Synthesis and evaluation of a new non-fluorescent quencher in fluorogenic oligonucleotide probes for real-time PCR

Article abstract of DOI:10.1039/b504759e

A non-fluorescent quencher, based on the diaminoanthraquinone Disperse Blue 3, has been incorporated into oligonucleotides at the 5′-end, the 3′-end and internally as a thymidine derivative. Fluorimetry and fluorogenic real-time PCR experiments demonstrat

Full text of DOI:10.1039/b504759e

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A R T I C L E
Synthesis and evaluation of a new non-fluorescent quencher in
fluorogenic oligonucleotide probes for real-time PCR
Jonathan P. May,a Lynda J. Brown,a Ian van Delft,a Nicola Thelwell,b Kate Harleya and
Tom Brown*a
a School of Chemistry, University of Southampton, Highfield, Southampton, SO17 1BJ, UK.
E-mail: tb2@soton.ac.uk; Fax: 44 2380 592991; Tel: 44 2380 592974
b DxS Ltd Manchester Incubator Building, 48 Grafton Street, Manchester, M13 9XX
Received 6th April 2005, Accepted 11th May 2005
First published as an Advance Article on the web 20th June 2005
A non-fluorescent quencher, based on the diaminoanthraquinone Disperse Blue 3, has been incorporated into
oligonucleotides at the 5ꢀ-end, the 3ꢀ-end and internally as a thymidine derivative. Fluorimetry and fluorogenic
real-time PCR experiments demonstrate that the quencher is effective with a wide range of fluorescent dyes. The
anthraquinone moiety increases the melting temperature of DNA duplexes, thus allowing shorter, more
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discriminatory probes to be used. The quencher has been used in Scorpion primers and TaqManꢀ probes for human
DNA sequence recognition and mutation detection.
Introduction
Results and discussion
Fluorescence methods are used widely to detect specific nucleic
acid sequences in homogeneous closed-tube genetic assays.
Typically an oligonucleotide probe is doubly labelled with a fluo-
rophore and a proximal quencher molecule and hybridisation of
the probe to the target nucleic acid leads to signal generation via
a conformational change in the probe. Examples are Scorpion
Dark quencher phosphoramidite: synthesis and evaluation
Commercially available 1-((2-hydroxyethyl)amino)-4-(methyl-
amino)anthraquinone (Disperse Blue 3) was purchased from
Aldrich as a crude mixture (20% purity). It was purified by silica
gel column chromatography, and converted to phosphoramidite
1 for 5ꢀ-labelling of oligonucleotides during solid-phase oligonu-
cleotide synthesis (Scheme 1).11,12
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primersTM1–4 and molecular beacons.5,6 TaqManꢀ probes have a
different mode of action that relies upon enzymatic cleavage
to separate the fluorophore and quencher.7,8 The quenching
mechanism in Scorpions and molecular beacons is primarily
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collisional in nature, whereas in TaqManꢀ probes, “through-
space” Fo¨rster quenching predominates. Many conventional
dye/quencher pairs used in fluorogenic primers and probes
suffer from drawbacks such as intrinsic fluorescence of the
quencher and poor spectral overlap between the fluorescent
dye and the quencher molecule. Both of these have an adverse
effect on the fluorescent signal-to-noise ratio. Indeed, the non-
fluorescent diazo-dyes DABCYL6 and methyl red,9 which are
frequently used in real-time PCR probes, do not efficiently
quench the fluorescence of several commonly used “long-
wavelength” fluorophores such as the cyanine dye, Cy5. These
dyes are widely used in fluorescent probes and are particularly
important in DNA microarrays. A new range of quenchers
have been developed based on azo dyes (BHQ 1, 2 and 3,
Biosearch Technologies) for use with commonly used fluorescent
dyes.
Scheme
1
Synthesis of Disperse Blue phosphoramidite for 5ꢀ-
oligonucleotide labelling. Reagents and conditions: i, 2-cyanoethyl-
N,N-diisopropyl chlorophosphine (1.1 eq.), DIPEA (4 eq.), CH2Cl2,
rt, 1 h, 64%.
Recently we described a new fluorescence quencher based on
1,4-diaminoanthraquinone.10 It is a non-fluorescent molecule,
thus eliminating any possibility of background signals which
can arise when fluorophores are used as quenchers. Importantly,
it has a broad absorption in the 550–700 nm region of the
visible spectrum and is thus able to quench a wide range of
fluorophores, especially those that emit at longer wavelengths.
Here, we report the synthesis of three versions of this quencher
suitable for incorporation into oligonucleotide probes at either
the 3ꢀ-end, 5ꢀ-end or internally in place of a thymidine. In each
case the degree of fluorescence quenching has been determined.
This monomer was used to synthesise oligonucleotide ODN
01 (Table 1) labelled with a 5ꢀ-Disperse Blue quencher. This
was compared with a 5ꢀ-methyl red labelled oligonucleotide of
the same sequence (ODN 07) which was synthesised using the
monomer shown in Fig. 1.9 Five complementary fluorescent
oligonucleotides (ODN 02–06) were also synthesised, each
labelled with a different fluorophore at the 3ꢀ-end. The following
fluorescent dye labels were chosen to span a broad range of
the visible fluorescence emission spectrum: FAM (514 nm), Cy3
(564 nm), TAMRA (584 nm), Cy5 (666 nm) and Cy5.5 (704 nm).
Fig. 2 shows the basis of the fluorescence quenching assay.
When the two oligonucleotides are annealed, the fluorophore
and quencher are held in close proximity causing the duplex
to be non-fluorescent. Heating causes the strands to dissociate,
separating the fluorophore from the quencher, and the change
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The use of the new dark quencher in TaqManꢀ and Scorpion
oligonucleotide probes in real-time PCR has been demonstrated
and its effect on duplex stability has also been studied by UV
thermal melting experiments.
