Hung S C, Ju J, Mathies R A, Glazer A N
Department of Molecular and Cell Biology, University of California, Berkeley, California, 94720-3206, USA.
Anal Biochem. 1996 Jul 1;238(2):165-70. doi: 10.1006/abio.1996.0270.
Energy-transfer (ET) fluorescent primers for DNA sequencing and multiplex genetic analysis (Ju, J., Ruan, C., Fuller, C. W., Glazer, A. N., and Mathies, R. A. (1995) Proc. Natl. Acad. Sci. USA 92, 4347-4351) are named according to the convention D-N-A, where D is the donor, N is the number of bases between the donor and the acceptor, and A is the acceptor. Thus, a primer that carries 6-carboxyfluorescein (FAM) at the 5'-end and 6-carboxy-4', 5'-dichloro-2',7'-dimethoxyfluorescein (JOE) attached to a modified thymidine 10 bases away is designated F10J. We describe here new ET primers, with 5- or 6-carboxyrhodamine-6G (G5 or G6) as acceptors (with FAM as the donor) in place of JOE, with improved match in the electrophoretic mobilities of the DNA fragments extended from the ET dye-labeled primers, and less overlap in the fluorescence emission of the various labeled DNA fragments. This reduced spectral overlap is most likely due to the narrower emission from G5 or G6 in F10G compared to that from JOE in F10J. With single-stranded M13mp18 DNA as the template, a typical run with F10G6 and three other ET primers on a capillary sequencer provided DNA sequences with 99% accuracy in the first 620 bases.
用于DNA测序和多重基因分析的能量转移（ET）荧光引物（Ju，J.，阮，C.，富勒，C.W.，格拉泽，A.N.，和马西斯，R.A.（1995年）《美国国家科学院院刊》92，4347 - 4351）根据D - N - A的惯例命名，其中D是供体，N是供体与受体之间的碱基数，A是受体。因此，一个在5'端携带6 - 羧基荧光素（FAM）且在距离修饰胸苷10个碱基处连接有6 - 羧基 - 4'，5' - 二氯 - 2'，7' - 二甲氧基荧光素（JOE）的引物被命名为F10J。我们在此描述新的ET引物，用5 - 或6 - 羧基罗丹明 - 6G（G5或G6）作为受体（以FAM作为供体）取代JOE，从ET染料标记引物延伸的DNA片段在电泳迁移率上有更好的匹配，并且各种标记DNA片段的荧光发射重叠更少。这种减少的光谱重叠很可能是由于与F10J中的JOE相比，F10G中G5或G6的发射更窄。以单链M13mp18 DNA为模板，在毛细管测序仪上用F10G6和其他三种ET引物进行的典型测序在前620个碱基中提供了准确率为99%的DNA序列。
