Near-Infrared Heavy-Atom-Modified Fluorescent Dyes for Base-Calling in DNA-Sequencing Applications Using Temporal Discrimination

Article abstract of DOI:10.1021/ac980018g

A series of near-IR fluorescent dyes were prepared which contained an intramolecular heavy atom for altering the fluorescence lifetimes to produce a set of probes appropriate for base-calling in a single-lane DNA sequencing format. The heavy-atom modifica

Full text of DOI:10.1021/ac980018g

Anal. Chem. 1998, 70, 2676-2684
Near-Infrared Heavy-Atom-Modified Fluorescent
Dyes for Base-Calling in DNA-Sequencing
Applications Using Temporal Discrimination
James H. Flanagan, Jr.,†,§ Clyde V. Owens,† Sarah E. Romero,† Emanuel Waddell,† Shaheer H. Kahn,‡
Robert P. Hammer,*,† and Steven A. Soper*,†
Department of Chemistry, Louisiana State University, Baton Rouge, Louisiana 70803-1804, and Perkin-Elmer Corporation,
Applied Biosystems Division, 850 Lincoln Centre Drive, Foster City, California 94404
A series of near-IR fluorescent dyes were prepared which
contained an intramolecular heavy atom for altering the
fluorescence lifetimes to produce a set of probes ap-
propriate for base-calling in a single-lane DNA sequencing
format. The heavy-atom modification consisted of an
intramolecular halogen situated on a remote section of
the chromophore in order to minimize the perturbation
on the lifetimes and fluorescence quantum yields. In
addition, the dye series possessed an isothiocyanate
functional group to allow facile attachment to sequencing
primers. The unconjugated dyes showed similar absorp-
rections would be required to correct for dye-dependent
mobility shifts.
The use of fluorescence detection in DNA sequencing has
become the preferred method due to the fact that it can provide
on-line analysis, possesses limits of detection comparable to those
of radiography, is easily integrated to the microgel separation
techniques, and can potentially allow the base-calling to be
performed in a single separation lane using spectral discrimin-
ation.1-3 The ability to perform the separation and base-calling
in a single lane has become critically important, in light of the
Human Genome Project, where throughput issues are a prime
concern.
tion and emission maxima (λabs ) 7 6 5 -7 6 8 nm; λem
)
7 9 4 -7 9 8 nm) as well as fluorescence quantum yields
that were invariant, within experimental error, with the
heavy atom. However, the lifetimes of these dyes were
found to vary with the identity of the halogen substitution
(I, τf ) 9 4 7 ps; F, τf ) 8 4 3 ps, measured in methanol),
with an average variation within the dye series of 3 5 ps.
The spectroscopic properties of the free dyes and the dyes
conjugated to sequencing primers on the 5 ′-end of the
oligonucleotide were determined in a DNA-sequencing
matrix (denaturing gels containing formamide). The
results indicated slight differences in the fluorescence
properties of the free dyes compared to those of the dye/
primer conjugates in this particular matrix. Inspection
of the ground-state absorption spectra showed significant
aggregation for the free dyes in this solution, but the
conjugated dyes exhibited no sign of aggregation due to
the highly anionic nature of the oligonucleotide. The
fluorescence lifetimes of the dye/ primer conjugates dem-
onstrated lifetimes which ranged from 735 to 889 ps, with
an average variation of 5 1 ps, an adequate difference to
allow facile discrimination of these dyes in DNA-sequenc-
ing conditions. In addition, the free solution electro-
phoretic mobilities of the native heavy-atom-modified dyes
were found to be very similar. When the dye/ primer
conjugates were electrophoresed in a cross-linked poly-
acrylamide gel electrophoresis capillary column, they
comigrated, indicating that, in single-lane sequencing
applications, when utilizing these dyes, no postrun cor-
The commonly used approach for single lane base-calling in
DNA sequencing applications using fluorescence is spectral
discrimination, where a set of four spectrally distinct chromo-
phores, which can be attached to either the sequencing primer
or the dideoxynucleotide, are identified on the basis of unique
emission maxima.1 The dyes typically used are the fluorescein
and/ or rhodamine derivatives, which contain structural modifica-
tions to alter the absorption and emission maxima to allow efficient
discrimination.1,3,4 An ideal property of the chromophore set is
similar absorption maxima, to allow excitation with a single
excitation source, but widely spaced emission maxima, to permit
efficient sorting onto the appropriate detection channel. A set of
commercial dyes are available (6-FAM, NED, TAMRA, and ROX)
which nearly possess the aforementioned characteristics.5 These
dyes can be excited with the 488- or 514-nm lines from an argon
ion laser and then spectrally isolated using a series of optical filters
onto appropriate photodetectors.3,6 Some of the potential difficul-
ties with spectral discrimination include the need for multiple
† Louisiana State University.
‡ PE-Applied Biosystems.
§ Current address: Transgenomic, Inc., Omaha, NE 68107.
(1) Smith, L. M.; Sanders, J. Z.; Kaiser, R. J.; Hughes, P.; Dodd, C.; Connell, C.
R.; Heiner, C.; Kent, S. B. H.; Hood, L. Nature 1 9 8 6 , 321, 674-678.
(2) Prober, G. M.; Trainor, G. L.; Dam, R. J.; Hobbs, F. W.; Robertson, C. W.;
Zagursky, R. J.; Cocuzza, A. J.; Jensen, M. A.; Baumeister, K. Science 1 9 8 7 ,
238, 336-341.
(3) Swerdlow, H.; Zhang, J. Z.; Chen, D. Y.; Harke, H. R.; Grey, R.; Wu, S.;
Dovichi, N. Anal. Chem. 1 9 9 1 , 63, 2835-2841.
(4) Trainor, G. L.; Hobbs, F. W.; Cocuzza, A. J.; Confalone, P. N. Nucleic Acids
Res. 1 9 8 8 , 16, 119-120.
(5) Applied Biosystems. Inc., Foster City, CA, 94404.
(6) Smith, L. M. Nature 1 9 9 1 , 349, 812-813.
2676 Analytical Chemistry, Vol. 70, No. 13, July 1, 1998
S0003-2700(98)00018-3 CCC: $15.00 © 1998 American Chemical Society
Published on Web 05/30/1998

excitation sources in some cases and multiple detection channels,
cross-talk between detection channels due to the broad emission
profiles associated with the chromophores, and dye-dependent
electrophoretic mobilities of the labeled oligonucleotides.
A method to eliminate the need for multiple detection channels
has been to use rotating filter wheels.3 Here, only one detection
channel is required, but, due to decreases in the duty cycle, losses
in the signal-to-noise ratio can result, producing errors in base-
calling. Losses in signal-to-noise ratio can be particularly trouble-
some when utilizing micro-CE-based systems for DNA sequencing,
where the injection volume can be on the order of several tens of
picoliters.7-9 In addition, this method still may require the need
for multiple excitation sources, and dye-dependent electrophoretic
mobilities are present. Another approach to eliminate the need
for multiple detection channels has been to incorporate only one
or two dyes and then use intensity variations to identify the
terminal base.3,10,11 The intensities of the resulting electrophoretic
peaks are controlled by adjusting the molar concentration of the
ddNTP during DNA polymerization. The difficulties associated
with this approach arise from nonuniformities in the incorporation
efficiencies of the ddNTPs during polymerization and the inability
to use dye-labeled terminators.
