Microwave-mediated synthesis of labeled nucleotides with utility in the synthesis of DNA probes

Article abstract of DOI:10.1021/bc100013b

A novel method of linking haptens to deoxycytidine 5′-triphosphate via microwave-mediated bisulfate-catalyzed transamination with hydrazine has been developed. This method enables the tethering of small molecule haptens to dCTP via a discrete polyethylene

Full text of DOI:10.1021/bc100013b

Bioconjugate Chem. 2010, 21, 1773–1778
1773
Microwave-Mediated Synthesis of Labeled Nucleotides with Utility in the
Synthesis of DNA Probes
Mark Lefever, Jerome W. Kosmeder II, Michael Farrell, and Christopher Bieniarz
Ventana Medical Systems, Inc., a member of the Roche Group, 1920 East Innovation Park Drive, Tucson, Arizona 85755.
Received January 7, 2010; Revised Manuscript Received July 22, 2010
A novel method of linking haptens to deoxycytidine 5′-triphosphate via microwave-mediated bisulfate-catalyzed
transamination with hydrazine has been developed. This method enables the tethering of small molecule haptens
to dCTP via a discrete polyethylene glycol (PEG) spacer, yielding N4-aminodeoxycytidine 5′-triphosphate-dPEG-
haptens. This synthetic approach employs microwave-catalyzed hydrazinolysis that enables the attachment
of spacers via hydrazine linkages. The microwave-mediated introduction of this hydrazine handle provides
a significant improvement in yield over those of published thermal methods. The microwave reaction was
shown to be scalable, and the final product was amenable to labeling with a wide variety of haptens. The
resulting nucleotide triphosphates, N4-aminodeoxycytidine 5′-triphosphate-dPEG-haptens, can serve as unique
substrates for the enzyme-mediated labeling of DNA probes. The efficacy of incorporation of one such
novel nucleotide, N4-aminodeoxycytidine 5′-triphosphate-dPEG4-DNP, has been demonstrated in nick
translation labeling of HER2 and HPV probes. The labeled probes have been shown to be effective in
visualizing their target genes in tissue.
nucleophile and release of ammonia, and base-catalyzed elimi-
nation of bisulfite to regenerate the double bond (20).
INTRODUCTION
Chemically modified nucleic acids are used as probes in
hybridization assays, e.g., Southern blots, Northern blots, dot blots,
and in situ hybridization (ISH), to detect RNA transcripts, gene
amplification, and translocations in cells, in microarray assays, and
in surface attachment chemistries and to generate various kinds of
bioconjugates (1-3). Recently, the use of ISH assays both by
fluorescent in situ hybridization detection (FISH) and by enzymatic
signal deposition for brightfield detection via silver in situ
hybridization (SISH) and chromogenic in situ hybridization (CISH)
has found important clinical applications (4, 5, 6, 7). The amplifica-
tion of the human epidermal growth factor 2 (HER2) gene in breast
and stomach cancers has been shown to identify patients who can
benefit from Herceptin treatment (8, 9). The amplification status
of other genes is being used increasingly to assist in diagnosis and
to profile tumors as the practice of personalized medicine progresses
(10). Detection of viral genomes in cells by ISH has many potential
clinical applications [e.g., human papilloma virus (HPV) in cervical
cancer] (11, 12).
For the generation of chemically modified nucleic acids,
uridine and cytidine are the most frequently used bases (13).
Enzymatic methods are frequently used to make probes by
incorporating modified nucleotide triphosphates to produce a
nucleic acid containing a label or reactive residue to which a
label, e.g., a hapten or fluorophore, can be subsequently coupled.
The bisulfite-mediated displacement of the C4 amino group
of cytosine, first reported in 1970 by Shapiro and Weisgras (21),
has become a widely reported method for the introduction of
bifunctional nucleophilic handles onto cytosine residues (14-22).
These newly introduced nucleophilic handles can then serve as
reaction centers for the introduction of reporter groups deriva-
tized with appropriate electrophilic handles. Reporter groups
that have been employed include immunoreactive groups,
photochemical cross-linking agents, fluorescent tags, alkylating
agents, and catalytically active metal chelates (14-22). The
bisulfite-catalyzed transamination of cytosine involves a number
of discrete steps: addition of bisulfite across the 5-6 double
bond of cytosine, transamination via attack of the nitrogen
In initial attempts to obtain cytosine derivatives tagged with
nucleophilic handles, the procedure described by Negishi was
employed (23). Although the detailed procedure was strictly
followed, the reaction produced more hydrolysis than product. The
initial failure led to the exploration of alternative conditions
employing microwave technology. This choice was based on a
number of studies indicating that microwaves are capable of
accelerating a wide range of thermally driven reactions, including
additions, cycloadditions, condensations, substitutions, rearrange-
ments, organometallic reactions, and a host of others (24-29). The
application of microwave energy results in a very rapid transfer
of energy to the reactant molecules enabling the activation
energy to be reached quickly (30, 31). Consequently, thermo-
dynamic products are produced more rapidly, often avoiding
the generation of unwanted side products that occur with
traditional thermal methods. The commercial microwave syn-
thesizers that are available allow for precise control of energy
and offer the option of simultaneous cooling. The ability to
maintain strict control of reaction conditions has proven to be
indispensible in achieving reproducibility. Failure to provide
sufficient microwave energy led to incomplete reactions,
whereas overheating led to product decomposition.
Herein is described the microwave-mediated synthesis of N4-
amino-dCTP and its use in labeling DNA. The assembly of these
reagents involved the tethering of various moieties to N4-amino-
dCTP via discrete water-soluble polyethylene glycol (PEG)
linkers. The resulting haptenylated nucleotide triphosphates can
serve as unique substrates for the enzyme-mediated labeling of
DNA probes via nick translation and/or random priming (32-35).
One such novel nucleotide, N4-aminodeoxycytidine 5′-triphos-
phate-(dPEG4-2,4-dinitrophenylamino)(N4-amino-dCTP-PEG4-
DNP) was used to label DNA for use as a probe.
EXPERIMENTAL PROCEDURES
Materials. Deoxycytidine 5′-triphosphate, hydrazine mono-
hydrate, sodium bisulfite, N-hydroxysuccinimide, 1-fluoro-2,4-
10.1021/bc100013b  2010 American Chemical Society
Published on Web 09/02/2010