2 5 3 4
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 2 5 3 4 – 2 5 4 2
T h i s j o u r n a l i s
T h e R o y a l S o c i e t y o f C h e m i s t r y 2 0 0 5
©

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Table 1 Oligonucleotides used in this study
Oligonucleotide
Sequence
ODN 01
ODN 02
ODN 03
ODN 04
ODN 05
ODN 06
ODN 07
ODN 08
ODN 09
ODN 10
ODN 11
ODN 12
ODN 13
ODN 14
ODN 15
ODN 16
ODN 17
ODN 18
ODN 19
ODN 20
ODN 21
ODN 22
ODN 23
ODN 24
ODN 25
ODN 26
ODN 27
ODN 28
ODN 29
ODN 30
ODN 31
ODN 32
D5GAAAAAACTTGGATCC
GGATCCAAGTTTTTTCTAMRA
GGATCCAAGTTTTTTCCy3T
GGATCCAAGTTTTTTCFAM
GGATCCAAGTTTTTTCCy5T
GGATCCAAGTTTTTTCCy5.5T
MeRedGAAAAAACTTGGATCC
FAMATGCCCTCCCCCATGCCATCCTGCGTTD3
FAMATGCCCTCCCCCATGCCATCCTGCGTTMeRed
TAMRAATGCCCTCCCCCATGCCATCCTGCGTTD3
TAMRAATGCCCTCCCCCATGCCATCCTGCGTTMeRed
JOEATGCCCTCCCCCATGCCATCCTGCGTTD3
JOEATGCCCTCCCCCATGCCATCCTGCGTTMeRed
ROXATGCCCTCCCCCATGCCATCCTGCGTTD3
ROXATGCCCTCCCCCATGCCATCCTGCGTTMeRed
TCCAAGTTTTTDUCTA
TCCAAGDUTTTTTCTA
TCCAAGTTTTTTCDUA
TCCAAGTTTTTTCTA
FAMTAGAAAAAACTTGGA
TAGAAAAAACTTGGA
ATTAGAAAAAACTTGGA
FAMCCCGCGCCTTTCCTCCACTGTTGCGCGCGGGMeRedHATGGTGTGTCTTGGGATTCA
FAMCCCGCGCCTTTCCTCCACTGTTGCGCGCGGGDUHATGGTGTGTCTTGGGATTCA
CAGTGGAGGAAAGMeRed
CAGTGGAGGAAAGD3
FAMCTTTCCTCCACTGTTGCHATGGTGTGTCTTGGGATTCA
AGGTGCGATTCTATGAGACGMeRedT
AGGTGCGATTCTATGAGACGD3
FAMCGTCTCATAGAATCGCACCTHAGATTGCCAAGGACGACATC
Cy3CGTCTCATAGAATCGCACCTHAGATTGCCAAGGACGACATC
Cy5CGTCTCATAGAATCGCACCTHAGATTGCCAAGGACGACATC
D5 = 5ꢀ Disperse Blue quencher; D3 = 3ꢀ Disperse Blue quencher; MeRed = methyl red quencher; FAM = fluorescein; TAMRA =
tetramethylrhodamine; JOE = 4ꢀ-5ꢀ-dichloro-2ꢀ,7ꢀ-dimethoxyfluorescein; ROX = rhodamine; DU = Disperse Blue quencher 2ꢀ-deoxyuridine; H =
hexaethyleneglycol.
Fig. 1 Methyl red phosphoramidite monomer.
Fig. 3 A comparison of the fluorescent signal-to-noise ratio of Disperse
Blue (blue) and methyl red (red) quenchers for five different fluorescent
dyes using the assay in Fig. 2.
efficient than Disperse Blue as a quencher for FAM (kem 514 nm),
but much less efficient in quenching the other four fluorescent
dyes TAMRA (kem 584 nm), Cy3 (kem 564 nm), Cy5 (kem 666 nm)
and Cy5.5 (kem 704 nm). The improved quenching by Disperse
Blue is likely to be a result of the relatively good overlap
between its absorption spectrum and the emission spectra of the
individual fluorescent dyes (Fig. 4). In contrast, the absorption
spectrum of methyl red does not overlap with the emission spec-
tra of the longer wavelength dyes used in fluorogenic assays and
it is noteworthy that methyl red does not quench Cy5 or Cy5.5
efficiently, even though the two molecules are in close proximity
and are presumably able to partake in collisional quenching.
Fig. 2 Assay to determine the relative efficiency of quenchers with a
range of fluorophores. When the fluorophore and quencher probes are
hybridised, the fluorescence is quenched. Fluorescence is observed when
the duplex is denatured by heating.
in fluorescence is measured. The efficiency of the quenching
is expressed as a ratio of the fluorescence of the single stranded
oligonucleotide (at 75 ◦C) relative to that of the duplex (at 25 ◦C).
The results (Fig. 3) were normalised to the values obtained
for Disperse Blue to allow a direct comparison between each
fluorophore-quencher pair. Methyl red was marginally more
3ꢀ-Labelling of oligonucleotides with Disperse Blue
In order to synthesise oligonucleotides labelled at the 3ꢀ-end
with Disperse Blue, a suitably functionalised CPG (controlled
pore glass) was prepared by the method in Scheme 2. The
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 2 5 3 4 – 2 5 4 2
2 5 3 5

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as quenchers of FAM, JOE, and TAMRA (Fig. 5). This improve-
ment in performance is manifested as an increase in fluorescent
signal, and in the case of TAMRA a decrease in Ct (cycle
number at which the fluorescent signal emerges from the back-
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ground). TaqManꢀ probes containing ROX fluorophore were
also studied, but it was not possible to use a passive reference and
consequently fluctuations in the PCR curves were observed, as
the software was unable to create a flat baseline (data not shown).
Synthesis of Disperse Blue dU phosphoramidite (10)
The synthesis of fluorogenic oligonucleotide probes sometimes
requires the quencher to be incorporated within the oligonu-
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cleotide sequence (e.g. Scorpion primers, certain TaqManꢀ
probes). In order to fulfil this requirement Disperse Blue
must be attached to a nucleoside that is suitable for Watson–
Crick base pairing and be converted to a phosphoramidite for
incorporation during automated oligonucleotide synthesis. The
modified 2ꢀ-deoxyuridine nucleoside 814 was chosen and coupled
to Disperse Blue succinic acid derivative 3 to give the Disperse
Blue 2ꢀ-deoxyuridine nucleoside 9. This was then converted to
the Disperse Blue phosphoramidite 10 for internal-labelling of
oligonucleotides at thymidine sites (Scheme 3).
Fig. 4 The visible absorption spectra of the Disperse Blue (blue) and
methyl red (red) quenchers.
2ꢀ-deoxyribose derivative 4 (a-anomer) was prepared by a
method previously reported in our laboratory9 and this provided
the backbone for attachment of the quencher moiety. In order to
couple Disperse Blue to backbone 4, it was necessary to convert
the primary alcohol to an amine. This was carried out in high
yield via a Mitsunobu reaction, followed by hydrazine cleavage of
the phthalimide protecting group. The primary amino group of
compound 2 was then treated with succinic anhydride to produce
the Disperse Blue succinic acid derivative 3. Amide coupling of 4
with the Disperse Blue derivative 3 produced compound 5, and
derivatisation of the 3ꢀ-hydroxyl group with succinic anhydride
gave 6 in good yield. Coupling of compound 6 to amino-
functionalised LCAA-CPG gave Disperse Blue CPG 7 suitable
for use in automated DNA synthesis (resin loading 27 lmol g−1).
Optimum conditions for deprotection of oligonucleotides con-
taining the Disperse Blue quencher have been determined.10
UV Thermal melting
A series of oligonucleotides (ODN 16-ODN 22 in Table 1)
was synthesised to investigate the effect of the Disperse Blue
dU quencher on duplex stability by UV thermal melting.
The oligonucleotides (ODN 16–18) contain the Disperse Blue
2ꢀ-deoxyuridine nucleoside at three different positions, and
ODN 19 is an unmodified control oligonucleotide of the same
sequence. Three complementary strands were prepared, all con-
taining the core sequence of ODN 21 (blunt-end complement).
ODN 20 differs by the addition of 5ꢀ-FAM and ODN 22 has two
additional bases (AT) at the 5ꢀ-end. In all cases incorporation
of the Disperse Blue quencher had a stabilising effect on the
duplex, causing an increase in Tm by up to 6.4 ◦C (Table 2).
