Singer V L, Jones L J, Yue S T, Haugland R P
Molecular Probes, Inc., Eugene, Oregon 97402, USA.
Anal Biochem. 1997 Jul 1;249(2):228-38. doi: 10.1006/abio.1997.2177.
A sensitive assay for detecting double-stranded (ds) DNA in solution is described. This assay employs a new dye, PicoGreen dsDNA quantitation reagent, which becomes intensely fluorescent upon binding nucleic acids. The brightness of this reagent is due to its high quantum yield (approximately 0.5, bound to ds calf thymus DNA) and large molar extinction coefficient (approximately 70,000 cm-1 M-1). The fluorescence enhancement of this dye upon binding dsDNA is > 1000-fold, with excitation and emission maxima near those of fluorescein. Unlike Hoechst 33258, PicoGreen reagent fluorescence intensity was the same upon binding to poly(dA).poly(dT) and poly(dG).poly(dC) homopolymers. The PicoGreen assay allowed the detection of 25 pg/ml dsDNA, surpassing the sensitivity achieved with Hoechst 33258 by 400-fold. The linear concentration range for DNA quantitation extended over four orders of magnitude-25 pg/ml to 1 microgram/ml-with a single dye concentration. Assay linearity was maintained even in the presence of salts, proteins, poly(ethylene glycol), urea, chloroform, ethanol, and agarose; some ionic detergents and heparin interfered. Linear DNAs yielded slightly brighter signals than supercoiled plasmids. Finally, the assay showed greater dsDNA:RNA selectivity than Hoechst 33258 in low ionic strength buffer and better dsDNA:single-stranded DNA selectivity in 1 M NaCl.
本文描述了一种用于检测溶液中双链（ds）DNA的灵敏检测方法。该检测方法使用了一种新型染料——PicoGreen双链DNA定量试剂，它在与核酸结合后会发出强烈荧光。这种试剂的高亮度归因于其高量子产率（与双链小牛胸腺DNA结合时约为0.5）和大的摩尔消光系数（约70,000 cm-1 M-1）。该染料与双链DNA结合后荧光增强超过1000倍，激发和发射最大值接近荧光素。与Hoechst 33258不同，PicoGreen试剂与聚（dA）·聚（dT）和聚（dG）·聚（dC）同聚物结合时荧光强度相同。PicoGreen检测方法能够检测到25 pg/ml的双链DNA，灵敏度比Hoechst 33258高400倍。DNA定量的线性浓度范围在四个数量级内——25 pg/ml至1微克/ml，只需单一染料浓度。即使存在盐、蛋白质、聚乙二醇、尿素、氯仿、乙醇和琼脂糖，检测的线性度也能保持；一些离子型去污剂和肝素会产生干扰。线性DNA产生的信号比超螺旋质粒略亮。最后，在低离子强度缓冲液中，该检测方法比Hoechst 33258表现出更高的双链DNA:RNA选择性，在1 M NaCl中双链DNA:单链DNA选择性更好。