To eliminate the need for multiple excitation sources, the use
of fluorescence-energy-transfer probes have been utilized in single-
lane DNA-sequencing applications.12-14 In this approach, a donor
(FAM) is attached to the 5′-end of a sequencing primer, and an
acceptor (FAM, JOE, TAMRA, ROX) is covalently bound to a
modified thymidine residue eight or nine bases down the
sequence. Since the primers utilize Fo¨ rster resonance energy
transfer, only a single excitation source is required (488 nm), with
the emission sorted onto appropriate detectors. Due to the
structural requirements for efficient energy transfer and the need
for two dyes per primer, this method will be difficult to adapt in
primer walking strategies, and the use of dye-labeled dideoxy-
nucleotides can be problematic. In addition, concerns with dye-
dependent mobility shifts are present.
proach will not easily be amenable to primer walking strategies
or dye-labeled dideoxynucleotide DNA sequencing.
As an alternative to spectral discrimination, various groups
have suggested that fluorescence lifetimes can potentially serve
as a viable method for base-calling in DNA-sequencing applica-
tions.16-18 The principal advantages associated with lifetime
discrimination for base-calling are the following: (1) since the
calculated lifetime is immune to concentration differences, dye-
labeled terminators can potentially be used as well as dye-
primers, with a wide choice in polymerase enzymes to suit the
particular sequencing application; (2) lifetime values can be
determined with higher precision than fluorescence intensities
under appropriate conditions, improving the accuracy in base-
calling; (3) lifetime determinations do not suffer from broad
emission profiles associated with spectral discrimination; and (4)
the fluorescence can potentially be processed on a single detection
channel without the need for spectral sorting to multiple detection
channels. Several problems do arise when considering lifetime
discrimination for DNA sequencing, especially when utilizing
microseparation techniques such as capillary gel electrophoresis.
The most pervasive problem is associated with the complex
instrumentation required for lifetime determinations. For ex-
ample, in time domain techniques, a pulsed laser is required with
a fast detector, typically a microchannel plate photomultiplier tube,
and sophisticated counting electronics. In addition, poor photon
statistics (low number of photocounts in decay profile) produced
from low loading masses and the transient nature of the signal,
determined by the width of the electrophoretic band (1-10 s),
can produce poor precision in the measurement. The poor
precision would also be compounded by the presence of large
amounts of scattering and impurity photons included in the decay
profile. Finally, complex algorithms are required for abstracting
the lifetime from the decay profile, making on-line determinations
during electrophoresis difficult.
Many of these concerns associated with lifetime determinations
for base-calling in DNA sequencing have been addressed using
near-IR fluorescence. For example, several groups have demon-
strated that semiconductor diode lasers, which can be operated
in a pulsed mode and lase between 680 and 800 nm in conjunction
with single-photon avalanche diodes (SPADs), can produce a time-
correlated single-photon-counting apparatus that is simple to
operate, with performance characteristics comparable to those of
visible devices, using mode-locked Nd:YAG lasers and micro-
channel plates.19-22 In addition, we have recently demonstrated
that lifetime measurements can be acquired in the near-IR using
a solid-state device and counting electronics situated on a PC
Recently, a set of electrophoretically uniform fluorescent dyes
for DNA sequencing have been reported.15 The dyes, which are
BODIPY derivatives, are attached to sequencing primers (5′-end)
via a unique linker structure that produces excellent sequencing
data without software correction for dye-dependent mobility shifts.
In addition, the dye-primer set yields narrower spectral emission
bandwidths compared to those of conventional dye-primer sets,
resulting in smaller amounts of cross-talk between detection
channels. However, as with the energy-transfer dyes, this ap-
(7) Jacobson, S. C.; Hergenroder, R.; Koutny, L. B.; Ramsey, J. M. Anal. Chem.
1 9 9 4 , 66, 1114-1118.
(8) Jacobson, S. C.; Moore, A. W.; Ramsey, J. M. Anal. Chem. 1 9 9 5 , 67, 2059-
2063.
(9) Woolley, A. T.; Mathies, R. A. Anal. Chem. 1 9 9 5 , 67, 3676-3680.
(10) Chen, D.; Harke, H. R.; Dovichi, N. J. Nucleic Acids Res. 1 9 9 2 , 20, 4873-
4880.
(16) Seeger, S.; Bahteler, G.; Drexhage, K. H.; Arden-Jacob, J.; Deltau, G.; Galla,
K.; Han, K. T.; Muller, R.; Kollner, M.; Rumphorst, A.; Sauer, M.; Schulz,
A.; Wolfrum, J. Ber. Bunsen-Ges. Phys. Chem. 1 9 9 3 , 97, 1542-1548.
(17) Soper, S. A.; Legendre, B. L.; Williams, D. C. Anal. Chem. 1 9 9 5 , 67, 4358-
4365.
(11) Huang, X. C.; Quesada, M. A.; Mathies, R. A. Anal. Chem. 1 9 9 2 , 64, 2149-
2154.
(12) Ju, J.; Glazer, A. N.; Mathies, R. A. Proc. Natl. Acad. Sci. U.S.A. 1 9 9 5 , 92,
(18) Li, L.; McGown, L. B. Anal. Chem. 1 9 9 6 , 68, 2737-2743.
(19) Imasaka, T.; Yoshitake, A.; Hirata, K.; Kawabata, Y.; Ishibashi, N. Anal. Chem.
1 9 8 5 , 57, 947-949.
4347-4351.
(20) Bachteler, G.; Drexhage, K. H.; Arden-Jacob, J.; Han, K. T.; Kollner, M.;
Muller, R.; Sauer, M.; Seeger, S.; Wolfrum, J. J. Lumin. 1 9 9 4 , 62, 101-
108.
(21) Soper, S. A.; Davidson, Y. Y.; Flanagan, J. H. J.; Legendre, B. L.; Owens, C.;
Williams, D. C.; Hammer, R. P. Proc. SPIE-Int. Soc. Opt. Eng. 1 9 9 6 , 2680,
235-246.
(13) Ju, J.; Kheterpal, I.; Scherer, J. R.; Ruan, C.; Fuller, C. W.; Glazer, A. N.;
Mathies, R. A. Anal. Biochem. 1 9 9 5 , 231, 131-140.
(14) Kheterpal, I.; Ju, J.; Radhakrishnan, A.; Brandt, G. S.; Ginther, C. L.; Scherer,
J. R.; Sensabaugh, G. F.; Mathies, R. A. Proc. SPIE-Int. Soc. Opt. Eng. 1996,
2680, 204-213.
(15) Metzker, M. L.; Jing, L.; Gibbs, R. A. Science 1 9 9 6 , 271, 1420-1422.
(22) Farrens, D. L.; Song, P.-S. Photochem. Photobiol. 1 9 9 1 , 54, 313-317.
Analytical Chemistry, Vol. 70, No. 13, July 1, 1998 2677

board.23 We have also shown that simple algorithms can be used
to calculate fluorescence lifetimes on-line during free solution or
gel capillary electrophoresis using near-IR time-resolved fluores-
cence.17 The standard deviation in the lifetime measurement of
C-terminated fragments labeled with a near-IR dye was found to
be approximately (9 ps with decay profiles constructed from as
few as 5000 photocounts. The high precision resulted primarily
from the fact that, in the near-IR, the low scattering cross sections
and the minimal number of intrinsically fluorescent components
produced low numbers of interfering photocounts in the decay.
Recently, a set of rhodamine chromophores have been pre-
pared which possess appropriate characteristics for lifetime-based
base-calling in DNA-sequencing applications.24-26 These dyes
absorb radiation in the deep red (∼650 nm) and have lifetimes
which range from 1.7 to 4.0 ns. However, the individual dyes in
the series are structurally unique, resulting in slightly different
absorption and emission maxima as well as unique mobilities,
requiring postrun correction in the electrophoresis for dye-
dependent mobility shifts on the oligonucleotide.