The increase in Tm was greatest when the quencher was
placed close to the end of the duplex (ODN 18) and least when
the quencher was in the middle (ODN 17). In the latter case
changing the nature of the complementary oligonucleotide had
very little effect on Tm. However, if the complementary sequence
was labelled at the 5ꢀ-end with FAM there was significant
stabilisation when the quencher and the fluorophore were in
close proximity. The Disperse Blue moiety protrudes into the
major groove from the 5-position of the uracil base, and with
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Synthesis and evaluation of Disperse Blue TaqManꢀ probes
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In the Taqmanꢀ method, fluorescence quenching occurs by
FRET13 within the intact probe. During the extension phase
of PCR the probe binds to its specific target and is degraded by
the 5ꢀ→3ꢀ exonuclease activity of Taq polymerase. This releases
the quencher and fluorophore into solution, where they are
spatially separated, resulting in an increase in fluorescent signal.
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TaqManꢀ probes targeting a 27-mer sequence in the human b-
actin gene (ODN 08 to ODN 15) were synthesised using the
3ꢀ-Disperse Blue quencher in combination with a number of
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different 5ꢀ-fluorophores. The Disperse Blue TaqManꢀ probes
performed significantly better than the methyl red counterparts
Scheme 2 Synthesis of Disperse Blue quencher CPG. Reagents and conditions: i, DEAD (1.1 eq.), PPh3 (1.1 eq.), phthalimide (1.1 eq.), THF, rt,
1.5 h, then hydrazine monohydrate (5 eq.), CH2Cl2–MeOH (1 : 1), rt, 15 h, 90% over two steps; ii, succinic anhydride (1.1 eq.), DMAP (0.1 eq.),
pyridine, rt, 1.5 h, 90%; iii, DIPEA (6 eq.), HOBT (1.1 eq.), EDC (1.1 eq.), 3 (1.1 eq), DMF, rt, 15 h, 60%; iv, succinic anhydride (1.2 eq.), DMAP
(0.1 eq.), pyridine, rt, 18 h then 60 ◦C, 5 h, 94%; v, LCAA-CPG, EDC (5 eq.), DIPEA (5 eq.), 1% DIPEA in CH2Cl2, rt, 4 h, loading = 27 lmol g−1
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 2 5 3 4 – 2 5 4 2
.
2 5 3 6

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to the general duplex-stabilising effect of the Disperse Blue
dU monomer; the presence of the propyne moiety at the 5-
position of the heterocyclic base (which is known to stabilise
DNA duplexes), stabilisation from the anthraquinone moiety by
intercalation,15 stacking of the anthraquinone at the 5ꢀ- or 3ꢀ-end
of the duplex to reduce fraying,16 and/or binding in the minor
groove.17 Factors that stabilise probe:target duplexes can be
utilised in genetic analysis to facilitate the use of shorter probes
and thereby make it possible to achieve greater discrimination
between the wild-type and single point mutations.
Scorpion primer analysis
Stem-loop Scorpion primers consist of a probe sequence held
in a hairpin loop conformation by a short complementary stem
duplex at the 5ꢀ- and 3ꢀ-termini of the probe.3 A fluorophore is
attached to the 5ꢀ-end of one arm of the stem and a quencher
to the 3ꢀ-end. The quencher is attached to the 5ꢀ-end of a PCR
primer via a PCR stopper. Scorpion primers anneal to the target
DNA in the cooling phase of the PCR cycle post-denaturation,
and the primer element is extended during the amplification
step. The resultant amplicon contains a sequence that is
complementary to the probe, which is rendered single-stranded
during the denaturation stage of each PCR cycle. On cooling,
the probe is free to bind to this complementary sequence,
producing an increase in fluorescence, as the quencher is no
longer in the vicinity of the fluorophore.
We utilised the W1282X locus18 of the human ABCC7 gene19,20
as the Scorpion test system. This gene resides on chromosome
7 and produces the cystic fibrosis transmembrane conductance
regulator protein. Specific mutations cause cystic fibrosis, an
autosomal recessive disease that is estimated to occur in 1/2000
live births and is carried by 5 percent of the population. These
mutations affect the transport of chloride ions across the apical
membrane of epithelial cells, producing a number of clinical
symptoms.
Two FAM Scorpion primers with either methyl red (ODN
23) or Disperse Blue (ODN 24) quenchers were synthesised
targeting a 17-mer wild-type sequence in the W1282X locus.
For the Disperse Blue Scorpion the derivatised 2ꢀ-deoxyuridine
phosphoramidite 10 was used. W1282X is a single point
mutation (G to A transition), so the wild-type Scorpions form a
duplex with the mutant target that contains a CA mismatch. The
Disperse Blue Scorpion gave comparable results to the methyl
red Scorpion in terms of Ct value and fluorescent signal (Fig. 6),
and both Scorpions discriminated clearly between the wild-type
(homozygote) and heterozygote.
The alternative “duplex” Scorpion primer format was also
evaluated.4 In this format there is no stem-loop and instead the
probe element has a fluorophore attached at its 5ꢀ-end, which is
annealed to a separate complementary oligonucleotide bearing
a quencher at its 3ꢀ-end. The mechanism of action is essentially
the same as in the stem-loop format, but the quenching
mechanism is inter-molecular as opposed to intra-molecular.
Two FAM duplex Scorpion primers with methyl red (ODN
25) or Disperse Blue (ODN 26) as quencher were synthesised
and compared in real-time PCR on the ABI PrismTM 7700
Fig. 5 Comparison of Disperse Blue and methyl red in a TaqManꢀR
PCR assay using the fluorophores FAM, JOE and TAMRA. The relative
fluorescence vs. the number of PCR cycles is shown.
Disperse Blue dU:dA as the penultimate base pair in the duplex,
the anthraquinone moiety is able to come into close contact
with the 5ꢀ-FAM. There are several factors that could contribute
Table 2 UV-Melting data (all temperatures in ◦C); DU = Disperse Blue quencher 2ꢀ-deoxyuridine
TmODN20 (5ꢀ
Oligonucleotide FAM complement) DTm
TmODN21 (blunt
complement)
TmODN22 (long
complement)
a
a
a
Sequence
DTm
DTm
TCCAAGTTTTT(DU)CTA ODN 16
TCCAAG(DU)TTTTTCTA ODN 17
TCCAAGTTTTTTC(DU)A ODN 18
56.7
54.2
57.5
5.6
3.1
6.4
55.1
55.0
55.8
3.4
3.3
4.1
56.3
55.4
57.2
4.1
3.2
5.0
TCCAAGTTTTTTCTA
(unmodified)
ODN 19
51.1
—
51.7
—
52.2
—
a DTm = Tm of duplex containing quencher − Tm of unmodified duplex.
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Scheme 3 Synthesis of Disperse Blue 2ꢀ-deoxyuridine phosphoramidite. Reagents and conditions: i, EDC (1.4 eq.), HOBt (1.4 eq.), DIPEA (1.4 eq.),
3 (0.9 eq.), DMF, rt, 18 h, 45%; ii, 2-cyanoethyl-N,N-diisopropyl chlorophosphine (1.5 eq.), DIPEA (4.0 eq.), THF, rt, 2.5 h, 93%.
Fig. 7 Comparison of Disperse Blue and methyl red as quenchers of
FAM in a duplex Scorpion primer assay. The relative fluorescence vs.
number of PCR cycles is shown.
