Hecker K H, Green S M, Kobayashi K
Transgenomic Inc., 2032 Concourse Drive, San Jose, CA 95131, USA.
J Biochem Biophys Methods. 2000 Nov 20;46(1-2):83-93. doi: 10.1016/s0165-022x(00)00133-0.
Sizing of DNA fragments is a routine analysis traditionally performed on agarose or polyacrylamide gels. Electrophoretic analysis is labor-intensive with only limited potential for automation. Recovery of DNA fragments from gels is cumbersome. We present data on automated, size-based separation of DNA fragments by ion-pair reversed-phase high performance liquid chromatography (IP RP HPLC) - DNA chromatography - on the WAVE DNA Fragment Analysis System with the DNASep cartridge. This system is suitable for accurate and rapid sizing of double-stranded (ds) DNA fragments from 50 to ca. 2000 base pairs (bp). Fluorescently labeled DNA fragments are compatible with the technology. Length-dependent separation of dsDNA fragments is sequence independent and retention times are highly reproducible. The resolving capabilities of DNA chromatography are illustrated by the analysis of multiple DNA size markers. Resolved dsDNA fragments are easily collected and are suitable for downstream applications such as sequencing and cloning. DNA chromatography under denaturing conditions with fluorescently labeled DNA fragments offers a means for the separation and purification of individual strands of dsDNA. Analysis of DNA fragments on the WAVE System is highly automated and requires minimal manual intervention. DNA chromatography offers a reliable and automated alternative to gel electrophoresis for the analysis of DNA fragments.
DNA片段大小测定是一项传统上在琼脂糖或聚丙烯酰胺凝胶上进行的常规分析。电泳分析劳动强度大，自动化潜力有限。从凝胶中回收DNA片段很麻烦。我们展示了在配备DNASep柱的WAVE DNA片段分析系统上，通过离子对反相高效液相色谱法（IP RP HPLC）——DNA色谱法，对DNA片段进行基于大小的自动化分离的数据。该系统适用于对50至约2000个碱基对（bp）的双链（ds）DNA片段进行准确、快速的大小测定。荧光标记的DNA片段与该技术兼容。dsDNA片段的长度依赖性分离与序列无关，保留时间高度可重复。通过对多个DNA大小标记物的分析展示了DNA色谱法的分离能力。分离出的dsDNA片段易于收集，适用于测序和克隆等下游应用。在变性条件下用荧光标记的DNA片段进行DNA色谱分析，为dsDNA单链的分离和纯化提供了一种方法。在WAVE系统上对DNA片段进行分析具有高度自动化，只需极少的人工干预。DNA色谱法为DNA片段分析提供了一种可靠且自动化的凝胶电泳替代方法。
