A versatile microfabricated platform for electrophoresis of double- and single-stranded DNA.
A versatile microfabricated platform for electrophoresis of double- and single-stranded DNA.
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用于双链和单链 DNA 电泳的多功能微加工平台。
DOI：
10.1002/elps.200390007
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发表时间：
2003
期刊：
影响因子：
2.9
通讯作者：
Burns,MarkA
中科院分区：
文献类型：
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作者：
Ugaz,VictorM;Lin,Rongsheng;Srivastava,Nimisha;Burke,DavidT;Burns,MarkA
我们展示了一个多功能的微制造电泳台，集成了集成的芯片上电极、加热器和温度传感器阵列。这种设计允许在同一设备中使用一系列不同的筛分凝胶，以在大约1厘米的距离内进行涉及单链和双链DNA的分离。我们使用该设备以100个碱基对的双链DNA梯形图作为标准样品，比较了线性和交联型聚丙烯酰胺、琼脂糖凝胶和热可逆性Pluronic-F127凝胶在凝胶浇注易用性、可重用性和整体分离性能方面的差异。虽然交联型聚丙烯酰胺基质在我们的系统中对各种DNA片段大小提供了一致的高质量分离，但Pluronic凝胶在去除和重新加载凝胶方面也提供了令人信服的优势。琼脂糖凝胶具有良好的分离性能，但是，由于需要更高的凝胶加载温度，必须额外小心以确保凝胶特性的一致性。我们还展示了使用浓度高达19%的变性交联聚丙烯酰胺凝胶来分离长度从18到400个碱基的单链DNA片段。在阅读长度为30个碱基的情况下，相差4个碱基的引物可以在20分钟内以0.9-1.0的分辨率分离。这一水平的性能足以进行各种基因分型分析，包括在微型制造平台中快速检测单核苷酸多态(SNPs)。使用单一的微电泳系统来满足广泛的分离应用的能力为分子生物学家提供了前所未有的便携和廉价形式的灵活性。
We demonstrate a versatile microfabricated electrophoresis platform, incorporating arrays of integrated on‐chip electrodes, heaters, and temperature sensors. This design allows a range of different sieving gels to be used within the same device to perform separations involving both single‐ and double‐stranded DNA over distances on the order of 1 cm. We use this device to compare linear and cross‐linked polyacrylamide, agarose, and thermo‐reversible Pluronic‐F127 gels on the basis of gel casting ease, reusability, and overall separation performance using a 100 base pair double‐stranded DNA ladder as a standard sample. While cross‐linked polyacrylamide matrices provide consistently high‐quality separations in our system over a wide range of DNA fragment sizes, Pluronic gels also offer compelling advantages in terms of the ability to remove and reload the gel. Agarose gels offer good separation performance, however, additional care must be exercised to ensure consistent gel properties as a consequence of the need for elevated gel loading temperatures. We also demonstrate the use of denaturing cross‐linked polyacrylamide gels at concentrations up to 19% to separate single‐stranded DNA fragments ranging in size from 18 to 400 bases in length. Primers differing by 4 bases at a read length of 30 bases can be separated with a resolution of 0.9–1.0 in under 20 min. This level of performance is sufficient to conduct a variety of genotyping assays including the rapid detection of single nucleotide polymorphisms (SNPs) in a microfabricated platform. The ability to use a single microelectrophoresis system to satisfy a wide range of separation applications offers molecular biologists an unprecedented level of flexibility in a portable and inexpensive format.