Fig. 6 Comparison of Disperse Blue and methyl red as quenchers of
FAM in a stem-loop Scorpion primer assay. The relative fluorescence vs.
number of PCR cycles is shown.
Studies were also made on the Rotor-Gene genetic analyser
(Fig. 8). This machine allows the longer wavelength Cyanine
Genetic analyser (Fig. 7). The Disperse Blue duplex Scorpion
performed better than the methyl red version, both in terms of
Ct and fluorescent signal. Each Scorpion discriminated between
homozygote and heterozygote in a cystic fibrosis mutation
assay, but the fluorescent signal-to-noise ratio was significantly
enhanced with the Disperse Blue quencher.
dyes to be used and so comparison was made between methyl
red (ODN 28) and Disperse Blue (ODN 29), with the fluorescent
dyes FAM (ODN 30), Cy3 (ODN 31) and Cy5 (ODN 32) in the
duplex Scorpion format. Good amplification was seen for both
quenchers with the FAM containing complement, although Ct
and fluorescent signal were better for Disperse Blue. The cyanine
2 5 3 8
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 2 5 3 4 – 2 5 4 2

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signal-to-noise ratios were observed, particularly with the longer
wavelength dyes. The Disperse Blue quencher significantly
stabilises DNA duplexes, and this may facilitate the design of
shorter, more specific probes.
Experimental
Preparation of compounds
Reagents were purchased from Aldrich, Avocado, Cruachem,
Fluka, Lancaster or Link Technologies and used without
purification, with the exception of the following solvents that
were purified by distillation: methanol (over iodine and magne-
sium), tetrahydrofuran (over sodium wire and benzophenone),
dichloromethane, diisopropylethylamine, pyridine and triethy-
lamine (over calcium hydride). All chemical reactions were
carried out under argon using oven dried glassware. Column
chromatography was performed under pressure using Fisher
Scientific DAVISIL 60A (35–70 micron) silica. Compounds
were visualised by irradiation at 254 nm or by staining with
anisaldehyde. Thin layer chromatography was performed using
Merck Kieselgel 60 F24 (0.22 mm thickness, aluminium backed).
1H NMR spectra were measured at 300 MHz on a Bruker AC300
spectrometer or 400 MHz on a Bruker DPX400 spectrometer.
13C NMR and 31P NMR spectra were measured at 75 MHz
and 90 MHz respectively on a Bruker AC300 spectrometer.
Chemical shifts are given in ppm relative to tetramethylsilane
and J values are given in Hz. Low-resolution mass spectra
were recorded using the electrospray technique on a Fisons
VG platform instrument in acetonitrile or a Waters ZMD
quadrupole mass spectrometer in methanol or water. High-
resolution mass spectra were recorded using the electrospray
technique on a Bruker APEX III FT-ICR mass spectrometer in
methanol, acetonitrile or water. Infrared spectra were recorded
on a BIORAD FT-IR using a Golden Gate adapter and
BIORAD WIN-IR software or a Satellite FT-IR using a Golden
Gate adapter and WIN FIRST-lite software. Absorptions are
described as strong (s) or medium (m). UV-vis data was recorded
on a Perkin–Elmer Lambda 2 spectrometer, using 1 mL cuvettes
at 25 ◦C. All extinction coefficients are quoted in parentheses
(cm2 mol−1). Melting points were measured on a Gallenkamp
electrothermal melting point apparatus and are uncorrected.
1-(((2-Cyanoethyl-N,N -diisopropylphosphoramidyl)ethyl)
amino)-4-(methylamino)anthraquinone (1). Crude 1-((2-hydroxy-
ethyl)amino)-4-(methylamino)anthraquinone (Disperse Blue
3, Aldrich) was purified by silica gel column chromatography
eluting with ethyl acetate–hexane (1 : 1). The major fraction
(pure Disperse Blue 3) (0.30 g, 1.00 mmol) was dissolved in dry
dichloromethane (20 mL) and diisopropylethylamine (0.65 g,
5.20 mmol) was added, followed by 2-cyanoethoxy-(N,N-
diisopropylamino)chlorophosphine (0.28 g, 1.20 mmol), under
argon. The reaction was stirred for 3 h then diluted with ethyl
acetate (50 mL), washed with saturated potassium chloride
(50 mL), dried (anhydrous sodium sulfate) and evaporated to
dryness in vacuo. Purification by silica gel column chromato-
graphy under argon, eluting with ethyl acetate, gave a solid
which was dissolved in anhydrous acetonitrile (5 mL) and
filtered. This gave the title compound as a blue/purple solid
that was sensitive to air (0.32 g, 64%). 1H NMR (CDCl3):
10.85–10.78 (m, 1H, CH2NH), 10.55–10.48 (m, 1H, CH3NH),
8.26–8.22 (m, 2H, CH Ar6,7), 7.62–7.57 (m, 2H, CH Ar5,8), 7.20
(d, J = 9.5 Hz, 1H, CH Ar3), 7.15 (d, J = 9.5 Hz, 1H, CH Ar2),
3.95–3.72 (m, 4H, CH, CH2N), 3.59–3.41 (m, 4H, CH2O),
3.00 (d, J = 5.2 Hz, 3H, CH3N), 2.55 (t, J = 6.6 Hz, 2H,
Fig. 8 Comparison of Disperse Blue and methyl red as quenchers of
FAM, Cy3 and Cy5 in a duplex Scorpion primer assay. The normalised
fluorescence vs number of PCR cycles is shown. Each PCR run was
carried out in duplicate.
dye Scorpions only showed good amplification when paired
with Disperse Blue. The relatively poor amplification observed
with the methyl red/cyanine dye combination is not just a
consequence of the improved signal-to-noise ratio produced by
Disperse Blue, and further investigations are needed to ascertain
the exact reason for this observation. Most importantly Disperse
Blue and cyanine dye combinations gave excellent signals relative
to background with the greatest fluorescence output seen for
Cy5.
Conclusion
A non-fluorescent quencher based on Disperse Blue has been
incorporated into synthetic oligonucleotides at the 5ꢀ- and 3ꢀ-
ends and internally within the DNA sequence. The quencher
absorbs over a broad region of the visible spectrum and has
been used to quench fluorophores that emit in the 514–704 nm
range. The utility of the quencher has been demonstrated in real-
i
CH2CN), 1.15–1.05 (m, 12H, Pr–CH3); 31P NMR (CDCl3):
148.96; ES+/MS: 497.3 (M + H)+, 519.3 (M + Na)+
1-((2-Aminoethyl)amino)-4-(methylamino)anthraquinone (2).
