Rapid pulsed field separation of DNA molecules up to 250 kb.
Rapid pulsed field separation of DNA molecules up to 250 kb.
复制标题
快速脉冲场分离高达 250 kb 的 DNA 分子。
DOI：
10.1093/nar/22.24.5366
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发表时间：
1994
影响因子：
14.9
通讯作者：
Lai,E
中科院分区：
文献类型：
--
作者：
Birren,B;Lai,E
脉冲场凝胶电泳（PFGE）能够分辨大范围的DNA分子，这些分子在常规琼脂糖凝胶中会全部共迁移。我们描述了脉冲场凝胶条件，允许高达250 kb的DNA片段在仅3.5小时内分离。这种分离采用市售的凝胶盒，使用明显偏离传统脉冲场条件的条件来实现。PFGE分离被认为需要大于90°的重新定向角度才能有效。然而，在90°甚至更小的重定向角度下，解析DNA片段的速度比标准脉冲场条件下快了几百kb和更小的∑5倍。以90°重定向角分离的DNA片段的迁移率与开关时间相关，如通常使用120°重定向角的DNA运行所见。对于几百kb或更小的DNA片段，可以使用更高的场强，从而进一步提高分离速度。所描述的条件允许在一个工作日内制备、消化和分析来自大型Insert细菌克隆的DNA，例如使用cosmld、Fosmld、P1、细菌人工染色体（BAC）或P1衍生人工染色体（PAC）载体的DNA。
Pulsed field gel electrophoresis (PFGE) Is capable of resolving a wide size range of DNA molecules which would all co-migrate in conventional agarose gels. We describe pulsed field gel conditions which permit DNA fragments of up to 250 kllobases (kb) to be separated in only 3.5 h. The separations, which employ commercially available gel boxes, are achieved using conditions which deviate significantly from traditional pulsed field conditions. PFGE separations have been thought to require reorientation angles greater than 90° to be effective. However, reorientation angles of 90° and even less will resolve DNA fragments a few hundred kb and smaller ∑5 × faster than with standard pulsed field conditions. The mobility of DNA fragments separated with 90° reorientation angles Is switch time-dependent, as is seen for DNA run with the commonly used reorientation angle of 120°. With DNA fragments of several hundred kb and smaller, higher field strengths may be used, resulting in still greater increases in separation speed. The conditions described allow DNA from large Insert bacterial clones, such as those using cosmld, Fosmld, P1, bacterial artificial chromosome (BAC), or P1-derived artificial chromosome (PAC) vectors, to be prepared, digested and analyzed on gels within a single working day.