In this work, we report on the preparation, photophysical
characterization, and electrophoretic mobility studies of a set of
unique fluorophores appropriate for single-lane base-calling for
DNA sequencing using lifetime discrimination. The dyes devel-
oped for this application are near-IR tricarbocyanine dyes which
possess an intramolecular heavy-atom modification. The heavy-
atom modification consisted of covalently inserting a halogen (I,
Br, Cl, F) into the molecular framework of the dye. The
intramolecular heavy atom results in perturbations in the singlet-
state photophysics (fluorescence quantum yield, Φf, and fluores-
cence lifetime, τf) due to enhanced intersystem crossing into the
triplet state resulting from spin-orbit coupling.27-29 The heavy
atom, however, does not alter the absorption or emission maxi-
mum of the base chromophore, allowing the fluorescence to be
excited with a single source and processed on a single detection
channel. In addition, we will show that the heavy-atom modifica-
tion to the base chromophore for these dyes produces uniform
electrophoretic mobilities in capillary gel conditions, negating the
need for postrun corrections due to dye-dependent mobility shifts.
Figure 1. Synthetic scheme for the preparation of the isothiocyanate
phenol intermediates (2-6) and the near-IR heavy-atom-modified
dyes (7-11).
sodium hydride were purchased from Aldrich Chemical Co.
(Milwaukee, WI). Phosphorous oxychloride and 2,3,3-trimethyl-
indoline were purchased from Kodak Co. (Rochester, NY).
Riboflavin and tyramine were obtained from Sigma Chemical Co.
(St. Louis, MO). All other solvents were purchased from Fisher
Scientific Co. (Pittsburgh, PA) and used as received.
EXPERIMENTAL SECTION
Chemicals. Aniline, cyclohexanone, N,N-dimethylformamide
(DMF), propane sultone, 2-iodophenol, 2-bromophenol, 2-chloro-
phenol, 2-fluorophenol, 1,1-thiocarbonyldiimidazole, m-chloroper-
benzoic acid, tetrabutylammonium tribromide, 1-(chloromethyl)-
4-fluoro-1,4-diazoniabicyclo[2.2.2]octane bis(tetrafluoroborate), and
Dye Synthesis and P urification. The structures of the near-
IR heavy-atom-modified dyes are shown in Figure 1, along with a
general scheme for their synthesis. The synthesis of dye 1 was
carried out using previously published procedures.30-32 The
heavy-atom-modified near-IR dyes containing the isothiocyanate
functional group were synthesized by first preparing the appropri-
ate phenol and adding this adduct to 1 . The synthesis of each
heavy-atom-modified p-isothiocyanatoethylphenol along with the
characteristic MS and NMR data are outlined below.
Synthesis of 2-(p-Hydroxyphenyl)ethyl Isothiocyanate (2). Tyramine
(400 mg, 2.9 mmol) was dissolved in anhydrous DMF (10 mL)
under an N2 atmosphere. 1,1-Thiocarbonyldiimidazole (520 mg,
2.9 mmol) was added, and the pale yellow solution turned an
(23) Legendre, B. L.; Williams, D. C.; Soper, S. A.; Erdmann, R.; Ortmann, U.;
Enderlein, J. Rev. Sci. Instrum. 1 9 9 6 , 67, 3984-3989.
(24) Bachteler, G.; Drexhage, K.-H.; Arden-Jacob, J.; Han, K.-T.; Kollner, M.;
Muller, R.; Sauer, M.; Seeger, S.; Wolfrum, J. J. Lumin. 1 9 9 3 , 62, 101-
108.
(25) Sauer, M.; Han, K.-T.; Muller, R.; Nord, S.; Schulz, A.; Seeger, S.; Wolfrum,
J.; Arden-Jacob, J.; Deltau, G.; Marx, N. J.; Drexhage, K. H. J. Fluoresc. 1995,
5, 247-250.
(26) Sauer, M.; Arden-Jacob, J.; Drexhage, K. H.; Marx, N. J.; Karger, A. E.;
Lieberwirth, U.; Muller, R.; Neumann, M.; Nord, S.; Paulus, A.; Schulz, A.;
Seeger, S.; Zander, C.; Wolfrum, J. Biol. Chromatogr. 1 9 9 7 , 11, 81-82.
(27) McGlynn, S. P.; Azumi, T.; Kinoshita, M. Molecular Spectroscopy of the Triplet
State; Prentice Hall: Englewood Cliffs, NJ, 1969.
(30) Makin, S. M.; Boiko, I. I.; Shavrygina, O. A. Zh. Org. Khim. 1 9 7 7 , 13, 2440-
2443.
(28) Kavarnos, G.; Cole, G.; Scribe, P.; Dalton, J. C.; Turro, N. J. J. Am. Chem.
Soc. 1 9 7 1 , 93, 1032-1034.
(31) Lipowska, M.; Patonay, G.; Strekowski, L. Synth. Commun. 1 9 9 3 , 23, 3087-
3094.
(29) Davidson, R. S.; Bonneau, R.; Joussot-Dubien, J.; Trethewey, K. R. Chem.
(32) Strekowski, L.; Lipowska, M.; Patonay, G. J. Org. Chem. 1 9 9 2 , 57, 4578-
Phys. Lett. 1 9 8 0 , 74, 318-320.
4580.
2678 Analytical Chemistry, Vol. 70, No. 13, July 1, 1998

amber red. After 30 min, the solvent was taken off by rotary
evaporation at 40 °C to produce an orange oil. The oil was
dissolved in CH3OH/ H2O (1:3, 4 mL) to precipitate the isothio-
cyanate. The product was filtered and washed with cold water,
to afford 320 mg of 2 (62%). GC/ MS: calcd for C9H9NOS, 180.0
(M+), found, 180.0. 1H NMR (DMSO-d6, 250 MHz): δ 7.67 (s,
1H), 7.05 (d, 2H, J ) 8.1 Hz), 6.70 (d, 2H, J ) 8.6 Hz), 3.80 (t, 2H,
J ) 6.6 Hz), 2.81 (t, 2H, J ) 6.6 Hz). IR: 2081 cm-1 (NdCdS).
Synthesis of 2-(3-Iodo-4-hydroxyphenyl)ethyl Isothiocyanate (3).
According to a procedure outlined by Evangelatos et al.,33 tyramine
(1.34 g, 0.01 mol) was dissolved in NH4OH (200 mL) with stirring.
A 0.96 N solution of I2 (21 mL, 0.02 mol) was slowly added into
the reaction solution from a pressure-equalizing addition funnel.
After 1.5 h, the solvent was concentrated to 50 mL, and the
solution was kept at 4 °C overnight to precipitate a white powder.
The precipitate was filtered and dried in vacuo to give 2-(3-iodo-
4-hydroxyphenyl)ethylamine. A 750-mg sample of this compound
(2.8 mmol) was dissolved in anhydrous DMF (5 mL) under an
N2 atmosphere. 1,1-Thiocarbonyldiimidazole (606 mg, 3.4 mmol)
was then added, and the pale yellow solution turned amber. After
24 h, H2O (20 mL) was added to the mixture, and the product
was extracted into CH2Cl2 (7 × 50 mL). The extract was dried
with MgSO4 and filtered, and the solvent was removed in vacuo
to produce an orange, oily mixture which was purified by flash
chromatography (solvent, CH2Cl2). Pure fractions were pooled
and dried in vacuo. The oily residue of 3 was crystallized at 4 °C
overnight. Yield: 282 mg (33%). GC/ MS: calcd for C9H8INOS,
304.9 (M+), found, 305.0. 1H NMR (CD3OD, 250 MHz): δ 7.59
(s, 1H), 7.08 (d, 1H, J ) 8.2 Hz), 6.78 (d, 1H, J ) 8.4 Hz), 3.67 (t,
2H, J ) 6.8 Hz), 2.85 (t, 2H, J ) 6.6 Hz).
