End-capped HyBeacon probes for the analysis of human genetic polymorphisms related to warfarin metabolism

Article abstract of DOI:10.1039/c001177k

HyBeacon probes have been used to characterise SNPs in the CYP2C9 and VKORC1 genes associated with variations in the efficiency of warfarin metabolism. PCR amplification of genomic DNA and probe target melting analysis provided a robust and reliable method to differentiate polymorphic sequences at these loci. Probes capped with 5′-trimethoxystilbene were found to exhibit larger fluorescence differences between hybridised and dissociated states than uncapped probes, generating melting peaks of considerably improved height. 3′-Pyrene modifications enhanced probe signal-to-noise and melting peak heights with the additional benefit of acting as PCR stoppers, and 5′,3′-doubly capped probes gave the best analytical results.

Full text of DOI:10.1039/c001177k

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PAPER
www.rsc.org/obc | Organic & Biomolecular Chemistry
End-capped HyBeacon probes for the analysis of human genetic
polymorphisms related to warfarin metabolism†
Nouha Ben Gaied,a James A. Richardson,a Daniel G. Singleton,a Zhengyun Zhao,a David Frenchb and
Tom Brown*a
Received 20th January 2010, Accepted 23rd March 2010
First published as an Advance Article on the web 14th April 2010
DOI: 10.1039/c001177k
HyBeacon probes have been used to characterise SNPs in the CYP2C9 and VKORC1 genes associated
with variations in the efficiency of warfarin metabolism. PCR amplification of genomic DNA and
probe target melting analysis provided a robust and reliable method to differentiate polymorphic
sequences at these loci. Probes capped with 5¢-trimethoxystilbene were found to exhibit larger
fluorescence differences between hybridised and dissociated states than uncapped probes, generating
melting peaks of considerably improved height. 3¢-Pyrene modifications enhanced probe signal-to-noise
and melting peak heights with the additional benefit of acting as PCR stoppers, and 5¢,3¢-doubly
capped probes gave the best analytical results.
base pair, among them stilbenes,5 pyrenes,6–8 and anthraquinones.9
Although 5¢-stabilization is effective, the use of simple, readily
Introduction
R
HyBeaconꢀ probes1,2 are linear oligonucleotides devoid of sec-
available 3¢-stabilizers in genetic probes would be preferable as
ondary structure, possessing one or more fluorescent dyes attached
such modifications will also act as PCR blockers. This negates the
to internal nucleotides and a 3¢-blocker to prevent PCR extension
of the probe. The inherent fluorescence quenching properties of
the DNA bases and attached fluorophores cause the level of
fluorescence emission to be low in the single-stranded state. Upon
hybridisation to the target DNA sequence, the resultant rigid
double-stranded conformation removes the fluorophore from the
vicinity of the DNA bases and increases the separation between
the dyes. This results in a significant increase in fluorescence, which
can be used to discriminate between similar DNA target sequences
by melting curve analysis.3 This is a powerful method of mutation
detection particularly as high-resolution DNA melting platforms
need for a separate 3¢-phosphate modification, thus simplifying
oligonucleotide synthesis by allowing all probes to be assembled
from a common solid support. There is a precedent for this in the
use of 3¢-minor groove binders (MGB probes).10,11 There is also
the possibility of using 5¢,3¢- dual-modified probes with enhanced
DNA-binding properties. To investigate the suitability of these
options in the context of HyBeacons we have synthesized several
3¢- pyrene and anthraquinone-modified probes, and examined
their effects on mismatch discrimination in the presence and in
the absence of 5¢-trimethoxystilbene (TMS) capping. The same 3¢-
capping monomers (Fig. 1A, B) have previously been incorporated
into oligonucleotides and shown to greatly increase the stability of
triplex forming oligonucleotides.12 This encouraged us to explore
their use in HyBeacon probes.