1774 Bioconjugate Chem., Vol. 21, No. 10, 2010
Lefever et al.
dinitrobenzene, and all organic solvents were commercial
products of Sigma Aldrich. Discrete dPEG amino acids were
products of Quanta Biodesign Ltd. (Powell, OH). Concentrated
HCl from VWR was utilized to make pH adjustments.
unit fitted with a 40 g silica gel column (MeOH/DCM, 0 to
20% MeOH over 25 min), affording the aniline as a yellow oil
(748 mg, 92%): 1H NMR (250 MHz, CDCl3) δ 2.58 (t, J ) 6.0
Hz, 2 H), 3.57-3.84 (m, 18 H), 6.94 (d, J ) 9.5 Hz, 1 H), 8.25
(dd, J ) 9.5, 2.6 Hz, 1 H), 8.81 (br s, 1 H), 9.12 (d, J ) 2.7
Hz, 1 H); 13C NMR (126 MHz, CD3OD) δ 36.0, 44.2, 67.9,
70.0, 71.5, 71.6, 71.71, 71.72, 71.74, 71.77, 116.2, 124.8, 131.1,
131.6, 137.1, 150.0, 175.4; ESI-MS m/z 430 (M - H); TOF-
HRMS calcd for C17H24N3O10 (M - H) 430.1462, found
430.1441.
Microwave and Thermal Hydrazinolysis. We prepared reac-
tion solutions by combining sodium bisulfite (203 mg, 2.85
mmol) and hydrazine monohydrate (1.0 mL, 20.6 mmol),
cooling the mixture on an ice bath, and adjusting the pH to 7.2
with the careful addition of concentrated HCl (1.48 mL). In
this premade hydrazine/bisulfite solution was dissolved deoxy-
cytidine 5′-triphosphate (300 mg, 0.587 mmol). The pH was
adjusted to 7.2 with additional hydrazine hydrate, and the
solution was transferred to a 10 mL glass microwave tube. The
tubes were either heated in a CEM Discover microwave reactor
at 55 °C (5 W max), with cooling, for times ranging from 1 to
60 min or placed in a 55 °C water bath for the appropriate length
of time. Aliquots (20 µL) were withdrawn at intervals and
diluted with 100 µL of methanol, and the extent of the reaction
was determined by immediate HPLC analysis. Analysis was
performed on a Waters BioAlliance HPLC system fitted with a
2996 photodiode array detector and a XBridge 5 µm, C18(2),
100A, 150 mm × 4.6 mm column (10 µL injection per run).
The column was eluted with 125 mM triethylammonium
carbonate and 1-4% ACN over 10 min at pH 8.5 and a flow
rate of 1 mL/min.
Synthesis of 3-[2-(2-{2-[2-(2,4-Dinitrophenylamino)ethoxy]-
ethoxy}ethoxy)ethoxy]propanate-N-hydoxysuccinimide Ester
(3). The acid 1 (748 mg, 1.73 mmol) was dissolved in 1.9 mL
of anhydrous dichloromethane (DCM); 1.0 M dicyclohexylcar-
bodiimide (DCC) in DCM (1.92 mL, 1.92 mmol) and N-
hydroxysuccinimide (NHS) (221 mg, 1.92 mmol) were added.
The solution was blanketed with dry argon and shaken at
ambient temperature for 16 h. After the solvent had been
