Characterization of PicoGreen reagent and development of a fluorescence-based solution assay for double-stranded DNA quantitation
Characterization of PicoGreen reagent and development of a fluorescence-based solution assay for double-stranded DNA quantitation
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DOI：
10.1006/abio.1997.2177
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发表时间：
1997-07-01
影响因子：
2.9
通讯作者：
Haugland, RP
中科院分区：
文献类型：
--
作者：
Singer, VL;Jones, LJ;Haugland, RP
本文介绍了一种灵敏的双链DNA定量分析方法。该方法采用了一种新的染料PicoGreen双链DNA定量试剂，它与核酸结合后发出强烈的荧光。该试剂的亮度是由于它的高量子产率（与小牛胸腺DNA结合时约为0.5）和大的摩尔消光系数（约为70，000 cm（-1）M-1）。该染料在结合dsDNA时的荧光增强>1000倍，激发和发射最大值接近荧光素的激发和发射最大值。与Hoechst 33258不同，PicoGreen试剂荧光强度在与聚（dA）、聚（dT）和聚（dG）、聚（dC）均聚物结合时相同。PicoGreen分析允许检测25 pg/ml dsDNA，超过Hoechst 33258所达到的灵敏度400倍。DNA定量的线性浓度范围扩展到四个数量级- 25 pg/ml至1 μ g/ml -使用单一染料浓度。线性DNA产生比超螺旋质粒稍亮的信号。最后，该测定在低离子强度缓冲液中显示出比Hoechst 33258更大的dsDNA：RNA选择性，并且在IM NaCl中显示出更好的dsDNA：单链DNA选择性。(C)北京：科学出版社.
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 (similar to 0.5, bound to ds calf thymus DNA) and large molar extinction coefficient (similar to 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 mu g/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. (C) 1997 Academic Press.