R
such as the LightCycler 480ꢀ, Rotor-Gene Q and CFX96TM are
now available. Unlike many other fluorogenic probes, HyBeacons
do not require internal secondary structure for their mode of
action, so the melting temperature (Tm) depends only on the degree
of homology between the probe and the target sequence, enabling
targets with single or multiple polymorphisms to be identified.4
A key aim in the area of SNP and point mutation analysis is
to achieve the greatest possible discrimination between similar
sequences, and for this it is desirable to use short probes in order
to widen the Tm-gap between allelic variants by increasing the ratio
of mismatch to Watson–Crick base pairs. For this to be feasible,
suitable methods are required to stabilize short DNA duplexes.
A number of 5¢- and internal modifications have previously
been incorporated into oligonucleotides to stabilize Watson–Crick
duplexes by electrostatic and aromatic stacking on the terminal
Fig. 1 Chemical structure of oligonucleotide 5¢-3¢ end caps.
aSchool of Chemistry, University of Southampton, Highfield, Southampton,
UK SO17 1BJ. E-mail: tb2@soton.ac.uk; Fax: +44(0)2380 592991;
Tel: +44(0)2380 592974
We elected to analyze genes associated with warfarin
metabolism to evaluate modified HyBeacons.13 Warfarin is an
important anticoagulant drug that is used to treat and pre-
vent thrombosis. Genetic polymorphisms within the human
cytochrome P450 (CYP2C9) and vitamin K epoxide reductase
complex subunit 1 (VKORC1) genes are associated with the
bInnovation and Development, LGC, Queens Road, Teddington, UK TW11
0LY
† Electronic supplementary information (ESI) available: Synthetic meth-
ods, supplementary figures include UV and fluorescent meltings. See DOI:
10.1039/c001177k
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Table 1 PCR primers, probes and oligonucleotide targets. FP: forward primer; RP: reverse primer; MT: matched target: MMT: mismatched target. F =
fluorescein dT residue (6-FAM dT)
Oligonucleotides
Sequence
CYP2C9*2 (430 C > T)
2C9*2-phos
2C9*2-AnthdR
2C9*2-AnthdRNH2
2C9*2-AmBuPyr
2C9*2-ThrPyr
GCATFGAGGACCGFGTTCAAG-P
GCATFGAGGACCGFGTTCAAG-AnthdR
GCATFGAGGACCGFGTTCAAG-AnthdR-NH2
GCATFGAGGACCGFGTTCAAG-AmBuPyr
GCATFGAGGACCGFGTTCAAG-ThrPyr
TMS-GCATFGAGGACCGFGTTCAAG-P
TMS-GCATFGAGGACCGFGTTCAAG-AnthdR
TMS-GCATFGAGGACCGFGTTCAAG-AnthdRNH2
TMS-GCATFGAGGACCGFGTTCAAG-AmBuPyr
TMS-GCATFGAGGACCGFGTTCAAG-ThrPyr
GTTTCGTTTCTCTTCCTGTTAGGAATT
GAAGATAGTAGTCCAGTAAGGTCAGTGATATG
CTTGAACACGGTCCTCAATGC
2C9*2-TMS/phos
2C9*2-TMS/AnthdR
2C9*2-TMS/AnthdRNH2
2C9*2-TMS/AmBuPyr
2C9*2-TMS/ThrPyr
2C9*2F2 (FP)
2C9*2R2 (RP)
2C9*2RC1 (MT)
2C9*2RC2 (MMT)
CYP2C9*3 (1075 A > C)
2C9*3-phos
CTTGAACACAGTCCTCAATGC
TCCAGAGATACCTFGACCTFCTCCC-P
TMS-TCCAGAGATACCTFGACCTFCTCCC-P
CATGCAAGACAGGAGCCACAT
2C9*3-TMS/phos
2C9*3F (FP)
2C9*3R (RP)
ACAAACTTACCTTGGIAATGAGA
2C9*3RC1 (MT)
2C9*3RC2 (MMT)
VKORC1 (-1639 G > A)
VKORC1-phos
GGGAGAAGGTCAAGGTATCTCTGGA
GGGAGAAGGTCAATGTATCTCTGGA
CATFGGCCAGGFGCGGT-P
TMS-CATFGGCCAGGFGCGGT-P
GCCAGCAGGAGAGGGAAATA
GCCTCCCAAAATGCTAGGATT
ACCGCACCTGGCCAATG
VKORC1-TMS/phos
VKOF1 (FP)
VKOR2 (RP)
VKORC1 (MT)
VKORC2 (MMT)
ACCGCACCCGGCCAATG
variations in the efficiency of warfarin metabolism.14–16 Since
natural blood clotting is inhibited by this drug, high serum
concentrations can cause severe side effects therefore careful
monitoring of patients on warfarin medication is critical. In
parallel it is important to develop improved methods of screening
for genetic variants associated with warfarin metabolism so that
patients at risk can be identified at the onset of treatment.
