McIntosh S L, Nunnally B K, Nesbit A R, Deligeorgiev T G, Gadjev N I, McGown L B
Department of Chemistry, P. M. Gross Chemical Laboratory, Duke University, Durham, North Carolina 27708, USA.
Anal Chem. 2000 Nov 1;72(21):5444-9. doi: 10.1021/ac000235t.
This paper describes the first use of frequency-domain fluorescence lifetime for multiplex detection of DNA restriction fragments in capillary electrophoresis (CE). The fragments were labeled with monomeric intercalating dyes that can be excited by either the 488- or 514-nm line of an argon ion laser and have lifetimes in the range of 0.5-2.5 ns. We were able to achieve multiplex lifetime detection in the CE separation of a restriction fragment digest and a DNA size ladder in the same run, for fragments shorter than 700 bp. Different gel buffer systems, including a modified polyacrylamide gel and several tris-borate-EDTA/hydroxyethylcellulose (TBE/HEC) gels, were investigated for separation and detection of the dye-labeled DNA fragments. Best results for both electrophoretic resolution and lifetime detection were obtained using a gel containing 1% high molecular weight (90,000-105,000) HEC and 0.3% low molecular weight (24,000-27,000) HEC in TBE buffer.
本文描述了首次在毛细管电泳（CE）中利用频域荧光寿命对DNA限制性片段进行多重检测。这些片段用单体嵌入染料标记，该染料可被氩离子激光的488或514纳米谱线激发，寿命在0.5 - 2.5纳秒范围内。对于短于700 bp的片段，我们能够在同一次运行中对限制性片段消化产物和DNA分子量标准进行CE分离时实现多重寿命检测。研究了不同的凝胶缓冲系统，包括改良聚丙烯酰胺凝胶和几种三羟甲基氨基甲烷 - 硼酸盐 - 乙二胺四乙酸/羟乙基纤维素（TBE/HEC）凝胶，用于分离和检测染料标记的DNA片段。在TBE缓冲液中使用含有1%高分子量（90,000 - 105,000）HEC和0.3%低分子量（24,000 - 27,000）HEC的凝胶，在电泳分辨率和寿命检测方面均获得了最佳结果。