1-((2-Hydroxyethyl)amino)-4-(methylamino)anthraquinone
(Disperse Blue 3) (0.10 g, 0.33 mmol), phthalimide (0.06 g,
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time PCR using simple hybridisation probes, TaqManꢀ probes
and two types of Scorpion primer. In most cases much improved
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2 5 3 9

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0.37 mmol) and triphenylphosphine (0.10 g, 0.37 mmol) were
dissolved in dry tetrahydrofuran (20 mL). To this was added
a solution of diethylazodicarboxylate (0.07 g, 0.37 mmol) in
dry tetrahydrofuran (2 mL). The reaction was stirred for 1.5 h,
evaporated to dryness in vacuo and the residue was dissolved
in dichloromethane–methanol (1 : 2, 20 mL). Hydrazine
monohydrate (0.083 g, 1.65 mmol) was added and the mixture
was stirred for 15 h, after which the solvent was removed
in vacuo. The blue solid was dissolved in dichloromethane
(50 mL), washed with sodium hydroxide (2 M, 2 × 50 mL)
and the product was removed from the organic layer by
washing with aqueous citric acid (10% w/v, 100 mL). The blue
aqueous layer was neutralised (saturated sodium bicarbonate,
2 × 100 mL) and the product extracted by washing with
dichloromethane (150 mL). The organic layer was dried
(anhydrous sodium sulfate) and evaporated to dryness in vacuo.
Purification by silica gel column chromatography, eluting with
dichloromethane–methanol (9 : 1) gave the title compound
a blue solid (0.14 g, 60%). 1H NMR (CDCl3): 10.68–10.62
(m, 1H, NH), 10.53–10.48 (m, 1H, NH), 8.23–8.19 (m, 2H,
CH Ar5,8), 7.62–7.56 (m, 2H, CH Ar6,7), 7.39–7.09 (m, 11H,
CH Ar), 6.72 (d, J = 9.0 Hz, 4H, CH Ar), 6.69–6.64 (m, 1H,
NH), 6.01–5.96 (m, 1H, NH), 5.12 (d, J = 4.5 Hz, 1H, H1ꢀ ),
4.16–4.09 (m, 2H, H3ꢀ , H4ꢀ ꢀ), 3.70 (s, 6H, OCH3), 3.69–3.60 (m,
1H, CHO), 3.49–3.44 (m, 4H, CH2), 3.33–3.27 (m, 1H, CHO),
3.12–3.01 (m, 4H, NCH2, CH25ꢀ ), 3.00 (d, J = 5.0 Hz, 3H,
NCH3), 2.52–2.41 (m, 4H, CH2CO), 2.15–1.90 (m, 2H, H2ꢀ ),
1.50–1.21 (m, 8H, CH2); 13C NMR (CDCl3): 182.55, 182.09,
171.93, 171.86 (CO), 158.31 (COMe), 146.93, 145.71 (CNH),
144.66, 135.87, 135.78, 134.36, 134.16 (C), 132.00, 131.84,
130.06, 129.90, 128.16, 128.00, 127.64 (CH), 126.60 (C), 125.89,
123.24, 123.02, 112.95 (CH), 110.10, 1ꢀ09.68 (C), 104.17 (CH1ꢀ ),
86.57 (CH4ꢀ ), 85.91 (C) 73.33 (CH3 ), 67.20 (CH2O), 63.94
(CH5ꢀ ), 55.04 (CH3), 45.98, 41.79, 40.79 (CH2N), 39.34 (CH22ꢀ ),
31.68, 29.33 (CH2), 29.26, 29.27 (CH2CO, CH3), 26.40, 25.71
(CH2); ES+/MS 913.0 (M + H)+, 935.0 (M + Na)+; HRMS
(ES+) for C53H60N4O10 (M + H)+: calculated 913.4382, found
913.4364. IR mmax/cm−1: 3293 (s), 2927 (m), 2868 (m), 1636 (m),
1572 (s), 1551 (s), 1513 (s). UV/vis (CH2Cl2): 562 nm (11.1 ×
103 dm3 mol−1 cm−1), 595 nm (19.6 × 103 dm3 mol−1 cm−1),
642 nm (20.7 × 103 dm3 mol−1 cm−1). Mp 150–152 ◦C.
1
as a blue amprphous solid (0.09 g, 90%). H NMR (CDCl3):
10.79 (br, 1H, CH2NH), 10.51 (br, 1H, CH3NH), 8.30–8.20
(m, 2H, CH Ar6,7), 7.65–7.55 (m, 2H, CH Ar5,8), 7.12 (d, J =
9.5 Hz, 1H, CH Ar3), 7.08 (d, J = 9.5 Hz, 1H, CH Ar2),
3.39–3.28 (m, 4H, CH2N), 3.00 (d, J = 5.0 Hz, 3H, CH3N),
1.32 (br s, 2H, NH2); 13C NMR (CDCl3): 182.89, 182.68 (CO),
147.31, 146.45 (CNH), 134.89, 134.85 (C Ar), 132.39, 132.35,
126.43, 126.33, 123.82, 123.33 (CH Ar), 110.45, 110.31 (C Ar),
46.31, 37.88 (CH2), 29.86 (CH3); ES+/MS: 296.0 (M + H)+;
HRMS (ES+) for C17H17N3O2 (M + H)+: calculated 296.1394,
found 296.1388. IR mmax/cm−1: 3325 (s), 2916 (m), 2857 (m),
1647 (m), 1561 (s), 1518 (s). UV/vis (CH2Cl2): 560 nm (4.3 ×
103 dm3 mol−1 cm−1), 598 nm (7.9 × 103 dm3 mol−1 cm−1),
645 nm (8.5 × 103 dm3 mol−1 cm−1).
1-(2-Succinamidoethylamino)-4-(methylamino)anthraquinone
(3). Compound 2 (0.09 g, 0.34 mmol), succinic anhydride
(0.04 g, 0.38 mmol) and N-dimethylaminopyridine (0.01 g,
0.09 mmol) were dissolved in dry pyridine (20 mL). The
reaction was stirred for 1.5 h, evaporated to dryness in vacuo,
and the product purified by silica gel column chromatography,
eluting with dichloromethane–methanol (9 : 1) to give the title
compound as a blue solid (0.09 g, 90%). 1H NMR (DMSO-d6):
10.81 (br, 1H, CH2NH), 10.62 (br, 1H, CH3NH), 8.36–8.27
(m, 2H, CH Ar6,7), 8.30–8.20 (m, 1H, NHCO), 7.78–7.65 (m,
2H, CH Ar5,8), 7.59 (d, J = 9.4 Hz, 1H, CH Ar3), 7.42 (d,
J = 9.4 Hz, 1H, Ar2), 3.61–3.20 (m, 4H, CH2N), 3.08 (d, J =
5.2 Hz, 3H, CH3N), 2.45 (t, J = 7.5 Hz, 2H, CH2CO), 2.34
(t, J = 7.5 Hz, 2H, CH2CO); 13C NMR (DMSO-d6): 180.89,
179.85, 173.07, 170.85 (CO), 146.07, 145.16 (CNH), 134.89,
134.85, 131.51, 124.93, 123.77, 123.45 (CH Ar), 107.85, 107.31
(C Ar), 54.13, 41.77, 39.28, 29.31 (CH2), 28.50 (CH3); ES+/MS:
396.0 (M + H)+; HRMS (ES+) for C21H21N3O5 (M + H)+:
calculated 396.1554, found 396.1545. IR mmax/cm−1: 3302 (s),
2921 (m), 1650 (m), 1564 (s). UV/vis (CH2Cl2): 561 nm (4.2 ×
103 dm3 mol−1 cm−1), 595 nm (7.8 × 103 dm3 mol−1 cm−1),
642 nm (8.7 × 103 dm3 mol−1 cm−1). Mp 179–182 ◦C.