Data from SNPs is of real clinical value, as it can be included
in dosing algorithms to achieve therapeutic anticoagulation for
treatment of arterial and venous thromboembolic disorders.17
More than 50 single nucleotide polymorphisms (SNPs) have been
identified in the regulatory and coding regions of the CYP2C9
gene, and more than 12 allelic genetic variants are known, among
them are CYP2C9*1 (Arg144/Ile359), CYP2C9*2 (Cys144/Ile359) and
CYP2C9*3 (Arg144/Leu359).14,18 The single amino acid replacement
that is present in CYP2C9*3 is known to be involved in significant
changes in substrate affinity, while the effects of CYP2C9*2 are
not well determined.
fluorophore-fluorophore quenching. This produces probes with
increased signal-to-noise ratios and improved melting peak
heights.
Polymorphic target sequences are detected through melting
peak analysis, using melting temperatures (Tms) to identify the
alleles present. The 2C9*2 probes were designed to analyse the
CYP2C9 430 C > T polymorphism (rs1799853) and possess
a C nucleotide at the polymorphic site, generating higher Tm
melting peaks with the complementary ‘wild-type’ (CYP2C9*1)
allele compared with the CYP2C9*2 target which produces a C/A
mismatch. Samples of *1/*2 genotype generate both matched and
mismatched melting peaks.
2C9*3 probes were designed to analyse the CYP2C9 1075
A > C polymorphism (rs1057910) and possess a C nucleotide
at the polymorphic site, generating higher melting peaks with
the complementary *3 allele compared with the ‘wild-type’ *1
sequence which exhibits a C/T mismatch. Samples of *1/*3
genotype yield both matched and mismatched melting peaks.
Samples that generate *2 and *3 melting peaks with the above tests
are typed as *2/*3. VKORC1 probes were designed to analyse the -
1639 G > A polymorphism (rs9923231) and are complementary to
target sequences possessing T at the polymorphic site, generating
melting peaks of reduced Tm when hybridized to the C target
variant.
Results and discussion
Several probes, each with two internal fluorescein dT (6-FAM dT)
residues, were designed to interrogate polymorphic CYP2C9 or
VKORC-1 target sequences (Table 1).
Before commencing the PCR and genotyping analysis of the
end-capped probes, we measured their fluorescence properties
(Table 2). The individual anthraquinones 1 and 2, and pyrenes
3 and 4 (Fig. 1) exhibit low fluorescence with excitation and
emission wavelengths well separated from those of fluorescein.
This is important, as these monomers are not intended to behave
Probes were either uncapped, 5¢-capped with TMS, 3¢-capped
with anthraquinone or pyrene derivatives, or capped at both
5¢ and 3¢-ends (Fig. 1). Previous work has demonstrated the
advantage of using HyBeacon probes containing two fluorescent
dyes rather than single-labelled probes,4 as the former results
in much less fluorescence in the (dark) single-strand due to
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Table 2 UV and fluorescence melting of 2C9*2 HyBeacon probes with matched and mismatched oligonucleotide targets. End modifications in Fig. 1.
