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Comparison of Fluorescence Energy Transfer Primers with Different Donor–Acceptor Dye Combinations

✍ Scribed by Su-Chun Hung; Richard A. Mathies; Alexander N. Glazer

Book ID
102560824
Publisher
Elsevier Science
Year
1998
Tongue
English
Weight
131 KB
Volume
255
Category
Article
ISSN
0003-2697

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✦ Synopsis

Fluorescence energy transfer (ET) primers are far superior to single dye-labeled primers as labels for DNA sequencing and polymerase chain reaction amplification. We compare here ET primers with different donor and acceptor dye combinations with respect to the relative acceptor fluorescence emission intensity and the amount of residual donor fluorescence emission. Primers with the following donor/acceptor pairs were synthesized: 6carboxyfluorescein/6-carboxy-X-rhodamine (FAM-ROX), 3-(⑀-carboxypentyl)-3-ethyl-5,5-dimethyloxacarbocyanine/6-carboxy-X-rhodamine (CYA-ROX), and the 4,4difluoro-4-bora-3␣,4␣-diaza-s-indacene-3-propionic acid (BODIPY) derivatives, 5,7-dimethyl-BODIPY/5-(4-phenyl-1,3-butadienyl) BODIPY (BODIPY 503/512 -BODIPY 581/591 ). Variables examined included the length of the 5-amino linker arm, the number of base pairs between the donor and acceptor, and the excitation wavelength (488 or 514 nm). Of the primers examined, CYA-ROX primers offer the best combination of acceptor fluorescence emission intensity and spectral purity.

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