Rapid pulsed field capillary electrophoretic separation of megabase nucleic acids.
Rapid pulsed field capillary electrophoretic separation of megabase nucleic acids.
复制标题
兆碱基核酸的快速脉冲场毛细管电泳分离。
DOI：
10.1021/ac00101a002
复制
发表时间：
1995
影响因子：
7.4
通讯作者：
Morris,MD
中科院分区：
文献类型：
--
作者：
Kim,Y;Morris,MD
在筛分聚合物超稀溶液中的脉冲场毛细管电泳已被用于分离长达160万碱基对的核酸片段。羟乙基纤维素溶液用于8000- 50000碱基对大小范围内的分离。在混合的羟乙基纤维素/聚（环氧乙烷）溶液中分离较长链的核酸。分离速度快（12-13分钟），但用恒电场（dc）毛细管凝胶电泳（CGE）分离长度大于约20-30 kbp（千碱基对）的双链（ds）DNA片段是困难的。只有部分分辨率的片段长达48 kbp已被证明。123.4约50 kbp以上的片段共迁移而不分离。为了改进CGE分离，一些实验室已经提出使用脉冲场毛细管凝胶电泳（PFCGE）。5 '7该技术来源于琼脂糖平板凝胶脉冲场电泳，其将经典的核酸分离从约20 kbp的大小限制扩展到至少10 Mbp（兆碱基对）。关于PFCGE的最初报道主要针对技术开发，并描述了分离相对较短的ds-DNA片段（0.5-23 kbp）的应用，其中预期或观察到仅适度增加分辨率和减少分离时间。在这项工作中，开发了几个重要的实验技术，包括线性聚丙烯酰胺筛分缓冲液的初始应用，5正弦调制的驱动电压，8延时场反转，7和随机脉冲持续时间场反转9在超稀聚合物溶液7以及在更传统的缓冲液。5· 6· 89尽管大多数报告都是关于核酸分离的，但PFCGE已被证明可以提高多糖分离的分辨率，
Pulsed field capillary electrophoresis in ultradilute solu-tions of sieving polymers has been used to separate nucleic acid fragments as long as 1.6 million base pairs. Hydroxyethyl cellulose solutions are used for separations in the size range 8000-50 000 base pairs. Longer chain nucleic acids are separated in mixed hydroxyethyl cel-lulose/poly (ethylene oxide) solutions. Separations are rapid (12-13 min).It is difficult to separate double-stranded (ds) DNA fragments longer than about 20-30 kbp (thousand base pairs) by constant electric field (dc) capillary gel electrophoresis (CGE). Only partial resolution of fragments as long as 48 kbp has been demonstrated. 1 23'4 Above about 50 kbp fragments comigrate without separation. To improve CGE separations, several laboratories have proposed the use of pulsed field capillary gel electrophoresis (PFCGE). 5'7 The technique is derived from agarose slab gel pulsedfield electrophoresis, which extends classical nucleic acid separation from a size limit of about 20 kbp to at least 10 Mbp (mega base pairs). The initial reports on PFCGE were aimed mostly at technique development and described applications to separation of relatively short ds-DNA fragments (0.5-23 kbp), where only modest increases in resolution and decreases in separation time were expected or observed. In this work, several important experimental techniques were developed, including theinitial application to linear polyacrylamide sieving buffers, 5 sinusoidal modulation of the driving voltage, 8 time-delayedfield inversion, 7 and random pulse duration field inversion9 in ultradilute polymer solutions7 as well as in more conventional buffers. 5· 6· 89 Although most reports have been to nucleic acid separations, PFCGE has been shown to improve resolution in polysaccharide separations as