Investigations on DNA intercalation and surface binding by SYBR Green I, its structure determination and methodological implications
Investigations on DNA intercalation and surface binding by SYBR Green I, its structure determination and methodological implications
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DOI：
10.1093/nar/gnh101
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发表时间：
2004-07-01
影响因子：
14.9
通讯作者：
Vitzthum, F
中科院分区：
文献类型：
--
作者：
Zipper, H;Brunner, H;Vitzthum, F
SYBR绿色I（SG）检测双链DNA（dsDNA）在凝胶电泳、dsDNA定量、实时荧光PCR等分子生物学方法中具有重要意义。生物物理学研究在定义的染料/碱基对比例（dbbn）被用来确定的结构-性能的关系，影响应用SG的方法。这些研究揭示了嵌入的发生，然后在dbm以上类似于0.15的表面结合。只有后者导致荧光显着增加。使用poly（dA）的研究。聚（dT）和聚（dG）。poly（dC）均聚物显示SG的序列特异性结合。此外，盐对SG荧光具有显著影响。我们还注意到SG与单链DNA的结合，尽管SG/ssDNA的荧光至少比dsDNA低11倍。为了进行这些研究，我们通过质谱和NMR分析确定SG的结构为[2-[N-（3-二甲基氨基丙基）-N-丙基氨基]-4-[2，3-二氢-3-甲基-（苯并-1，3-噻唑-2-基）-亚甲基]-1-苯基-喹啉鎓]。为了比较，还测定了PicoGreen（PG）的结构，其为[2-[N-双-（3-二甲氨基丙基）-氨基]-4-[2，3-二氢-3-甲基-（苯并-1，3-噻唑-2-基）-亚甲基]-1-苯基-喹啉鎓]。这些结构-性质关系有助于设计使用SG的方法，特别是溶液中的dsDNA定量和实时PCR。
The detection of double-stranded (ds) DNA by SYBR Green I (SG) Is important in many molecular biology methods including gel electrophoresis, dsDNA quantification In solution and real-time PCR. Biophysical studies at defined dye/base pair ratios (dbprs) were used to determine the structure-property relationships that affect methods applying SG. These studies revealed the occurrence of Intercalation, followed by surface binding at dbprs above similar to0.15. Only the latter led to a significant increase in fluorescence. Studies with poly(dA) . poly(dT) and poly(dG) . poly(dC) homopolymers showed sequence-specific binding of SG. Also, salts had a marked impact on SG fluorescence. We also noted binding of SG to single-stranded (ss) DNA, although SG/ssDNA fluorescence was at least similar to11-fold lower than with dsDNA. To perform these studies, we determined the structure of SG by mass spectrometry and NMR analysis to be [2-[N-(3-dimethylaminopropyl)-N-propylamino]-4-[2,3-dihydro-3-methyl-(benzo-1,3-thiazol-2-yl)-methylidene]-1-phenyl-quinolinium]. For comparison, the structure of PicoGreen (PG) was also determined and is [2-[N-bis-(3-dimethylaminopropyl)-amino]-4-[2,3dihydro-3-methyl-(benzo-1,3-thiazol-2-yl)-methylidene]-1-phenyl-quinolinium]. These structure-property relationships help In the design of methods that use SG, in particular dsDNA quantification in solution and real-time PCR.