Synthesis of 2-(3-Bromo-4-hydroxyphenyl)ethyl Isothiocyanate (4).
Following a modification of the procedure of Kajigaeshi et al.,34
tyramine (1.03 g, 7.5 mmol) and CaCO3 (830 mg, 8.3 mmol) were
dissolved in CH2Cl2/ CH3OH (7:3, 60 mL). After the tyramine had
completely dissolved, tetrabutylammonium tribromide (4.0 g, 8.3
mmol) was added with stirring. Immediately, the orange solution
turned brown. The reaction was followed by TLC (silica, CH2Cl2/
CH3OH 7:3; plates were stained with a ninhydrin solution (1% in
butanol)) and was complete after 5 min. The reaction was then
filtered and the filtrate washed with Et2O (5 × 20 mL). The ether
was removed, and the residue, 2-(3-bromo-4-hydroxyphenyl)-
ethylamine, was used without further purification. This amine
was dissolved in anhydrous DMF (5 mL) under an N2 atmosphere
with 1,1-thiocarbonyldiimidazole (1.44 g, 7.5 mmol) added, turning
the orange solution an amber color. The reaction was followed
by TLC (silica, CH2Cl2/ CH3CN 4:1). After 30 min, the solvent
was removed on a rotary evaporator at 40 °C. The residue was
repeatedly washed with Et2O (7 × 10 mL). The Et2O was removed
to afford an orange oil which was purified by flash chromatography
(silica, CH2Cl2/ hexane 3:1). The pure fractions were collected,
pooled, and dried in vacuo to yield 4 . Yield: 400 mg (21%). GC/
MS: calcd for C9H8BrNOS, 257.0 (M+), found, 257.0. 1H NMR
(CD3OD, 250 MHz): δ 7.38 (s, 1H), 7.05 (d, 1H, J ) 8.5 Hz), 6.85
(d, 1H, J ) 8.6 Hz), 3.69 (t, 2H, J ) 6.7 Hz), 2.86 (t, 2H, J ) 6.6
Hz).
Synthesis of 2-(3-Chloro-4-hydroxyphenyl)ethyl Isothiocyanate (5).
Following a modification of a procedure outlined by Chung et al.,35
tyramine hydrochloride (1.0 g, 5.8 mmol) was dissolved in a
solution of 1 M hydrochloric acid in anhydrous DMF (9 mL) with
stirring. m-Chloroperbenzoic acid (1.09 g, 6.3 mmol) was added
slowly with stirring, and the solution turned a pale yellow color.
After 30 min, the reaction was poured into CH2Cl2 (50 mL) with
stirring. The resulting precipitate was collected by filtration and
washed with CH2Cl2 (3 × 10 mL). The resulting white product
was dried at 70 °C to produce 2-(3-chloro-4-hydroxyphenyl)-
ethylammonium chloride. This product was dissolved in anhy-
drous DMF (3 mL) under N2, and 1,1-thiocarbonyldiimidazole (644
mg, 3.6 mmol) was added with stirring. After 30 min, the DMF
was removed in vacuo, and the residue was dissolved in CH2Cl2
(20 mL). This solution was washed with H2O (3 × 50 mL) in a
250-mL separatory funnel. The CH2Cl2 layer was collected, dried
with MgSO4, and filtered. The CH2Cl2 was removed in vacuo to
afford an oil which was purified by flash chromatography (silica,
CH2Cl2/ CH3CN 10:1). Pure fractions from the column were
collected, pooled, and concentrated in vacuo. Pure product 5 was
crystalized at 4 °C. Yield: 167 mg (33%). GC/ MS: calcd for C9H8-
ClNOS, 213.0 (M+), found, 213.0. 1H NMR (CD3OD, 250 MHz):
δ 7.20 (s, 1H), 7.00 (d, 1H, J ) 7.9 Hz), 6.87 (d, 1H, J ) 8.5 Hz),
3.67 (t, 2H, J ) 6.8 Hz), 2.84 (t, 2H, J ) 6.7 Hz).
Synthesis of 2-(3-Fluoro-4-hydroxyphenyl)ethyl Isothiocyanate (6).
Following a modification of a procedure outlined by Banks et al.,36
tyramine hydrochloride (1.0 g, 5.8 mmol) was dissolved in H2O/
CH3OH (1:1, 26 mL) with stirring. 1-(Chloromethyl)-4-fluoro-1,4-
diazoniabicyclo[2.2.2]octane bis(tetrafluoroborate) (2.06 g, 5.8
mmol) was added, and the solution turned a dark brown color.
After 18 h, the solvent was taken off in vacuo, and the residue
was dissolved in CH3OH (20 mL) and filtered. The solvent was
then removed from the filtrate, and the crude product, 2-(3-fluoro-
4-hydroxyphenyl)ethylammonium chloride, was precipitated from
CH3CN (10 mL). This was dissolved in anhydrous DMF (3 mL)
under N2 with stirring, and 1,1-thiocarbonyldiimidazole (286 mg,
1.6 mmol) was added. After 30 min, the reaction was removed
from the stirrer, and H2O (20 mL) was added. The product was
extracted from the solution with CH2Cl2 (3 × 50 mL). The CH2Cl2
layer was collected, dried with MgSO4, and filtered. The CH2Cl2
was then removed from the filtrate to give an orange oil. The oil
was purified by flash chromatography (silica, CH2Cl2/ CH3OH 10:
1). The pure fractions were collected, pooled, and dried in vacuo
to afford 6 as an oil. Yield: 127 mg (40% of crude). GC/ MS:
calcd for C9H8FNOS, 197.0 (M+), found, 197.0. 1H NMR (CD3OD,
250 MHz): δ 6.98 (d, 1H, J ) 11.9 Hz), 6.88 (m, 2H), 3.69 (t, 2H,
J ) 6.6 Hz), 2.87 (t, 2H, J ) 6.7 Hz).
Synthesis of Near-IR, Heavy-Atom-Modified Dyes 7-11. Follow-
ing a procedure outlined by Narayanan and Patonay,37 a 60% oil
dispersion of NaH (14 mg, 0.3 mmol) was added to anhydrous
DMF (4 mL) under an N2 atmosphere at 0 °C. In a separate flask,
the appropriate phenol (2 -6 , 58 mg, 0.3 mmol) was dissolved in
anhydrous DMF (1 mL) under an N2 atmosphere at 0 °C with
this solution added to the slurry of NaH. After 30 min, the
(35) Chung, K. H.; Kim, H. J.; Kim, H. R.; Ryu, E. K. Synth. Commun. 1 9 9 0 , 20,
2991-2997.
(36) Banks, R. E.; Besheesh, M. K.; Mohialdin-Khaffaf, S. N.; Sharif, I. J. J. Chem.
(33) Evangelatos, S. T.; Kakabakos, S. E.; Evangelatos, G. P.; Ithahkissios, D. S.