Oligonucleotide sequences in Table 1. Matched target = 2C9*2RC1 with CG base pair, mismatched target = 2C9*2RC2 with CA mismatch
Fluorescence melting
UV melting
Quantum yield
Single strand
End mods 5¢- 3¢
Tm/◦Ca
Tm/◦Cb
Tm/◦Cc
Double strand
Phos
AnthdR
AnthdRNH2
AmBuPyr
ThrPyr
TMS-phos
TMS-AnthdR
TMS-AnthdRNH2
TMS-AmBuPyr
TMS-ThrPyr
67.5/58.7
73.1/64.3
71.9/63.0
73.0/64.5
73.6/65.2
68.3/59.7
74.0/65.4
72.5/63.6
74.1/65.7
74.7/66.3
58.8/55.3
62.3/54.6
60.6/53.3
61.7/56.1
62.2/56.4
60.1/55.9
63.2/56.1
61.4/53.5
62.8/56.5
63.3/56.8
62.4/54.5
65.0/56.9
63.8/55.4
64.9/57.1
65.4/57.2
63.8/55.8
65.8/57.8
64.6/56.1
66.1/57.9
66.5/58.7
0.219
0.067
0.054
0.318
0.281
0.214
0.054
0.053
0.286
0.245
0.807
0.242
0.233
0.902
0.773
0.616
0.274
0.199
0.958
0.890
a TaKaRa PCR buffer pH 8.3, 1 M NaCl. b 10 mM Na2HPO4/NaH2PO4 buffer, 200 mM NaCl, pH 7. c 10 mM Na2HPO4/NaH2PO4 buffer, 200 mM
NaCl, pH 7.
Table 3 Comparison of 5¢ and 3¢-capped probes with oligonucleotide
targets. 1 ¥ TaKaRa PCR buffer containing 1 M NaCl, pH 8.3 (TaKaRa
Bio Inc.)
Probe cap
Uncapped
TMS
Target
N
Tm/◦C
Area
Height
S/N
A
G
A
G
A
G
A
G
A
G
A
G
6
6
6
6
6
6
6
6
6
6
6
6
50.1
57.7
52.6
60.8
51.0
59.6
49.1
58.0
51.3
59.5
51.2
59.4
2.6
7.8
5.4
10.4
2.1
3.2
1.6
2.4
3.7
8.6
3.9
10.3
0.4
0.9
1.0
1.8
0.4
0.5
0.3
0.4
0.6
1.4
0.7
1.7
1.3
1.9
2.3
3.5
2.1
2.8
2.1
2.8
1.7
2.7
1.6
2.5
3¢-AnthdR
3¢ AnthdRNH2
3¢-AmBuPyr
3¢-ThrPyr
as fluorophores, but as PCR stoppers and duplex stabilizers. To
evaluate the effect of the end-caps on fluorescence, we measured
the quantum yields of probes in single and double-stranded
states at the excitation and emission wavelengths of fluorescein
(Table 2).
A decrease of the overall fluorescence quantum yield relative to
the uncapped probe was observed with anthraquinones at the 3¢-
end of single-stranded oligonucleotides, presumably due to FRET
or electron transfer between fluorescein and the anthraquinone
chromophores, with a smaller but significant decrease in the
double-stranded state. This quenching was also observed by
fluorescence melting curve analyses through the generation of
melting peaks of reduced height (Fig. 2B, Table 3).
Fig. 2 (A) Comparison of uncapped (blue), 5¢-TMS capped (red) and
3¢-ThrPyr capped (green) CYP2C9*2 probe melting peaks with oligonu-
cleotide targets. (B) Comparison of uncapped (blue), 3¢-AnthdR capped
(red) and 3¢-AnthdR-NH2 capped (green) probes with PCR amplified
targets. (C) Comparison of uncapped (blue), 3¢-AmBuPyr (red) and
3¢-ThrPyr (green) capped probes with PCR amplified targets. No-template
controls in black. LightCyclerꢀR , excitation at 470 nm.
In contrast, when 3¢-pyrenes were used, either in single or
double-stranded DNA, no reduction was observed in the quantum
yield and melting peak heights compared to the uncapped probe;
in fact there was a slight increase in both. The 3¢-ThrPyr and
5¢-TMS caps provided similar improvements to the sharpness
(inverse of width) and intensity (height) of melting peaks relative
to the uncapped probe (Fig. 2A). We also systematically evaluated
the effects of the end-caps for their ability to increase duplex
melting temperature against single-stranded oligonucleotide tar-
gets (Tables 2 and 3). The 2C9*2 probes were evaluated using
oligonucleotides which were fully complementary (2C9*2RC1) or
possessed a single nucleotide mismatch (2C9*2RC2). Fluorescence
and ultraviolet melting was measured in buffers of varying salt
composition and pH (500 mM, 200 mM NaCl at pH 7, 8.1
and 8.7). The modifications generally produced a slight increase
in Tm.
