Electrophoretically uniform fluorescent dyes for automated DNA sequencing

Science

Volumn 271, Issue 5254, 1996, Pages 1420-1422

  Metzker, Michael L ^a   Lu, Jing ^a   Gibbs, Richard A ^a  

^a BAYLOR COLLEGE OF MEDICINE   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

FLUORESCEIN; FLUORESCENT DYE; RHODAMINE;

ARTICLE; DNA SEQUENCE; ELECTROPHORETIC MOBILITY; FLUORESCENCE; PRIORITY JOURNAL;

EID: 0029969377     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.271.5254.1420     Document Type: Article

References (25)

1. L. M. Smith et al., Nature 321, 674 (1986).
2. 3 buffer (pH 9.0) and divided into four portions. To each tube, 3 μl of either FAM-NHS, JOE-NHS, TAMRA-NHS, or ROX-NHS were added. For BODIPY additions, see (21).
3. BODIPY 503/512-R930, BODIPY 523/547-R931, BODIPY 564/570-R930, and BODIPY 581/591-R930 termination reactions mimicked the spacing pattern of R865 primer labeled with conventional dyes.
4. M. L Metzker, unpublished results
5. BODIPY 503/512, BODIPY 523/547, and BODIPY 564/570 contained a six-carbon linker, and BODIPY 581/591 contained a three-carbon linker.
6. The half-bandwidth of BODIPY 503/512 was 59% that of FAM, BODIPY 523/547 was 75% that of JOE, BODIPY 564/570 was 71% that of TAMRA, and BODIPY 581/591 was 89% that of ROX.
7. The signal strength of BODIPY dye primers was evaluated by fluorescence spectroscopy (range 71 to 10094) with a Hitachi Model F-4010 fluorescence spectrophotometer in 1 × tris-borate EDTA buffer containing 7 M urea and by the 373A DMA sequencer (range 54 to 133%) Spectroscopy measurements were done in duplicate and determined by comparing the fluorescence intensity at λ maximum of BODIPY dye primers to conventional dye primers FAM and BODIPY 503/512 were excited at 488 nm, and all remaining dyes were excited at 514 nm. The 373A measurements were determined by M13 cycle sequencing reactions of four different molecular clones The relative intensity values were determined by normalizing the BODIPY dye signal to the remaining dye signals and comparing it to its normalized conventional dye signal
8. The donor dye for FET primers was added with 6-FAM amidite. The leader sequences for FET-0 to FFT-3 primers were 5′-FAM-T*GT, 5′-FAM-TT*GT, 5′-FAM-GTT*GT, and 5′-FAM-CGTT*GT followed by the primer sequence AAAACGACGGCCAGT. Primers were synthesized (0.2 μmol) with C6dT (T*) and were ethanol-precipitated The leader sequences for BET-1 to BET-10 primers were 5′-NTT*GT, 5′-NTGT*, 5′-NTTGT*, 5′-NGTTGT*. 5′-NCGTTGT*, 5′-NACGTTGT*, 5′-NTTGTTTGT*, 5′-MTTGTGTTGT*. 5′-NTTGTCGTTGT*, and 5′- NTTGTACGTTGT* followed by the primer sequence AAAACGACGGCCAGT. Primers were synthesized (0.2 μmol) with either C3 or C6 amino link (N) and C6dT (T*) and resuspended in 400 μl of 0 01 N NaOH. To each tube, 20 μl of BODIPY 503/512-SE were added, incubated at 25°C for 10 min, ethanol-precipitated, incubated in 200 μl of 80% acetic acid for 20 min, and ethanol-precipitated (21).
9. T Förester, Modern Quantum Chemistry, Istanbul Lectures, Part III, O. Sinanoglu, Fd (Academic Press, New York, 1965), pp. 93-137.; L. Stryer, Annu. Rev. Biochem. 47, 819 (1978).
10. T Förester, Modern Quantum Chemistry, Istanbul Lectures, Part III, O. Sinanoglu, Fd (Academic Press, New York, 1965), pp. 93-137.; L. Stryer, Annu. Rev. Biochem. 47, 819 (1978).
11. J. Ju, C. Ruan, C. W Fuller, A. N. Glazer, R A Mathies, Proc. Natl Acad. Sci. U.S.A. 92, 4347 (1995)
12. p) in the presence of the acceptor dye (excitation = 488 nm) (7). 12. Although BET-3-BODIPY 576/589 showed better mobility in the first 50 bases of the chromatogram than BET-3-BODIPY 581/591, its λ maximum did not overlap with the instrument's filter.
13. R. A. Gibbs, P. N Nguyen, A. Edwards, A. B Civitello, C. T. Caskey, Genomics 7, 235 (1990).
14. M L. Metzker, K. M. Allain, R. A. Gibbs, Comput. Appl. Biosci. 11, 187 (1995).
15. M. L. Metzker, M. A. Ansari-Lan, X. M. Liu, R A Gibbs, in preparation
16. S. Tabor and C. C. Richardson, Proc. Natl. Acad. Sci U.S.A. 92, 6339 (1995).
17. Other dyes examined were BODIPY 530/550 (5,7-diphenyl-BODIPY), BODIPY 558/568 [5-(2-thienyl)-BODIPY], BODIPY 576/589 [5-(2-pyrrolyl)-BODIPY], and BODIPY 589/616 {6-[({4-[5-(2-thienyl)-BODIPY-3- yl]phenoxy)acetyl)amino]hexanoic acid).
18. A. C. Pease et al , ibid. 91, 5022 (1994).
19. J. S Chamberlain, R. A. Gibbs, J E. Ranier, P. N. Nguyen, C. T. Caskey, Nucleic Acids Res. 16, 11141 (1988)
20. P. M. Holland, R D. Abramsom, R. Watson, D. Gelfand, Proc. Natl. Acad. Sci. U.S.A. 88, 7276 (1991)
21. 3 (pH 9.0) buffer and 30 μl of BODIPY 523/547-SE were added. Dye-labeling reactions were incubated at 25°C for 4 to 16 hours. After ethanol precipitation, dye-labeled primers were purified by reversed-phase high performance liquid chromatography (RP-HPLC), evaporated to ∼20 μl, resuspended in deionized water. and diluted to 0.4 pmol/μl The RP-HPLC hardware system was as described elsewhere (22) with the exception of a Beckman (Fullerton, CA) model 127 gradient solvent module and an ABI aquapore RP-300 column (4.6 mm by 250 mm). Gradient conditions for single and FET dye primers were as follows: 20% B, 5 min, 20% B to 40% B, 30 min, 40% B to 100% B, 18 min; and 100% B, 5 min. BET primers. 30% B, 5 min; 30% B to 100% B, 40 min; and 100% B, b min at a flow rate of 1.0 ml/min
22. M. L Metzker et al , Nucleic Acids Res. 22, 4259 (1994).
23. Reactions were run on a 4.75% polyacrylamide gel and analyzed by the version 210 analysis software program with the AB150 base caller.
24. B. A. Larder et al., Nature 365, 671 (1993)
25. We thank M. Ali Ansan-Lari for critical review of the manuscnpt. Supported in part by grants 1 P30 HG00210 and 1 R01 HG00823 from the National Institutes of Health MLM is supported in part by a Basic Research on HIV and AIDS training grant A107483 from the National Institute of Allergy and Infectious Diseases.