iFRET: an improved fluorescence system for DNA-melting analysis
iFRET: an improved fluorescence system for DNA-melting analysis
复制标题
DOI：
10.1101/gr.297202
复制
发表时间：
2002-09-01
期刊：
影响因子：
7
通讯作者：
Brookes, AJ
中科院分区：
文献类型：
--
作者：
Howell, WM;Jobs, M;Brookes, AJ
荧光共振能量转移(FRET)是检测大分子间空间关系的有力工具，其用途之一是跟踪DNA杂交状态。这一过程包括测量FRET供体和受体部分由于DNA 11杂交/变性而靠近或远离时的荧光变化。在本研究中，我们介绍了一种新的FRET，我们称之为诱导FRET(IFRET)，它非常适合于熔化曲线分析。这项创新需要使用双链DNA特异性插层染料(例如，SYBR Green 1)作为FRET供体，并将传统的FRET受体固定在其中一个DNA分子上。以SNP基因分型技术动态等位基因特异性杂交(DASH)为平台，比较了iFRET与两种不同的荧光策略，即单独使用嵌入染料和使用标准FRET对(荧光素为供体，6-罗丹明为受体)。IFRET配置结合了插层染料的优点，如高信号强度和低成本，并保持了传统FRET检测系统所提供的特异性和多重潜力。因此，iFRET为监测DNA分子之间的相互作用提供了一种新颖而有吸引力的模式。
Fluorescence resonance energy transfer (FRET) is a powerful tool for detecting spatial relationships between macromolecules, one use of which is the tracking of DNA hybridization status. The process involves measuring changes in fluorescence as FRET donor and acceptor moieties are brought closer together or moved farther apart as a result of DNA 11 hybridization/denaturation. In the present study, we Introduce a new version of FRET, which we term induced FRET (iFRET), that is ideally suited for melting Curve analysis. The innovation entails using a double-strand, DNA-specific intercalating dye (e.g., SYBR Green 1) as the FRET donor, with a conventional FRET acceptor affixed to one of the DNA molecules. The SNP genotypin technique dynamic allele specific hybridization (DASH) was used as a platform to compare iFRET to two alternative fluorescence strategies, namely, the use of the intercalating dye alone and the use of a standard FRET pair (fluorescein as donor, 6-rhodamine as acceptor). The IFRET configuration combines the advantages of intercalating dyes, such as high signal strengths and low cost, with maintaining the specificity and multiplex potential afforded by traditional FRET detection systems. Consequently, iFRET represents a fresh and attractive schema for monitoring interactions between DNA molecules.