High-speed parallel separation of DNA restriction fragments using capillary array electrophoresis.
High-speed parallel separation of DNA restriction fragments using capillary array electrophoresis.
复制标题
使用毛细管阵列电泳高速平行分离 DNA 限制性片段。
DOI：
10.1006/abio.1993.1571
复制
发表时间：
1993
影响因子：
2.9
通讯作者：
Mathies,RA
中科院分区：
文献类型：
--
作者：
Clark,SM;Mathies,RA
开发了一种用于进行DNA的高速、高通量大小测定的新方法。含有长度在70和10，000个碱基对之间的DNA限制性片段的样品使用毛细管阵列在20分钟内进行电泳分离，并使用荧光检测系统以高灵敏度进行检测。在7个100 μm i.d.的阵列上进行ΦX174/HaeIII片段的分离，350 μm外径使用可替换的羟乙基纤维素溶液作为筛分介质的毛细管。在运行缓冲液中使用溴化乙锭对片段进行荧光标记，并用激光激发共焦荧光扫描仪进行检测。检测限为每条带0.1 pg DNA，进样稀释至0.1 ng/μl的DNA样品可检测到分离。从微量离心管盒中电动进样样品，采用的格式将有助于毛细管电泳在分子生物学分离中的应用。将该技术扩展到较低的DNA浓度和1000毛细管阵列的可行性进行了讨论。
A new method for performing high-speed, high-throughput sizing of DNA has been developed. Samples containing DNA restriction fragments between 70 and 10,000 base pairs in length are electrophoretically separated using capillary arrays in ∼20 min and detected with high sensitivity using a fluorescence detection system. The separations of ΦX174/HaeIII fragments are performed on an array of seven 100-μm i.d., 350-μm o.d. capillaries using replaceable hydroxyethyl-cellulose solutions as the sieving medium. The fragments are fluorescently labeled using ethidium bromide in the running buffer and detected with a laser-excited, confocal-fluorescence scanner. The limit of detection is ∼1 pg of DNA per band and separations can be detected injecting DNA samples as dilute as 0.1 ng/μl. Samples were electrokinetically injected from a cassette of microcentrifuge tubes using a format that will facilitate the application of capillary electrophoresis to separations in molecular biology. The feasibility of extending this technique to lower DNA concentrations and to ∼100 capillary arrays is discussed.