Cyanine dyes with high absorption cross section as donor chromophores in energy transfer primers
- Univ. of California, Berkeley, CA (United States)
Energy transfer (ET) fluorescent primers are significantly superior to single dye-labeled primers for DNA sequencing and multiplex genetic analyses. We describe here ET primers in which a donor chromophore with a large absorption cross section but a low fluorescence quantum yield is exploited to increase the Stokes-shifted fluorescence emission of acceptor dyes. The new ET primers have 3-({var_epsilon}-carboxy-pentyl)-3{prime}-ethyl-5,5{prime}-dimethyloxacarbocyanine (CYA; {var_epsilon}{sub M}{sup 488 nm} 142,000 M{sup -1} cm{sup -1}) at the 5{prime}-end as a common energy donor, and fluorescein or rhodamine derivatives (FAM, R6G, TAMRA, and ROX), attached to a modified thymidine 10 bases away within the primer sequence, as acceptors. With 488-nm excitation, the fluorescence emission intensity of these four ET primers is 1.4- to 24-fold stronger than that of the corresponding primers labeled only with the single acceptor dye. When compared with the corresponding ET primers with a fluorescein derivative (FAM; {var_epsilon}{sub M}{sup 488 nm} 60,000 M{sup -1} cm{sup -1}) as donor, the fluorescence emissions of primers with CYA as donor and FAM, R6G, TAMRA, and ROX as acceptors are respectively 0.8-, 1.0-, 1.7-, and 1.7-fold as intense. The low fluorescence quantum yield of the CYA donor resulted in distinct fluorescence signals for the DNA-sequencing fragments with much lower crosstalk between the four detection channels than that seen with ET primers based on a FAM donor. With single-stranded M13mp18 DNA as the template, the CYA ET primers provided DNA sequences on a four-color capillary sequencer with 100% accuracy in the first 500 bases. 15 refs., 7 figs., 1 tab.
- OSTI ID:
- 535195
- Journal Information:
- Analytical Biochemistry, Vol. 243, Issue 1; Other Information: PBD: 1 Dec 1996
- Country of Publication:
- United States
- Language:
- English
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