Next we compared the uncapped and 5¢-TMS-capped 2C9*2,
2C9*3 and VKORC1 probes using oligonucleotide targets and
R
melting curve analysis performed with a LightCyclerꢀ instrument
(Table 4). The melting peak heights, areas, Tm values and signal-
to-noise were all increased relative to the uncapped probes. The
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Table 4 Melting curve analysis of 5¢-trimethoxystilbene capped probes
with oligonucleotide targets (where N is the number of melting peaks
analysed and S/N the signal-to-noise ratio). 1 ¥ TaKaRa PCR buffer
containing 1 M NaCl, pH 8.3 (TaKaRa Bio Inc.)
Table 5 Melting curve analysis of 5¢-trimethoxystilbene capped probes
and PCR amplified targets
Probe
Target
N
Tm/◦C
Area
Height
Probe
Target
N
Peak Tm/◦C Area Height S/N
C9*2
C9*2TMS
2C9*3
G
G
T
G
T
G
C
T
C
T
7
7
7
7
7
7
7
7
7
7
59.9
61.4
56.4
64.4
58.9
66.0
48.4
58.4
51.9
60.7
7.4
10.9
0.4
1.4
2.1
3.4
7.2
7.1
11.2
9.5
1.0
1.8
0.4
0.6
0.6
1.1
1.1
1.1
1.8
1.6
C9*2
A (*2)
G (*1)
A (*2)
G (*1)
T (*1)
G (*3)
T (*1)
G (*3)
C
6
6
6
6
6
6
6
6
6
6
6
6
50.6
57.9
53.0
60.9
54.9
61.9
57.4
64.2
48.4
58.4
52.6
61.3
2.2
6.3
6.3
11.7
5.1
5.1
7.6
7.4
8.9
8.7
12.7
13.0
0.3
0.7
0.9
1.7
0.6
0.6
1.0
0.9
1.0
0.9
1.5
1.5
1.4
2.0
2.2
3.4
1.4
1.5
1.6
1.7
2.0
2.2
4.1
4.3
C9*2TMS
2C9*3
2C9*3TMS
VKORC1
2C9*3TMS
VKORC1
VKORC1TMS
VKORC1TMS
T
C
T
Table 6 Melting curve analysis of 3¢-capped probes and PCR amplified
targets (where N is the number of melting peaks analysed).
Probe cap
Target
N
Tm/◦C
Area
Height
3¢-pyrene probes performed in a similar manner to the TMS probes
(Table 3).
Uncapped
G
G
G
G
G
14
7
7
7
7
60.4
62.6
61.6
62.2
62.5
9.8
3.4
3.6
11.2
12.7
1.3
0.6
0.6
1.6
1.8
3¢-AnthdR
3¢-AnthdRNH2
3¢-AmBuPyr
3¢-ThrPyr
A series of asymmetric PCR amplifications and post-PCR
melting experiments were then performed. These assays generated
an excess of the target DNA strand, limiting the amount of
complementary strand that would compete for probe hybridis-
ation. The TMS caps increased the amount of fluorescent signal
generated during target amplification, possibly by improving the
thermodynamics and kinetics of probe-target binding, but it did
not change the cycle threshold at which target amplification was
detected (Fig. 3).
Fig. 3 Real time PCR analysis of 2C9*2-phos (blue) and 2C9*2-TMS/
phos (red) probes. The TMS cap increases the fluorescence signal generated
during amplification without affecting the cycle threshold of detection.
No-template controls are presented (black).
Fig.
4 (A) Melting curve analysis of 2C9*2-phos (blue) and
2C9*2-TMS/phos (red) probes with PCR-amplified targets. (B) Melt-
ing curve analysis of 2C9*3-phos (blue) and 2C9*3-TMS/phos (red)
probes with heterozygous DNA samples. (C) Melting curve analysis
of VKORC1-phos (blue) and VKORC1-TMS/phos (red) probes with
heterozygous DNA samples. Seven replicate positive controls and two
No-template controls (black) were analysed for each capped and uncapped
probe. LightCyclerꢀR , excitation at 470 nm.
