Sherwood C S, Haynes C A, Turner R F
University of British Columbia, Vancouver, Canada.
Biotechniques. 1995 Jan;18(1):136-41.
Several commercially available DNA-staining dyes can yield highly sensitive fluorimetric assays under optimized conditions. However, the high cost of most dyes, coupled with the need for elaborate or expensive instrumentation and/or mL sample volumes, makes assays of this type very costly for routine use. We present a rapid, highly sensitive double-stranded DNA assay based on the new and relatively inexpensive monomeric cyanine dye PO-PRO-3. This dye exhibits maximum excitation/emission wavelengths that are compatible with one of the standard filter sets available on multi-well plate fluorimeters such as the Pandex FCA. The assay does not depend on DNA tertiary structure and is relatively insensitive to protein contamination (up to 1 mg/mL protein), although the bound dye fluorescence does diminish significantly at ionic strengths above approximately 25 mM. Under the assay conditions described here, subnanogram detection limits (in 60-microL sample volumes) are achievable. This assay makes high-sensitivity DNA quantification cost-effective and convenient for many routine analytical applications.
几种市售的DNA染色染料在优化条件下可产生高灵敏度的荧光测定法。然而，大多数染料成本高昂，再加上需要精密或昂贵的仪器设备和/或毫升级别的样品体积，使得这类测定法在常规使用中成本极高。我们基于新型且相对廉价的单体花青染料PO-PRO-3提出了一种快速、高灵敏度的双链DNA测定法。这种染料的最大激发/发射波长与多孔板荧光计（如Pandex FCA）上可用的标准滤光片组之一相兼容。该测定法不依赖于DNA三级结构，并且对蛋白质污染（高达1 mg/mL蛋白质）相对不敏感，不过在离子强度高于约25 mM时，结合染料的荧光会显著减弱。在此处描述的测定条件下，（在60微升样品体积中）可实现亚纳克级的检测限。这种测定法使高灵敏度DNA定量对于许多常规分析应用而言具有成本效益且方便易行。
