DNA purification on a lab-on-valve system incorporating a renewable microcolumn with in situ monitoring by laser-induced fluorescence
DNA purification on a lab-on-valve system incorporating a renewable microcolumn with in situ monitoring by laser-induced fluorescence
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DOI：
10.1007/s00216-007-1196-0
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发表时间：
2007-05-01
影响因子：
4.3
通讯作者：
Wang, Jian-Hua
中科院分区：
文献类型：
--
作者：
Chen, Xu-Wei;Xu, Zhang-Run;Wang, Jian-Hua
采用微珠注射法，通过微固相萃取（SPE）纯化DNA，并将可再生的硅胶微柱填充在阀上实验室（LOV）中。通过选择合适的上样和洗脱介质，可以很好地去除人全血中复杂的基质成分，包括蛋白质。DNA纯化过程进行了在线监测，通过使用激光诱导的荧光在可拆卸的侧部的LOV单元纳入光纤。整个系统的实际适用性被证明通过分离/纯化在一个模拟的矩阵和人类血液遗传DNA的DNA，通过执行SPE，在原位监测的纯化产物，和柱后PCR扩增的DNA。当DNA在一个模拟的矩阵中在本系统中处理（10.0 ng μ l（-1）DNA，50 ng μ l（-1）牛血清白蛋白，1.0%Triton X-100），在610 nm处监测激光诱导的荧光，通过采用相同的样品加载量和0.5 μ l/s（-1）的洗脱流速，实现了70%的总提取/收集效率，沿着，相对标准偏差为3.8%。在类似的条件下进行人全血样品的DNA分离和纯化。
Bead injection in a lab-on-valve (LOV) system was adopted for DNA purification via micro solid-phase extraction (SPE) with a renewable silica microcolumn packed in a channel of the LOV unit. The complex matrix components in human whole blood, including proteins, were well eliminated by choosing properly the sample loading and elution media. The DNA purification process was monitored on-line by using laser-induced fluorescence in a demountable side part of the LOV unit incorporating optical fibers. The practical applicability of the entire system was demonstrated by separation/purification of lambda-DNA in a simulated matrix and human blood genetic DNA by performing SPE, in situ monitoring of the purified products, and postcolumn PCR amplification. When DNAs in a simulated matrix (10.0 ng mu l(-1) lambda-DNA, 50 ng mu l(-1) bovine serum albumin, 1.0% Triton X-100) were processed in the present system and laser-induced fluorescence was monitored at 610 nm, an overall extraction/collection efficiency of 70% was achieved by employing identical sample loading and an elution flow rate of 0.5 mu l s(-1), along with a precision of 3.8% relative standard deviation. DNA separation and purification from human whole-blood samples were performed under similar conditions.