The melting peak heights and areas of TMS capped probes
were improved relative to the uncapped probes (Fig. 4, Table 5),
and this increased the ability of the analysis software to detect
and identify target alleles. The uncapped 2C9*3 probe generated
small mismatched melting peaks (Fig. 4B) and analysis software
occasionally failed to detect the allele with a small number (<1%)
of samples of *1/*3 genotype, yielding an incorrect *3/*3 call.
The improved peak height generated with the 2C9*3TMS
capped probe ensures assay accuracy and robustness.
When 3¢-capped probes were used in PCR amplification,
anthraquinone analogues generated melting peaks of reduced
height and area compared with the uncapped probe (Fig. 2B,
Table 6), whereas the AmBuPyr and ThrPyr caps increased the
heights and areas of melting peaks (Fig. 2C, Table 6). As with
TMS, none of the 3¢-caps had an effect on cycle threshold. The
3¢-ThrPyr cap provides the best alternative to 5¢-TMS, exhibiting
equivalent improvements to melting peak height, with the added
benefit that the 3¢-cap modification replaces the 3¢-phosphate
typically employed to prevent PCR extension from probes.
Probes capped at both 5¢ and 3¢ ends were also found to enhance
the fluorescence difference between hybridised and dissociated
probe states, increasing the heights and areas of melting peaks.
The 2C9*2 probes capped at the 5¢ end with TMS and at the
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Table 7 Properties of HyBeacon probes capped at both 5¢ and 3¢ ends
(where N is the number of melting peaks analysed and S/N the signal-to-
noise ratio).
Conclusions
End-capped HyBeacons have been developed to reliably geno-
type single nucleotide polymorphisms associated with warfarin
metabolism by melting curve analysis. Probe melting temperatures,
signal-to-noise ratios and melting peak heights are enhanced using
5¢ and 3¢-cap modifications, increasing the efficiency, accuracy
and robustness of the assay. This is likely to be of benefit in
the analysis of unpurified clinical samples (such as saliva, swabs
and blood), which may yield melting peaks of reduced height
compared with extracted DNA due to low DNA concentrations
and the presence of PCR inhibitors.2,4 Clinical decisions could
be made on the basis of the assay results, so a high level of
reliability is important. End-cap modifications are now routinely
used by us, and are particularly useful with target sequences that
form secondary structures which would otherwise prevent efficient
probe hybridisation. The 3¢-caps have the additional benefit of
acting as PCR stoppers and can be used in place of 3¢ phosphate.
Probe cap
Target N Area Tm/◦C Height S/N DTm/◦C
Uncapped
A
G
A
G
A
G
A
G
A
G
A
G
6
6
6
6
6
6
6
6
6
6
6
6
2.2
6.7
4.8
9.2
2.2
3.0
1.4
2.0
4.9
9.0
5.5
50.4
58.0
53.0
61.1
53.5
61.9
51.5
60.3
53.8
62.0
53.8
0.3
0.8
0.9
1.5
0.4
0.5
0.2
0.3
0.8
1.5
0.9
1.8
1.3
2.0
TMS
2.3 2.6
3.5 3.1
3.0 3.1
3.6 3.9
2.6 1.2
3.1 2.2
2.2 3.5
3.5 3.9
2.5 3.4
3.9 4.0
TMS-AnthdR
TMS-AnthdRNH2
TMS-AmBuPyr
TMS -ThrPyr
10.2 62.0
3¢ end with AmBuPyr or ThrPyr provided a small Tm and peak
height increase with oligonucleotide targets compared with the
probe capped with TMS only (Table 7, Fig. 5A). The improvement
to peak heights, however, was not as pronounced with PCR-
amplified targets (Fig. 5B), although the dual-capped probes did
produce the highest melting temperatures. As anticipated, the
2C9*2 probes capped at the 5¢-end with TMS and at the 3¢-end
with AnthdR or AnthdRNH2 generated melting peaks of reduced
height compared with the probe capped with TMS only (Table 7).