J. Pharm. Sci. 1 9 9 3 , 82, 1228-1231.
(34) Kajigaeshi, S.; Kakinami, T.; Okamoto, T.; Kakamura, H.; Fujikawa, M. Bull.
Soc., Perkin Trans. 1 9 9 6 , 1, 2069-2076.
Chem. Soc. Jpn. 1 9 8 7 , 60, 0, 4187-4189.
(37) Narayanan, N.; Patonay, G. J. Org. Chem. 1 9 9 5 , 60, 2391-2395.
Analytical Chemistry, Vol. 70, No. 13, July 1, 1998 2679

phenoxide isothiocyanate solution was added to chlorodye 1 (100
mg, 0.14 mmol) dissolved in anhydrous DMF (4 mL) under N2.
The reaction was followed by HPLC. After 18 h, the reaction was
quenched with dry ice, and the solvent was removed on a rotary
evaporator at 40 °C. The residue was dissolved in CH3OH/ H2O
(1:1, 3 mL), filtered, purified by preparatory HPLC, and dried in
vacuo overnight to give 7 -1 1 . Typical yield: 30-50 mg (25-
45%). The compounds were characterized and checked for purity
using FAB-MS, NMR, and FT-IR. The following data were
collected for the FAB-MS results: molecular weight calculated
for 8, 966.2 (protonated form, M+), found, 965.8; calcd for 9, 920.2,
found, 920.8; calcd for 1 0 , 876.3, found, 876.7; calcd for 1 1 , 860.3,
found, 860.4. The data obtained from the proton NMR charac-
terization were as follows. 8 (CD3OD, 250 MHz): δ 7.92 (s, 1H),
7.86 (D, 2H, J ) 14.1 Hz), 7.37 (m, 5H), 7.19 (m, 4H), 6.73 (d, 1H,
J ) 8.7 Hz), 6.35 (d, 2H, J ) 14.3 Hz), 4.32 (t, 4H, J ) 7.3 Hz),
3.71 (t, 2H, J ) 5.9 Hz), 2.95 (m, 6H), 2.77 (br t, 2H), 2.21 (m,
4H), 2.04 (br t, 2H), 1.66 (s, 6H), 1.12 (s, 6H). 9 (CD3OD, 250
MHz): δ 7.89 (d, 2H, J ) 14.1 Hz), 7.71 (s, 1H), 7.36 (m, 5H),
7.20 (m, 4H), 6.83 (d, 1H, J ) 8.0 Hz), 6.36 (d, 2H, J ) 14.0 Hz),
4.32 (t, 4H, J ) 7.5 Hz), 3.74 (t, 2H, J ) 5.9 Hz), 2.95 (m, 6H),
2.80 (br t, 4H), 2.21 (m, 4H), 2.05 (br t, 2H), 1.63 (s, 6H), 1.13 (s,
6H). 1 0 (CD3OD, 250 MHz): δ 7.89 (d, 2H, J ) 14.2 Hz), 7.56
(s, 1H), 7.37 (m, 5H), 7.20 (m, 4H), 6.86 (d, 1H, J ) 8.1 Hz), 6.36
(d, 2H, J ) 14.3 Hz), 4.33 (t, 4H, J ) 7.5 Hz), 3.75 (t, 2H, J ) 5.8
Hz), 2.95 (m, 6H), 2.81 (br t, 4H), 2.21 (m, 4H), 2.04 (br t, 2H),
1.29 (br s, 12 H). 1 1 (CD3OD, 250 MHz): δ 7.96 (d, 2H, J )
14.0 Hz), 7.37 (m, 5H), 7.23 (m, 4H), 7.00 (d, 1H, J ) 8.7 Hz),
6.90 (d, 1H, J ) 8.1 Hz), 6.36 (d, 2H, J ) 14.0 Hz), 4.33 (t, 4H, J
) 7.5 Hz), 3.76 (t, 2H, J ) 6.5 Hz), 2.94 (m, 6H), 2.80 (br t, 4H),
2.21 (m, 4H), 2.04 (br t, 2H), 1.35 (s, 12H). The isothiocyanate
near-IR dyes were stored as dry powders at -20 °C in the dark
until required for labeling reactions.
Labeling and Purification of Sequencing Primers with Near-IR,
Heavy-Atom-Modified Dyes. The M13mp18 universal sequencing
primers (17mer) containing a 6-carbon alkyl linker terminated with
an amino group on the 5′-end were derivatized with the near-IR
dyes according to procedures outlined by Li-COR.38 Briefly, 50
nmol of DNA (2× precipitated from NaOAc buffer in cold ethanol)
was added to 25 µL of carbonate buffer (400 mM, pH 9.5), 25 µL
of EDTA (2 mM), and 100 µL of the near-IR dye (5 mM) dissolved
in DMF, giving a 10-fold molar excess of dye over DNA. After
the reaction was allowed to proceed at room temperature for
approximately 4 h, 10 µL of NaOAc and 480 µL of cold ethanol
were added to the reaction mixture. The solution was centrifuged
for 20 min at 10 °C and 15 000 rpm. The supernatant was
discarded and the ethanol precipitation step repeated again. The
DNA/ dye conjugate was then dried, and 200 µL of water was
added to the pellet. The DNA/ dye conjugate was finally puri-
fied using preparatory HPLC under the following conditions:
column, C18 (10 cm × 4.6 mm, Brownlee); flow rate, 1.7 mL/ min;
mobile phase A, 0.1 M triethylammonium acetate, 4% CH3CN, 96%
H2O; mobile phase B, 0.1 M triethylammonium acetate, 80%
CH3CN, 20% H2O. The gradient conditions were 90/ 10 to 55/ 45
A/ B over 5 min, 55/ 45 to 0/ 100 A/ B over 20 min, hold at 0/ 100
A/ B for 5 min. The collected fractions were pooled and taken to
dryness using a centrifugal evaporator and stored in the dark at
-20 °C. The yield of dye-labeled primer was estimated to
be 30%.
Spectroscopic Analysis. The absorbance spectra were
acquired on a Perkin-Elmer Lambda 3 spectrophotometer (Perkin-
Elmer, Norwalk, CT). The uncorrected fluorescence spectra were
collected on a Spex 3000 fluorometer (Spex, Edison, NJ). The
spectrofluorometer contained a red-sensitive photomultiplier tube
(R636, Hamamatsu Corp.) and emission gratings blazed for 750
nm. The fluorescence quantum yields were calculated relative
to IR-125 in DMSO (Φf ) 0.13) according to the procedure
outlined by Demas and Crosby.39
Time-resolved fluorescence measurements were performed
using a near-IR time-correlated single-photon-counting instrument
built in-house, which has been described previously.40 The system
basically consisted of a mode-locked Ti:sapphire laser pumped
by the all-lines output of an Ar ion laser (Coherent Lasers, San
Jose, CA) and a passively quenched single-photon avalanche diode
(EG&G Optoelectronics, Vaudreuil, Canada). The dye concentra-
tion used for lifetime determinations was 1 × 10-8 M in the
appropriate solvent system. The fluorescence lifetimes were
calculated using a reiterative nonlinear least-squares algorithm
written in-house, with decay profiles accumulated until ap-
proximately 10 000 photocounts were present in the channel with
the maximum number of counts.
Capillary Electrophoresis. Capillary zone electrophoresis
was performed on a Waters Quanta 4000 CE System (Millipore,
Marlborough, MA), with the output signals integrated on a Perkin-
Elmer LCI-100 laboratory computing integrator (Norwalk, CT).
