500-nm-radius bare fused-silica capillary. After a DNA sample is injected, the analytes are eluted out in a chromatographic separation format. The elution order of DNA molecules follows strictly with their sizes, with the longer DNA being eluted out faster than the shorter ones. High resolutions are obtained for both short (a few bases) and long (tens of thousands of base pairs) DNA fragments. Effects of key experimental parameters, such as eluent composition and elution pressure, on separation efficiency and resolution are investigated. We also apply this technique for DNA separations of real-world genotyping samples to demonstrate its feasibility in biological applications. PCR products (without any purification) amplified from Arabidopsis plant genomic DNA crude preparations are directly injected into the nanocapillary, and PCR-amplified DNA fragments are well resolved, allowing for unambiguous identification of samples from heterozygous and homozygous individuals. Since the capillaries used to conduct the separations are uncoated, column lifetime is virtually unlimited. The only material that is consumed in these assays is the eluent, and hence, the operation cost is low.