5ꢀ-O-(4,4ꢀ-Dimethoxytrityl)-1ꢀ-O-(6-N1-(2-((4-(methylamino)-
anthraquinone)amino)ethyl)succinamidohexyl)-2ꢀ-deoxy-3ꢀ-O-oxo-
butanoic acid-D-ribose (6). Compound 5 (0.15 g, 0.16 mmol)
was dissolved in dry pyridine (5 mL) and succinic anhydride
(0.04 g, 0.40 mmol) and 4-(dimethylamino)pyridine (20.00 mg)
◦
were added. The mixture was heated at 60 C for 8 h and the
solvent then removed in vacuo. The residue was dissolved in
dichloromethane (150 mL), washed with saturated potassium
chloride (150 mL), dried (anhydrous sodium sulfate) and
evaporated to dryness. Purification by column chromatography
eluting with dichloromethane–methanol–acetic acid (99 : 1 :
0 to 80 : 20 : 0 to 79 : 20 : 1) gave the title product as a blue
amorphous solid (0.15 g, 94%). 1H NMR (CDCl3): 10.50–10.40
(m, 2H, NH), 8.15–8.08 (m, 2H, CH Ar6,7), 7.55–7.49 (m, 2H,
CH Ar5,8), 7.35–7.06 (m, 11H, CH Ar), 6.90–6.79 (m, 2H, NH),
6.71 (d, J = 8.5 Hz, 4H, CH Ar), 5.14 (d, J = 4.5 Hz, 1H,
H1ꢀ ), 5.07 (d, J = 6.0 Hz, 1H, H3ꢀ ), 4.13–4.09 (m, 1H, H4ꢀ ),
3.68 (s, 6H, OCH3), 3.62–3.33 (m, 4H, CH2O, H5ꢀ ), 3.31–3.11
(m, 4H, NCH2), 3.12–3.02 (m, 3H, NCH3), 2.96–2.86 (m,
2H, NCH2), 2.60–2.42 (m, 8H, CH2CO), 2.30–1.85 (m, 2H,
H2ꢀ ), 1.70–1.00 (m, 8H, CH2); 13C NMR (CDCl3): 182.55,
182.09, 171.86, 171.93 (CO), 157.30 (COCH3), 145.84, 144.69
(CNH), 143.69, 134.87, 134.76, 133.23, 132.89 (C), 130.90,
130.66, 128.91, 127.02, 126.62 (CH), 125.60 (C), 124.92, 124.77,
122.25, 121.96, 111.95 (CH), 110.10, 1ꢀ09.68 (C), 102.69 (CH1ꢀ ),
84.94 (CH4ꢀ ), 82.09 (C) 74.42 (CH3 ), 65.61 (CH2O), 62.91
(CH5ꢀ ), 54.04 (CH3), 45.98, 41.79, 40.79 (CH2N), 39.34 (CH22ꢀ ),
31.68, 29.33 (CH2), 29.27, 29.26 (CH2CO, CH3), 26.40, 25.71
(CH2); ES+/MS: 1034.9 (M + Na)+, 1051.4 (M + K)+. IR
mmax/cm−1: 3322 (s), 2941 (m), 2606 (s), 1655 (s), 1574 (s).
UV/vis (CH2Cl2): 597 nm (10.6 × 103 dm3 mol−1 cm−1), 642 nm
(9.7 × 103 dm3 mol−1 cm−1).
5ꢀ-O-(4,4ꢀ-Dimethoxytrityl)-1ꢀ-O-(6-N1-(2-((4-(methylamino)-
anthraquinone)amino)ethyl)succinamidohexyl)-2ꢀ-deoxy-D-ribose
(5). Compound 3 (0.10 g, 0.26 mmol), 1-(3-dimethylamino-
propyl)-3-ethylcarbodiimide hydrochloride (EDC) (0.06 g,
0.29 mmol), 1-hydroxybenzotriazole (HOBt) (0.04 g, 0.29 mmol)
were dissolved in N,N-dimethylformamide (5 mL) and stirred
for 10 min. To this was added 1-(6-aminohexyloxy)-5-(4,4ꢀ-
dimethoxytrityl)-2-deoxy-a-D-ribose (compound 4) (0.16 g,
0.29 mmol) in N,N-dimethylformamide (5 mL) and diisopropyl-
ethylamine (0.5 mL). This solution was then added to the acidic
mixture and stirred for 15 h. The solvent was then removed
in vacuo and the solid was redissolved in dichloromethane
(100 mL), washed with water (100 mL), saturated sodium
bicarbonate (100 mL), dried (anhydrous sodium sulfate) and
evaporated to dryness in vacuo to give the title compound as
5ꢀ-O-(4,4ꢀ-Dimethoxytrityl)-1ꢀ-O-(6-N1-(2-((4-(methylamino)-
anthraquinone)amino)ethyl)succinamidohexyl)-2ꢀ -deoxy-3ꢀ -O-
oxobutanoic acid-D-ribose LCAA-CPG (7). All glassware
was soaked in trimethylsilyl chloride–dichloromethane
(9.5 : 0.5) for 0.5 h and then rinsed with acetone, water,
then acetone again, before drying in an oven. The resin
(0.50 g) was washed with a solution of dichloromethane–
diisopropylethylamine (99 : 1) to liberate the free amines and
then dried under argon. Compound 6 (0.05 g, 0.05 mmol) was
dissolved in dichloromethane–diisopropylethylamine (99 : 1)
(0.5 mL) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide
hydrochloride (47.00 mg, 0.25 mmol) was added. The
resin (long-chain alkylamino controlled pore glass, Link
Technologies, 0.50 g) was added and the mixture agitated gently
2 5 4 0
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 2 5 3 4 – 2 5 4 2

View Article Online
for 2 h. The resin was filtered and washed with dichloromethane–
diisopropylethylamine solution (99 : 1, 3 × 10 mL), soaked in a
mixture of acetic anhydride, 2,6-lutidine and 1-methylimidazole
in tetrahydrofuran for 1 h (DNA synthesis capping agents),
washed with dichloromethane–diisopropylethylamine (99 : 1,
3 × 10 mL) solution and finally with diethyl ether (3 × 20 mL).
A small sample of the resin (2 mg) was dissolved in conc.
hydrochloric acid–ethanol (3 : 2 v/v, 25 mL) and shaken for
5 min. The absorbance of the orange solution (1 mL, 1 cm path
length) was measured at 495 nm (abs = 0.156) and the loading
of the resin was then calculated as 27 lmol g−1 from the known
absorbance of 1 lmol of DMT cation in 25 mL volume (71.7).
Columns were prepared for 0.2 lmol scale oligonucleotide
synthesis by adding 9 mg of functionalised resin to each column
(Glen Research screw-fit columns).