Free solution separations were performed using a 75-µm-i.d.
capillary column (Polymicro Technologies, Phoenix, AZ) with a
total length of 58 cm and a detection window 50 cm from the
injection end. The running buffer consisted of 5 mM sodium
borate buffer (pH 9.3) dissolved in 50:50 water/ methanol. Dye
concentrations of 5 × 10-5 M dissolved in the running buffer were
electrokinetically injected onto the column for 20 s with an applied
voltage of 30 kV (517 V/ cm). The separations were performed
at an applied voltage of 25 kV (431 V/ cm). The analytes were
detected on-column using absorbance at 254 nm. Free solution
mobilities were calculated relative to the mobility of riboflavin
(neutral marker) in order to correct for the electroosmotic flow.
Capillary gel electrophoresis was performed in a 6%T/ 5%C
polyacrylamide gel column (75 µm i.d., J&W Scientific, Folsom,
CA) with a total length of 33 cm and a detection window 26 cm
from the injection end. A mixture of the dye (8 -1 1 )/ oligo-
nucleotide conjugates was electrokinetically injected onto the
column for 3 s at an applied voltage of -5 kV, with separations
performed in reverse mode at an applied voltage of -8.25 kV (250
V/ cm). The detection was performed using laser-induced fluo-
rescence, incorporating the system described above for lifetime
measurements, except that the laser was operated in a continuous
wave mode of operation.
RESULTS AND DISCUSSION
The absorption and emission spectra of the heavy-atom-
modified near-IR dyes measured in methanol are shown in Figure
2. As can be seen from this figure, the introduction of the heavy-
(39) Demas, J. N.; Crosby, G. A. J. Phys. Chem. 1 9 7 1 , 75, 991-1024.
(38) Draney, D. (Li-COR), private communication.
(40) Soper, S. A.; Mattingly, Q. L. J. Am. Chem. Soc. 1 9 9 4 , 116, 3744-3752.
2680 Analytical Chemistry, Vol. 70, No. 13, July 1, 1998

Table 2. Fluorescence Lifetimes of Near-IR Dyes (1,
7-11) in Pure Methanola
dye
τf (ps)b
ø2
SD (ps)
1
7
8
9
1 0
1 1
529
873
947
912
880
843
1.1
1.1
1.3
1.2
1.1
1.2
6
8
10
10
9
8
a
The lifetimes were measured at a dye concentration of 1 nM and
excited with 1 mW of average laser power tuned to 765 nm. b Fluo-
rescence lifetime data were collected in methanol and calculated using
a reiterative nonlinear least-squares deconvolution method. The decay
profiles were collected until approximately 10 000 counts were ac-
cumulated into the channel with the maximum number of counts.
Figure 2. Absorption and emission spectra of the near-IR heavy-
atom-modified dyes dissolved in methanol at a concentration of 1
µM. In the case of the fluorescence spectra, the data were collected
on a red-sensitive photon-counting spectrofluorometer using an
excitation wavelength of 710 nm, which allowed collection of the entire
emission spectrum without interference from the Rayleigh line.
< 1 1 , with dye 1 showing the smallest quantum yield since the
heavy atom (Cl) is attached directly to the chromophore and, in
8 -1 1 , it is spatially removed from the chromophore.
The fluorescence lifetime data for these dyes in pure methanol
are shown in Table 2, along with the ø2 values and the standard
deviation in the measurements. Relative to dye 1 , the insertion
of the heavy atom directly onto the chromophore (1 ) perturbs
the lifetime of the singlet state to a much larger degree than in
those cases where the heavy atom was spatially removed from
the chromophore (8 -1 1 ). In the latter cases, spin-orbit
coupling would be expected to occur predominately through-space
and not through-bond (as in 1) and, thus, exert a smaller influence
on the singlet-state lifetime, consistent with our data.41 Interest-
ingly, we found that τf increased with increasing molecular weight
of the intramolecular heavy-atom modification, contrary to what
is typically observed.24-26,38-41 We have recently carried out
photophysical measurements of the triplet state using flash
photolysis techniques to understand the observed trend.42 The
results of these studies indicated that the heavy-atom modification
does increase the efficiency of crossing into the triplet state, with
the heavier atom showing a larger rate of intersystem crossing.
However, the major nonradiative manifold in these dyes was not
intersystem crossing but internal conversion, which is typically
observed in these tricarbocyanine dyes.43 Both the intersystem
crossing and internal conversion rates are affected by the presence
of the heavy-atom modification, but to differing degrees, producing
the observed trend in the lifetimes of these dyes.42 However, it
should be pointed out that, for the present application (base-calling
in DNA sequencing), the order of the effect is inconsequential.
The only important criterion is that the lifetimes can be discrimi-
nated with high precision to affect high accuracy in the base-
calling. In the case of methanol, the average variation in this
series of dyes was found to be 35 ps ((3 ps).
Table 1. Absorption and Emission Maxima, Extinction
Coefficients and Fluorescence Quantum Yields for the
Heavy-Atom-Modified Near-IR Fluorescent Dyesa
dye
λabs (nm)
λ
em (nm)
ꢀ (M-1 cm-1
)
Φfb
1
7
8
9
1 0
1 1
778
765
766
768
768
768
802
794
796
798
797
797
181 000
230 000
216 000
254 000
239 000
221 000
0.05
0.07
0.15
0.14
0.14
0.14
a
All values were measured in methanol. b Precision in Φf measure-
ments was ∼15%.
atom modification onto these dyes produced only minor changes
in the absorption (λabs ) 765-768 nm) and emission (λem ) 794-
798 nm) maxima. Table 1 lists the absorbance and emission
maxima as well as the molar absorptivities and fluorescence
quantum yields (Φf) for dyes 1 , 7 -1 1 . The addition of the
intramolecular heavy atom produced only minor differences in
the extinction coefficients (ꢀS), with all extinction coefficients
>200 000 cm-1 M-1, typical values for these types of tricarbo-
cyanine dyes.40 In Table 1 are also shown the fluorescence
quantum yields that were calculated for the dye series; as can be
seen, the introduction of the heavy-atom modification had no
appreciable effect on Φf within the precision of the measurement,
with the quantum yields for the heavy-atom dyes ranging between
0.14 and 0.15. In the case of dye 7 , it can be seen that it had a
smaller quantum yield (0.07) compared to those of the heavy-
atom-modified dyes (8 -1 1 ). Also, for dye 1 , where the chloro
substituent was attached directly to the chromophore, the
fluorescence quantum yield was smaller than for those dyes where
the heavy-atom modification was spatially removed from the base
chromophore. In addition, its absorption and emission maxima
were red-shifted from those seen for dyes 7 -1 1 . If the intramo-
lecular heavy atom affected only the rate of intersystem crossing,
then it would be expected that the dyes containing a halogen
would show fluorescence quantum yields less than those for dye
7 , with the quantum yields decreasing in the order 8 < 9 < 1 0
We next carried out a spectroscopic analysis of the heavy-atom-
modified near-IR dyes and the dye-primer conjugates in a DNA
sequencing matrix. In this case, the absorption and fluorescence
spectra were measured in a nonpolymerized acrylamide solution
(6%T/ 5%C) containing 1× TBE and 40% formamide. The motiva-
tion for using formamide was to keep the organic content high,
(41) Fouassier, J. P.; Lougnot, D. J.; Faure, J. Opt. Commun. 1 9 7 6 , 18, 263-
267.
(42) Flanagan, J. H.; Legendre, B. L.; Soper, S. A., manuscript in preparation.
(43) Buettner, A. V. J. Chem. Phys. 1 9 6 7 , 46, 1398-1401.