The reduced performance of AnthdR and AnthdRNH2 probes
is likely to be due to FRET-based quenching of fluorescein by
the anthraquinone chromophore, which, unlike pyrene, absorbs at
the excitation frequency of fluorescein (UV/visible spectra in the
ESI†).
Experimental
DNA phosphoramidite monomers, solid supports including
phosphate-on synthesis columns, and additional reagents were
purchased from Link Technologies Ltd or Applied Biosystems
Ltd. Anthraquinone and pyrene analogues for 3¢-oligonucleotide
modification (Fig. 1) and the corresponding oligonucleotide
synthesis resins were prepared as described previously.12 Human
genomic DNA for the PCR experiments was sourced from Gentris
products, distributed by LGC Standards.
Determination of quantum yields
Quantum yields of fluorescein were calculated at an optical density
of 0.01 at the excitation wavelength of 460 nm for modified
R
HyBeaconsꢀ. A standard of fluorescein in 0.1 M NaOH was
used for quantum yield determination of oligonucleotides which
were dissolved in 10 mM Na2HPO4/NaH2PO4 buffer (pH 8)
containing 100 mM NaCl. Emission spectra were recorded using
a Perkin Elmer LS 50B luminescence spectrometer fitted with a
Perkin Elmer PTP1 Peltier temperature control device using a 90◦
collection angle.
Oligonucleotide synthesis and purification
Oligonucleotide synthesis was carried out on an Applied Biosys-
tems 394 automated DNA/RNA synthesizer using standard
0.2 mmol and 1.0 mmol phosphoramidite cycles of acid-catalyzed
detritylation, coupling, capping and iodine oxidation. All b-
cyanoethyl phosphoramidite monomers were dissolved in anhy-
drous acetonitrile to a concentration of 0.1 M immediately prior
to use. The coupling time for normal A, G, C and T monomers was
25 s, and this was extended to 10 min for fluorescein-dT (FAM) and
trimethoxystilbene (TMS), purchased from Link Technologies Ltd
and Glen Research respectively. For resins functionalized with L-
threoninol derivatives, coupling of the first monomer to the solid
support was carried out for 5 min to ensure an efficient reaction at
this hindered site. Stepwise coupling efficiencies and overall yields
were determined by automated trityl cation conductivity moni-
toring and in all cases were >98.0%. Cleavage of oligonucleotides
from the solid support and deprotection were achieved by exposure
Fig. 5 Comparison of 2C9*2-phos (blue), 2C9*2-TMS/phos (red),
2C9*2 TMS/AmBuPyr (green) and 2C9*2-TMS/ThrPyr probes (black).
Melting curve analysis was performed with (A) oligonucleotide and (B)
PCR-amplified targets. No-template controls are presented. LightCyclerꢀR ,
excitation at 470 nm.
Oligonucleotide probes melt in a non-cooperative manner, as
the termini dissociate from the target earlier than central regions
due to ingress of water. Stabilizing the probe by 5¢- or 3¢-capping
with aromatic groups prevents “end-fraying” of duplexes and
increase probe Tm. It also increases the signal of hybridised
probes relative to uncapped oligonucleotides of identical sequence,
yielding probes with melting peaks of increased height
2732 | Org. Biomol. Chem., 2010, 8, 2728–2734
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to concentrated aqueous ammonia for 60 min at room temperature
followed by heating in a sealed tube for 5 h at 55 ◦C. Purification
of oligonucleotides was carried out by reversed-phase HPLC on a
Gilson system using a Brownlee Aquapore column (C8, 8 mm ¥
PCR amplification and melting curve analysis
PCR volumes were 20 mL, containing 2 mL purified human
DNA (between 1–10 ng mL-1 concentration), 1¥ QIAGEN PCR
buffer, 1 unit HotStarTaq polymerase (QIAGEN, Crawley, UK),
1 mM dNTPs (0.25 mM each—GE Healthcare, Amersham, UK),
0.1 mM forward primer, 0.5 mM reverse primer, 150 nM probe and
10 ng mL-1 BSA (Roche Diagnostics, Lewes, UK). The sequences of
primers, probe and blocker oligonucleotides are detailed in Table 1.