2H, CH Ar6,7), 8.10–7.98 (m, 1H, NH), 7.64–7.50 (m, 2H, CH
Ar5,8), 7.41–7.01 (m, 14H, CH Ar, NH), 6.78 (d, J = 8.5 Hz,
4H, CH Ar), 6.61 (s, 1H, NHCO), 6.26 (t, J = 6.5 Hz, 1H, H1ꢀ ),
4.59–4.48 (m, 1H, H3ꢀ ), 4.27–4.16 (m, 1H, H4ꢀ ), 4.15–3.90 (m,
i
2H, CH2), 3.90–3.21 (m, 16H, CH3O, CH2N, PrH, CH2O),
3.19–3.08 (m, 2H, CH2N, H5ꢀ ), 2.98 (d, J = 5.0 Hz, 3H,
CH3N), 2.65–2.41 (m, 6H, CH2), 2.38–1.88 (m, 2H, CH2O,
H2ꢀ ), 1.61–0.97 (m, 18H, CH2, iPrCH3); ES+/MS: 1296.5 (M +
Na)+. 31P NMR (CDCl3): 149.60, 149.28.
Oligonucleotide synthesis
All oligonucleotides were synthesised on an ABI 394 DNA
synthesiser by standard automated solid-phase methods using
b-cyanoethyl phosphoramidite chemistry (0.2 lmol scale). All
coupling efficiencies (as measured by automated trityl analysis)
were >98%. For the synthesis of Cy3, Cy5, Cy5.5 and Disperse
Blue oligonucleotides, fast-deprotecting dmf dG and acetyl dC
phosphoramidites were used (Link Technologies Ltd.). Disperse
Blue-functionalised resin was packed into ABI 394 DNA syn-
thesis columns (Glen Research Inc.). Deprotection of oligonu-
cleotides containing the Disperse Blue quencher was carried
out by transferring the resin-bound oligonucleotides from the
columns into 3 mL vials and treating with water–methanol–tert-
butylamine (2 : 1 : 1) at 70 ◦C for 2.5 h. Oligonucleotides contain-
ing◦cyanine dyes were deprotected in conc. aqueous ammonia at
55 C for 1 h only, and all other oligonucleotides were cleaved
and deprotected using concentrated aqueous ammonia at 55 ◦C
for 5 h. Methyl red aminohexyl deoxyribose CPG and FAM
aminohexyl deoxyribose CPG were used to add 3ꢀ-MeRed and
3ꢀ-FAM respectively and were prepared by published methods.9
All fluorescent dyes except ROX, TAMRA, and JOE were added
as phosphoramidites during oligonucleotide synthesis. Oligonu-
cleotides with 3ꢀ-cyanine dyes were synthesised on a thymidine
column and the first addition during solid-phase synthesis was
the cyanine dye. This method was used due to the inavailability
of cyanine-dye CPG. ROX, TAMRA and JOE NHS esters
(1 mg in DMSO, 25 lL) were added post-synthetically to
the 5ꢀ-aminohexyl- or 3ꢀ-aminohexyldeoxyribose-functionalised
oligonucleotides which were made from the corresponding
aminolink monomer or CPG.9 The amino-modified oligonu-
cleotides were labelled by dissolving in 0.5 M sodium bicar-
bonate/carbonate buffer at pH 9.25 (60 lL) and the resultant
solution was set aside overnight at room temperature. Excess dye
was removed by Sephadex gel-filtration (Nap 10, Pharmacia),
and oligonucleotides were purified by reversed-phase Gilson
HPLC on a C8 (octyl) column, eluting with a gradient of 0% to
40% acetonitrile in 0.1 M ammonium acetate buffer.21
5ꢀ -O-(4,4ꢀ-Dimethoxytrityl)-5-(3-(6-N1-(2-((4-(methylamino)-
anthraquinone)amino)ethyl)succinamidohexamide)propynyl)-2ꢀ -
deoxyuridine (9). Compound 3 (0.3 g, 0.78 mmol), 1-(3-dim-
ethylaminopropyl)-3-ethylcarbodiimide hydrochloride (0.23 g,
1.2 mmol) and 1-hydroxybenzotriazole (0.16 g, 1.2 mmol)
were dissolved in dry N,N-dimethylformamide (20 mL) and
stirred for 10 min. To this was added 6-amino-N-(3-(5ꢀ-
(4,4ꢀ -dimethoxytrityloxy)-2ꢀ -deoxyuridin-5-yl)prop-2-ynyl)-
hexanamide 8 (0.6 g, 0.87 mmol) and diisopropylethylamine
(0.16 g, 1.2 mmol) in dry N,N-dimethylformamide (10 mL).
The reaction mixture was stirred at room temperature for
18 h then the solvent was removed in vacuo. The residue was
dissolved in dichloromethane (150 mL), washed with water
(150 mL), saturated sodium bicarbonate (150 mL), dried with
sodium sulfate, evaporated to dryness and purified by column
chromatography with silica gel (dichloromethane–methanol, 9 :
1) to yield the title compound (0.38 g, 45%) as a blue solid.
1H NMR (CDCl3): 10.64 (s, 1H, CH2NH), 10.50 (br, 1H,
CH3NH), 8.16–8.07 (m, 2H, CH Ar6,7), 7.98–7.93 (m, 1H,
NH), 7.55–7.45 (m, 2H, CH Ar5,8), 7.40–7.05 (m, 12H, CH
Ar, NH), 7.00–6.87 (m, 2H, CH Ar), 6.72 (d, J = 8.5 Hz, 4H,
CH Ar), 6.35 (s, 1H,ꢀ NHCO), 6.18 (t, J = 6.5 Hz, 1H, H1ꢀ ),
4.49–4.41 (m, 1H, H3 ), 4.09–3.96 (m, 1H, H4ꢀ ), 3.90–3.70 (m,
2H, CH2), 3.65 (s, 6H, CH3O), 3.42–3.31 (m, 4H, CH2N),
3.29–3.16 (m, 2H, H5ꢀ ), 3.12–2.99 (m, 2H, CH2), 2.87 (d, J =
5.0 Hz, 3H, CH3N), 2.45–2.31 (m, 4H, CH2CO), 2.25–1.85 (m,
4H, CH2CO, H2ꢀ ), 1.56–1.12 (m, 6H, CH2); 13C NMR (CDCl3):
181.73, 181.29, 172.90, 172.34, 172.23, 162.70 (CO), 158.14
(C), 149.53, 146.59, 145.50 (CNH), 144.18 (C), 142.48 (CH),
135.18, 133.97, 133.73 (C), 131.56, 131.45, 129.59, 127.60,
127.53, 126.52, 125.61, 125.50, 122.78, 112.79 (CH Ar), 109.42,
109.04 (C Ar), 98.96 (C), 89.05 (C), 86.49 (C), 86.21 (CH),
85.39 (CH), 73.92 (C), 71.49 (CH), 63.33 (CH2), 54.86 (CH),
52.50 (CH2), 41.48, 41.25, 39.06, 38.82, 35.39, 31.35, 29.41
(CH2), 28.96 (CH3), 28.51, 25.71, 24.51 (CH2); ES+/MS: 1096.5
(M + Na)+; HRMS (ES+) for C60H63N7O12 (M + H)+: calculated
1074.4636, found 1074.4607. IR mmax/cm−1: 3317 (s), 3073 (m),
2941 (m), 1650 (s), 1574 (m). UV/vis (CH2Cl2): 560 nm (6.3 ×
103 dm3 mol−1 cm−1), 596 nm (12.2 × 103 dm3 mol−1 cm−1),
644 nm (13.6 × 103 dm3 mol−1 cm−1). Mp 138–144 ◦C.
