Investigating DNA migration in pulsed fields using a miniaturized FIGE system.
Investigating DNA migration in pulsed fields using a miniaturized FIGE system.
复制标题
使用微型 FigE 系统研究脉冲场中的 DNA 迁移。
DOI：
10.1002/elps.200800113
复制
发表时间：
2008
期刊：
影响因子：
2.9
通讯作者：
Ugaz,VictorM
中科院分区：
文献类型：
--
作者：
Chen,Xiaojia;Ugaz,VictorM
PFGE是一种成熟的技术，用于分离长度从千碱基到兆碱基的DNA片段。但其中许多分离需要长的实验时间(通常接近几十个小时)和施加高电压(通常接近几十千伏)的不良组合。在这里，我们提出了一种简单的微型无花果仪器，能够在3 h内分离长度达32.5 kb的DNA片段，使用20 V的适度应用电位。该设备足够小，可以在荧光显微镜下成像，允许在分离过程中观察到迁移的DNA条带。我们利用这一能力来研究包括正反向电压比、脉冲时间和温度在内的参数对分离性能的影响。我们还表征了DNA迁移率对片段大小N的依赖关系，并在所研究的大小范围内观察到n−0.5附近的比例。该系统的高速度、低功耗和简单的设计有助于快速和廉价地进行未来在PFGE中的DNA迁移研究。
PFGE is a well‐established technique for fractionation of DNA fragments ranging from kilobases to megabases in length. But many of these separations require an undesirable combination of long experiment times (often approaching tens of hours) and application of high voltages (often approaching tens of kV). Here, we present a simple miniaturized FIGE apparatus capable of separating DNA fragments up to 32.5 kb in length within 3 h using a modest applied potential of 20 V. The device is small enough to be imaged under a fluorescence microscope, permitting the migrating DNA bands to be observed during the course of the separation run. We use this capability to investigate how separation performance is affected by parameters including the ratio of forward and backward voltage, pulse time, and temperature. We also characterize the dependence of DNA mobility on fragment sizeN, and observe a scaling in the vicinity ofN−0.5over the size range investigated. The high speed, low power consumption, and simple design of this system may help enable future studies of DNA migration in PFGE to be performed quickly and inexpensively.