Analytical Chemistry, Vol. 70, No. 13, July 1, 1998 2681

Table 3. Fluorescence Properties of the
Heavy-Atom-Modified Near-IR Dyes (8-11), Both Free
and Conjugated to a 17mer Sequencing Primer,
Measured in a Nonpolymerized Acrylamide Solution
Containing 40% Formamide and 6%T/5%Ca
absorbance
maxima (nm)
emission
maxima (nm)
fluorescence
lifetime (ps)
dye
free
conjugate
free
conjugate
free
conjugate
8
9
1 0
1 1
764
764
765
766
773
773
774
774
792
793
793
794
799
797
800
800
760
735
719
688
889
821
759
735
a
The absorption and emission spectra were obtained using a dye
concentration of 1 µM, while the lifetimes were measured using a dye
concentration of 10 nM. The free dyes and dye-primer conjugates
were excited with 1 mW of average laser power tuned to 765 nm.
since the tricarbocyanine near-IR dyes have been shown to
possess improved photophysical properties in high organic content
solutions.40 While urea gels are commonly used for sequencing,
formamide gels have been shown to offer some unique advan-
tages, such as improved stability when operating under high
electric field conditions.44-46 The absorbance and fluorescence
emission maxima are shown in Table 3 for both the free dyes
and the dye-primer conjugates. The covalent attachment of the
dye to the oligonucleotide resulted in minor perturbations to the
absorption and emission maxima. The absorption maxima for the
native dyes were found to be ∼765 nm, while the maxima for the
dye-primer conjugates were ∼773 nm, an 8-nm shift. We also
observed aggregation effects in the ground-state absorption
spectra for the free dyes in this matrix, while the dye-primer
conjugate spectra showed only monomeric species (see Figure
3). The peak seen at 765 nm for the free dyes can be assigned to
the monomer, since it was present in the case of methanol, where
it is expected that little, if any, aggregation should occur. The
broad peak centered at approximately 689 nm is most likely due
to a dimer or other higher order aggregate that forms at this dye
concentration (1 µM) in this particular solvent system but was
absent in the case of methanol. It can also be seen from these
data that the absorption of the monomeric form decreases in the
order 8 < 9 < 1 0 < 1 1 . In the case of the dye-primer
conjugates, there is an absence of this broad, blue-shifted band
arising from the dimer or other higher order aggregate. The lack
of aggregation for the dye-primer conjugates is likely due to the
increased solvation expected for the conjugate due to the highly
anionic nature of the oligonucleotide.
Figure 3. Absorption spectra of the free dyes (A) and the dye-
primer conjugates (B) measured in the nonpolymerized acrylamide
solution containing 40% formamide. The absorption spectra were
acquired using a dye concentration of approximately 1 µM.
dye due to the lack of aggregation effects observed for the
conjugates at this dye concentration.
The fluorescence lifetimes for the heavy-atom-modified dyes
and the dye-primer conjugates were next measured in this
nonpolymerized acrylamide solution with the data listed in Table
3. In Figure 4 is shown a prompt peak and decay profiles for
dyes 8 and 1 1 conjugated to the sequencing primer. All decays
were found to be adequately fit to a single-exponential function
with ø2 values which ranged from 1.1 to 1.7. As can be seen from
the data in Tables 2 and 3 for the free dyes, the lifetimes were
shorter in the nonpolymerized gel solution compared to those of
methanol, but the general order in the lifetime values was
preserved. In the case of methanol, dye 8 had a lifetime of 947
ps, while in the gel matrix it was found to be 760 ps. The
differences in lifetimes between the two solvent matrixes are
consistent with our previous work, which has shown that the
tricarbocyanines display solvent-dependent photophysics, with
generally poorer photophysics observed in more aqueous media.40
It was also noticed that the lifetime variation for the free dye series
was found to be 24 ( 8 ps, while in the case of methanol, it was
35 ( 3 ps.
In the fluorescence spectra, the emission maxima for the dye-
primer conjugates showed an approximate 6-nm shift compared
to the native dyes in this solvent system. In addition, the emission
intensities of the dye-primer conjugates were found to be
somewhat higher than those associated with the free dyes in this
gel matrix (data not shown). This effect was attributed to the
increased absorption of the monomeric or fluorescing state of the
(44) Swerdlow, H.; Dew-Jager, K. E.; Brady, K.; Grey, R.; Dovichi, N. J.; Gesteland,
R. Electrophoresis 1 9 9 2 , 13, 475-483.
(45) Rocheleau, M. J.; Grey, R. J.; Chen, D.; Harke, H. R.; Dovichi, N. J.
Electrophoresis 1 9 9 2 , 13, 484-486.
(46) Swerdlow, H.; Dew-Jager, K.; Gesteland, R. BioTechniques 1 9 9 4 , 16, 684-
693.
If these dyes are to be effectively used in a single-lane DNA
sequencing format for base-calling, both the absolute magnitude
and the variation in the lifetimes for the dye series are important.
When the dyes possess short lifetimes, the criteria imposed on
2682 Analytical Chemistry, Vol. 70, No. 13, July 1, 1998

indicated that, with the use of near-IR fluorescence monitoring,
these effects are minimal, and the precision in the measurement
is determined primarily by photon statistics.17
To evaluate the effects on the fluorescence lifetimes of these
dyes by conjugating them to sequencing primers, the lifetimes of
the dye-primer conjugates were determined in the nonpolymer-
ized matrix containing formamide, the results of which are shown
in Table 3. As can be seen from these data, the lifetime values
between the free dyes and the dye-primer conjugates were found
to differ, with the conjugates showing a longer lifetime than the
free dye. For example, in the case of dye 8 , the free dye showed
a lifetime of 760 ps, while the conjugate of dye 8 possessed a
lifetime of 889 ps. It was expected that the observed differences
were not due to aggregation effects, since the dye concentration
used (10 nM) was significantly less than those used to collect
the absorption and emission spectra and little aggregation would
be expected at this low dye concentration. Also, the average
variation in the lifetimes for this dye series was found to be 51 (
23 ps for the dye-primer conjugates, significantly larger than in
the case of the free dyes. However, the variation between the
dye-primer conjugates of 1 0 and 1 1 was only 24 ps, while those
for the other pairs, 8/ 9 and 9/ 10, were 68 and 62 ps, respectively.
Therefore, for base-calling applications, the photocounts necessary
to achieve the required precision in the measurement will be set
by the smallest variation within the series, which, in this case, is
24 ps. This means that at least 10 000 photocounts will be
required per decay profile or electrophoretic band to achieve
discrimination at 3σ.
We noticed that the observed lifetimes were sensitive to the
amount of formamide present in the acrylamide solution. For
example, when the amount of acrylamide and bisacrylamide was
kept constant (6%T/ 5%C) and the percentage of formamide was
reduced to 10%, the lifetimes were determined to be 621 ps for
dye 8 , 608 ps for dye 9 , 534 ps for dye 1 0 , and 513 ps for dye
1 1 . In addition, the ø2 values were also higher (>2.0). Attempts
to fit the data to double-exponential functions did not reduce the
value of ø2 (i.e., improve the goodness of the fit). We also
measured the lifetimes of these dyes in a nonpolymerized
denaturing solution containing 7 M urea and found that these
lifetimes were smaller than those found in the 40% formamide
solutions. Dye 8 possessed a lifetime of 586 ps in 7 M urea, and
dye 1 0 was found to have a lifetime of 483 ps in this same
medium. These data suggest that the lifetime values will be
sensitive to the composition of the fractionating medium. How-
ever, even though the actual lifetime values of the heavy-atom-
modified dyes did change with changes in the composition of the
medium, the relative order of the lifetime values for each dye in
the series was retained in any particular solvent matrix.
