Hung S C, Mathies R A, Glazer A N
Department of Molecular and Cell Biology, University of California, Berkeley 94720, USA.
Anal Biochem. 1997 Oct 1;252(1):78-88. doi: 10.1006/abio.1997.2287.
We have synthesized and characterized the spectroscopic properties of 56 energy transfer (ET) fluorescent dye-labeled primers differing in (i) the spacing between the donor and acceptor, (ii) the nature of the spacer (either oligonucleotide or polydideoxyribose phosphate), (iii) the primer sequence (M13 (-40), M13 (-21), M13 reverse, SP6, T3, and T7 priming sequences), and (iv) the dyes chosen as the donor (6-carboxyfluorescein, F; or 3-(epsilon-carboxypentyl)-3'-ethyl-5,5'-dimethyloxacarbocyanine, C) and acceptor (F; 5 & 6-carboxyrhodamine-110, R110; 6-carboxyrhodamine-6G, G; N,N,N',N'-tetramethyl-6-carboxyrhodamine, T; and 6-carboxy-X-rhodamine, R) chromophores. This study led to the development of two significantly improved ET primer sets for multiple-color analyses. These primers are named using the convention D-N-A, where D is the donor, A is the acceptor, and N is the number of nucleotides between the donor and the acceptor. The primer set C4R110, C4G, C4T, and C4R provides acceptor emissions of high spectral purity with donor:acceptor emission ratios of < 0.002 for C4G, < 0.004 for C4T, and < 0.005 for C4R and excellent matching in the electrophoretic mobilities of single-base extension DNA fragments. The C4R110, C4G, C4T, and C4R set is valuable for diagnostic applications where minimization of crosstalk between different labels is of particular importance. The set C10R110, C10G, C10T, and C10R, which uses only rhodamine dyes as acceptors, shows significantly improved matching in the electrophoretic mobilities of single-base extension DNA fragments over the previously described set C10F, C10G, C10T, and C10R and is the best available for sequencing.
我们合成并表征了56种能量转移（ET）荧光染料标记引物的光谱特性，这些引物在以下方面存在差异：（i）供体与受体之间的间距；（ii）间隔物的性质（寡核苷酸或聚脱氧核糖磷酸）；（iii）引物序列（M13（-40）、M13（-21）、M13反向、SP6、T3和T7引物序列）；以及（iv）作为供体（6-羧基荧光素，F；或3-（ε-羧基戊基）-3'-乙基-5,5'-二甲基氧杂碳菁，C）和受体（F；5和6-羧基罗丹明-110，R110；6-羧基罗丹明-6G，G；N，N，N'，N'-四甲基-6-羧基罗丹明，T；以及6-羧基-X-罗丹明，R）发色团所选择的染料。这项研究促成了两种显著改进的用于多色分析的ET引物组的开发。这些引物按照D-N-A的惯例命名，其中D是供体，A是受体，N是供体与受体之间的核苷酸数量。引物组C4R110、C4G、C4T和C4R提供了高光谱纯度的受体发射光，对于C4G，供体：受体发射光比率<0.002，对于C4T<0.004，对于C4R<0.005，并且单碱基延伸DNA片段的电泳迁移率匹配良好。C4R110、C4G、C4T和C4R引物组对于不同标记之间的串扰最小化特别重要的诊断应用很有价值。引物组C10R110、C10G、C10T和C10R仅使用罗丹明染料作为受体，与先前描述的引物组C10F、C10G、C10T和C10R相比，其单碱基延伸DNA片段的电泳迁移率匹配性有显著改善，是目前用于测序的最佳选择。