Asymmetric PCR was used to generate an excess of the target
strand such that probe hybridisation was favoured over annealing
of amplified sequences. Amplification of target sequences was
˚
250 mm, 300 A pore) with a gradient of acetonitrile in ammonium
acetate increasing from 0% to 50% buffer B over 30 min with
a flow rate of 4 mL min-1 (buffer A: 0.1 M ammonium acetate,
pH 7.0, buffer B: 0.1 M ammonium acetate with 50% acetonitrile
pH 7.0). Elution of oligonucleotides was monitored by ultraviolet
absorption at 295 nm. After HPLC purification, oligonucleotides
were desalted using NAP-10 Sephadex columns (GE Healthcare)
according to the manufacturer’s instructions.
R
performed using a LightCyclerꢀ instrument. Following an initial
denaturation to activate the hotstart enzyme (95 ◦C, 15 min),
targets were amplified using 55 cycles comprising denaturation
(95 ◦C, 5 s), primer annealing (55 ◦C, 10 s) and extension of
products (72 ◦C, 15 s). Following amplification, reactions were
incubated at 72 ◦C for 2 min prior to a denaturation (95 ◦C,
30 s) and cool (35 ◦C, 1 min) steps. Melting curve analysis was
performed by heating from 35 ◦C to 80 ◦C, at 0.2 ◦C per second.
The Tms and areas of melting peaks were obtained using version
Ultraviolet melting studies
To determine duplex melting temperatures (Tm), UV melting
studies were carried on a Varian Cary 400 scan UV-visible
spectrophotometer using Hellma SUPRASIL synthetic quartz
10 mm path length cuvettes, monitoring at 260 nm with a
DNA single strand concentration of 1.0 mM and a volume of
1.2 mL. Samples were prepared as follows: single strand of the
target and probe were mixed in a 1 : 1 ratio (concentrations
determined by UV absorbance and extinction coefficients) in 2 mL
Eppendorf tubes then lyophilized before resuspending in 1.2 mL
in 10 mM phosphate buffer, 200 mM or 500 mM NaCl at pH 7.0
and finally filtered through Kinesis cellulose 13 mm diameter
0.45 mM syringe filters. The UV melting◦protocol involved initial
R
3.5 of LightCyclerꢀ software. Peak height was determined as the
maximum -dF/dT value from exported melting peak data and
signal-to-noise ratio (S/N) was calculated using exported melting
curve data, as the fluorescence at Tm - 10 ◦C divided by the
fluorescence at Tm + 10 ◦C.
◦
denaturation by heating to 8◦0 C at 1◦0 C min-1 followed by an
annealing step cooling to 20 C at 0.5 C min-1. The temperature
was maintained at 20 ◦C for 20 min before starting the melting
exp◦eriment which involved heating from 20 ◦C to 80 ◦C at
0.5 ◦C min-1, holding at 80 ◦C for two min, then cooling to 20 ◦C at
0.5 C min-1. Two successive melting curves were measured before
Acknowledgements
This project was supported by the European Commission’s Sixth
framework Programme (Project reference ZNIP) [037783] and an
EPSRC research studentship to JAR. Oligonucleotide synthesis
was carried out by ATDBio Ltd.
◦
◦
◦
rapid annealing from 80 C to 20 C at 10 C min-1. Tm values
were calculated using Cary Win UV thermal application software,
taking an average of the two melting curves.
Notes and references
Fluorescence melting studies on probe-target duplexes
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R
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10 mM sodium phosphate buffer, pH 7.0 varying the salt
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3.5), taking an average of the two melting curves. For the data in
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QIAGEN PCR buffer, a total of 3 mM Mgl2, 150 nM fluorescent
probe, 150 nM target oligonucleotide and 10 ng mL-1 BSA (Roche
Diagnostics, Lewes, UK). Following a denaturation (95 ◦C, 15 s)
and cooling (35 ◦C, 30 s), samples were heated to 95 ◦C at 0.2 ◦C
per second.
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The Royal Society of Chemistry 2010
©