Figure 4. Fluorescence decay profiles for dyes 8 and 11 conjugated
to the M13mp18 sequencing primers measured in a nonpolymerized
acrylamide gel solution containing 40% formamide and 1× TBE along
with the prompt peak (instrument response function). In this case,
the dye concentration was set at 10 nM with approximately 1 mW of
laser power at 765 nm used for excitation. To minimize photobleach-
ing effects during accumulation of the decay profiles, the dye solution
was flowed through the laser beam at a volumetric flow rate of 0.1
mL/min.
the instrument become critical: namely, a short instrument
response function becomes necessary in order to adequately
determine the lifetime. This will be particularly important under
ultradilute conditions and short residence times (poor photon
statistics), since it may be necessary to gate out scattered photons
to perform the lifetime calculation over a time interval where the
scattered photons contribute insignificantly. In the present case,
our instrument showed a response function of 165 ps, significantly
less than the lifetime of dye 1 1 (688 ps) measured in this
nonpolymerized acrylamide solution. The variation or spread in
the lifetimes for the dye series will determine the accuracy in the
base-calling, since the bases are called by the lifetime calculated
for each band in the electropherogram. Basically, the lifetime
variation between dyes must be larger than the discrimination
criterion, which is based on the standard deviation in the
measurement. Using maximum likelihood estimators for deter-
mining fluorescence lifetimes, the standard deviation (σ) in the
measurement can be determined from47
σ ) τf(N)-(1/ 2)
(1)
where N is the total number of photocounts comprising the decay
profile. If each electrophoretic band consists of ∼5000 counts
and the lifetime of the base chromophore is 760 ps, then a lifetime
difference of approximately 31 ps is required for discrimination
at 3σ. If the number of counts in each band is doubled, then the
variation required in this case is only 22 ps (3σ). Therefore, the
average variation observed for the free dyes is well within the
criterion for facile discrimination when the number of photocounts
exceeds 10 000. However, it should be noted that interferences,
such as scattering and/ or fluorescent impurities, can degrade the
relative precision in the measurement. Our recent results have
Effective use of these dyes in a DNA-sequencing protocol
requires the electrophoretic mobilities (µem) be uniform in order
to minimize base-calling errors arising from dye-dependent mobil-
ity shifts. The apparent mobilities (µapp) of the heavy-atom-
modified chromophores were calculated in free solution capillary
zone electrophoresis using
µapp ) Leff/ (tmE)
(2)
(47) Hall, P.; Selinger, B. J. J. Phys. Chem. 1 9 8 1 , 85, 2941-2945.
where Leff is the length of the capillary column from the injection
Analytical Chemistry, Vol. 70, No. 13, July 1, 1998 2683

Table 4. Electrophoretic Mobilities of
Heavy-Atom-Modified Near-IR Fluorescent Dyes (7-11)
in Free Solutiona
dye
µem (cm2/ (V‚s), × 105)b
7
8
9
1 0
1 1
-5.5 ( 0.2
-5.1 ( 0.1
-5.0 ( 0.2
-4.9 ( 0.3
-5.2 ( 0.1
a
The carrier buffer consisted of borate (pH 9.3) and methanol, with
the electrophoresis carried out at a field strength of 431 V/ cm. b The
negative sign indicates that the mobility is associated with anionic
species that run counter to the direction of the electroosmotic flow.
to the detection window (cm), tm is the migration time of the
analyte (s), and E is the field strength (V/ cm). The electro-
phoretic mobilities (µem) were determined from the expression
Figure 5. Capillary gel electropherogram of the heavy-atom-
modified near-IR dyes (8-11) conjugated to a 17mer M13mp18
sequencing primer. In this case, the dye-primer concentration was
1 nM, and the column consisted of a 6%T/5%C cross-linked poly-
acrylamide sieving matrix with 7 M urea as the denaturant. The
sample was injected onto the column electrokinetically for 10 s at 5
kV, and the electrophoresis was carried out at a field strength of 250
V/cm.
µem ) µapp - µeo
(3)
where µeo is the electroosmotic flow. The calculated mobilities
are shown in Table 4. As can be seen from these data, the heavy-
atom-modified dyes 8 -1 1 demonstrated rather uniform electro-
phoretic mobilities (within experimental error), with these values
ranging between -4.9 and -5.2 × 10-5 cm2/ (V‚s). In the case
of the near-IR dye not containing the heavy-atom modification (dye
7 ), it showed a mobility that was larger than those of the heavy-
atom dyes, most likely a result of the smaller frictional contribution
to the mobility produced by H compared to the heavy-atom
modifications (I, Br, Cl, F), since all of these dyes possess similar
charges (monoanionic at this pH).
The heavy-atom dyes 8-11 conjugated to the 17mer sequenc-
ing primer were then electrophoresed in a gel-filled capillary
column to investigate mobility differences under these electro-
phoretic conditions. The resulting electropherogram is shown
in Figure 5. As can be seen from this figure, a single peak was
observed which migrated from the column at 1752 s. Shown in
the inset of this figure is an expanded view of the resulting
electrophoretic peak, which clearly shows only one peak, indicat-
ing that the heavy-atom-substituted dyes comigrated; therefore,
they possessed uniform mobilities under these gel conditions. Due
to the lack of dye-dependent mobilities observed for this dye
series, postrun corrections in the electropherogram will not be
required during DNA sequencing applications, simplifying the
base-calling and improving the accuracy in sequence reconstruc-
tion.
photophysical properties which differed from those of the free
dyes in a formamide/ acrylamide medium. Electrophoretic mobil-
ity studies in free solution carried out on these heavy-atom-
modified near-IR dyes indicated that they possess uniform
mobilities in free solution and, when conjugated to sequencing
primers, comigrate under gel electrophoresis conditions.
For DNA-sequencing applications, these dyes will serve as
excellent labels using lifetime discrimination, since only one
excitation source and one detection channel will be required to
process the fluorescence data since they possess similar absorp-
tion and emission maxima. In addition, the uniform mobilities
will eliminate the need for postrun corrections, typically necessary
for multidye approaches. While performing a time-resolved
measurement can be somewhat instrumentally prohibitive, the
dyes we have prepared absorb light in the near-IR, allowing the
use of solid-state pulsed-diode lasers to serve as excitation sources.
In conjunction with avalanche photodiodes, a simple time-cor-
related single-photon-counting apparatus can be constructed to
dynamically measure fluorescence lifetimes on-line during capillary
gel electrophoresis.23 In addition, the use of near-IR fluorescence
monitoring will significantly reduce matrix interferences, improv-
ing the precision in the lifetime measurement.
ACKNOWLEDGMENT
The authors thank the National Institutes of Health and
CONCLUSION
Louisiana Educational Quality Support Fund (LEQSF) for financial
support of this work, and Louisiana State University for an Alumni
Association undergraduate fellowship to S.E.R.
We have synthesized and characterized a series of chromo-
phores that contain an intramolecular heavy atom (halogen) which
does not perturb the absorbance or emission maximum of the
base chromophore but induces a difference in τf resulting from
influences on both the intersystem crossing and internal conver-
sion rates on the base chromophore. These dyes were conjugated
to sequencing primers, and the conjugates were observed to have
Received for review January 6, 1998. Accepted April 10,
1998.
AC980018G
2684 Analytical Chemistry, Vol. 70, No. 13, July 1, 1998