R
TaqManꢀ real-time PCR
TaqManꢀR PCR reactions were carried out on an ABI PrismTM
7700 Genetic analyser. All reagents were supplied by Eurogentec.
Total reaction volume was 50 lL and the reaction mix contained
1 × reaction buffer with ROX passive reference to normalise the
data. The ABI PrismTM 7700 uses ROX as a passive reference
to normalise the data and account for pipetting inaccuracies
between samples. No passive reference was used when testing
the ROX labelled probe and consequently baseline fluctuations
were observed, as the software was unable to create a flat baseline
(data not shown). The reaction mix also included a standard
dNTP mix (200 lM, including dUTP), 4 mM MgCl2, 1 unit
of HotGoldStarTM Taq polymerase and sterile water. Primers
and probes were used at a concentration of 0.5 lM and were
designed to detect human b-actin.22 Human genomic DNA
(30 ng, Roche) was added to each reaction and this was replaced
with sterile water for negative controls, run with each set of
5ꢀ -O-(4,4ꢀ-Dimethoxytrityl)-5-(3-(6-N1-(2-((4-(methylamino)-
anthraquinone)amino)ethyl)succinamidohexamide)propynyl)-3ꢀ -
O-(2-cyanoethyl-N,N-diisopropylphosphoramidyl)-2ꢀ-deoxyuridine
(10). To compound 9 (0.35 g, 0.33 mmol) in dry dichloro-
methane (30 mL) was added diisopropylethylamine (0.23 mL,
1.32 mmol) and 2-cyanoethyl-N,N-diisopropyl chlorophos-
phine (0.113 mL, 0.50 mmol). The reaction mixture was stirred
under argon for 2.5 h then concentrated in vacuo. The residue
was dissolved in ethyl acetate (degassed with argon), washed
with saturated potassium chloride, dried (anhydrous sodium
sulfate), reduced to dryness in vacuo then purified by silica
gel column chromatography under argon to yield the title
R
samples. Standard TaqManꢀ PCR cycling conditions were used,
namely 95 ◦C for 10 min (initial denaturation), then 40 cycles of
95 ◦C for 15 s, 60 ◦C for 1 min.
1
product as an oil (0.36 g, 93%). H NMR (CDCl3): 10.73 (s,
1H, CH2NH), 10.61–10.48 (m, 1H, CH3NH), 8.31–8.15 (m,
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 2 5 3 4 – 2 5 4 2
2 5 4 1

View Article Online
Scorpion primer real-time PCR
Acknowledgements
The Scorpion sequences used to evaluate the fluorescence
quenchers (Table 1) were targeted at a 17- mer region of the wild-
type W1282X locus of the human ABCC7 gene.3,4 The sequence
of the reverse primer was 5ꢀ-GGCTAAGTCCTTTTGCTCAC-
3ꢀ. Total reaction volumes were 25 lL, containing 12.5 ng of
Human genomic DNA (NA 11472 (wild-type) and NA 1723
(W1282X heterozygote) purchased from Coriell). DNA was
replaced with sterile water for the negative controls. Concen-
trations of reagents were as follows: 1 × PCR buffer containing
ROX passive reference (Eurogentec), 200 lM dNTPs (a mix
of dCTP, dGTP, dATP, dTTP and dUTP), 0.5 lM Scorpion
primer and reverse primer, 2.5 lM quencher oligonucleotide
for the duplex Scorpion, 0.5 units of HotGoldStar polymerase
(Eurogentec), 4 mM magnesium chloride. All Scorpion PCR
reactions were carried out on an ABI PRISMTM 7700 Genetic
analyser with the quencher option turned off. Cycling was as
Jonathan May was funded by an EPSRC CASE studentship
with Oswel Research Products Ltd., and Ian van Delft was the
recipient of an EPSRC Analytical Chemistry PhD studentship.
References
1 D. Whitcombe, S. Kelly, J. Mann, J. Theaker, C. Jones and S. Little,
Am. J. Human Genet., 1999, 65, 2333.
2 D. Whitcombe, J. Theaker, S. P. Guy, T. Brown and S. Little, Nat.
Biotechnol., 1999, 17, 804–807.
3 N. Thelwell, S. Millington, A. Solinas, J. A. Booth and T. Brown,
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4 A. Solinas, L. J. Brown, C. McKeen, J. M. Mellor, J. Nicol, N. Thelwell
and T. Brown, Nucleic Acids Res., 2001, 29, e96.
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49–53.
◦
7 K. J. Livak, S. A. J. Flood, J. Marmaro, W. Giusti and K. Deetz, PCR
Methods Appl., 1995, 357.
follows: 95 C for 10 min (initial denaturation), then 40 cycles
of 95 ◦C for 30 s, 53 ◦C for 30 s, 72 ◦C for 30 s. Fluorescence was
8 P. M. Holland, R. D. Abramson, R. Watson and D. H. Gelfand,
Proc. Natl. Acad. Sci. U. S. A., 1991, 88, 7276–7280.
9 C. M. McKeen, L. J. Brown, J. T. G. Nicol, J. M. Mellor and T.
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2003, 970–971.
monitored during the 53 ◦C step.
The duplex Scorpions containing the cyanine dyes (Fig. 8)
were studied on a Corbett Research Rotorgene Rg-3000. The
PCR curves were quantitised and show the relative fluorescence
with respect to the background. These PCRs were carried out
with 2 mM MgCl2, 2U Taq Polymerase, 0.5 lM primers and
2.5 lM quencher. Cycling conditions were: 95 ◦C for 5 min,
then 50 cycles of 95 ◦C for 30 s, 40 ◦C for 30 s, 72 ◦C for 30 s.
11 L. J. McBride and M. H. Caruthers, Tetrahedron Lett., 1983, 24,
245–248.
12 S. L. Beaucage and M. H. Caruthers, Tetrahedron Lett., 1981, 22,
1859–1862.
13 P. Wu and L. Brand, Anal. Biochem., 1994, 218, 1–13.
14 L. J. Brown, J. P. May and T. Brown, Tetrahedron Lett., 2001, 42,
2587–2591.
Spectrometric measurements
Fluorescence spectra were recorded on a PE LS50B lumines-
cence spectrometer with 5 nm slit widths using quartz glass
3 mL cuvettes. All hybridisation assays were performed in buffer
consisting of sodium phosphate (100 mM), EDTA (1 mM)
and sodium chloride (100 mM), pH 7.0. Fluorescent dyes
were excited at their absorption maxima: FAM (495 nm), Cy3
(550 nm), TAMRA (555 nm), Cy5 (650 nm), Cy5.5 (690 nm) and
emission intensity measured at the experimentally determined
maxima: FAM (514 nm), Cy3 (564 nm), TAMRA (584 nm),
Cy5 (666 nm), Cy5.5 (704 nm). UV/vis data were recorded on
a P◦erkin–Elmer Lambda 2 spectrometer, using 1 mL cuvettes at
25 C. UV thermal melting was performed on a Perkin–Elmer
Lambda 2 UV/vis spectrometer connected to a PTP-1 temper-
ature programmer, and data acquisition was recorded using the
PECSS software. All UV melts were performed in triplicate.
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2 5 4 2
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 2 5 3 4 – 2 5 4 2