removed in vacuo, the residue was taken up in dry DCM and
the urea byproduct was removed by filtration. Purification was
performed by silica gel chromatography utilizing an Isco
CombiFlash automated chromatography unit fitted with a 40 g
silica gel column (2-propanol/DCM, 0 to 20% 2-propanol over
25 min) and afforded the active ester as a thick yellow oil (561
1
mg, 61%): H NMR (600 MHz, CDCl3) δ 9.05 (d, J ) 2.6, 1
N4-Aminodeoxycytidine 5′-Triphosphate Purification. Reac-
tion mixtures were fractionated by HPLC utilizing a Waters
Delta 600 HPLC system fitted with a 2996 photodiode array
detector and a Waters Sunfire Prep 10 µ, C18(2), 100A, 250
mm × 50 mm column (350 µL of reaction mixture per run).
The column was eluted with 125 mM triethylammonium
carbonate and 1-4% acetonitrile (ACN) over 20 min at pH 8.5
and a flow rate of 20 mL/min. The N4-amino-dCTP fraction
eluted at 30-32 min with a λmax of 275 nm. The N4-amino-
dCTP fraction was lyophilized three times from deionized (DI)
water to ensure removal of all residual triethylammonium
carbonate and then stored at -80 °C until it was used. To obtain
the sodium salt, cation exchange was performed on a 50 mL
column packed with Sp Sephadex C-25 equilibrated with 0.2
N NaOH and washed with water until the pH of the eluant buffer
was 7.0. The lyophilized triethylammonium salt was taken up
in 1 mL (4 × 0.250 mL) of water, loaded on the cation exchange
column, and eluted with water. The UV-vis spectra of the
collected fractions were recorded on an Agilent 8453 UV-vis
spectrophotometer, and the N4-amino-dCTP-containing fraction
were pooled and lyophilized to give the sodium salt of N4-amino-
dCTP as a white crystalline solid (284 mg, 85%): 1H NMR (600
MHz, D2O) δ 2.22 (m, 1 H), 2.31 (m, 1 H), 3.99 (m, 1 H), 4.10
(m, 2 H), 4.53 (d, J ) 2.7 Hz, 1 H), 5.96 (br s, 1 H), 6.23 (m,
1 H), 7.74 (s, 1 H); 13C NMR (63 MHz, D2O) δ 42.42, 68.19
(d, J ) 5.5 Hz, 1 C), 73.53, 88.81 (d, J ) 9.1 Hz, 1 C), 89.32,
98.51, 146.42, 153.68, 163.84; 31P NMR (121 MHz, D2O) δ
-8.01 (d, J ) 48 Hz, 1 P), 10.22 (d, J ) 50 Hz, 1 P), -21.56
(t, J ) 45 Hz, 1 P); ESI-MS m/z 481.0 (M - H); ESI-HRMS
calcd for C9H17N4O13P3 (M - H) 480.9927, found 480.9943.
H), 8.75 (s, 1 H), 8.20 (dd, J ) 9.5, 2.6 Hz, 1 H), 6.94 (d, J )
9.5 Hz, 1 H), 3.87-3.75 (m, 5 H), 3.71-3.55 (m, 16 H), 2.84
(t, J ) 6.4 Hz, 3 H), 2.80 (dd, J ) 19.3, 7.8 Hz, 4 H), 1.99 (s,
1 H); 13C NMR (63 MHz, CDCl3) δ 25.27, 31.79, 42.96, 65.36,
68.27, 70.10, 70.27, 70.32, 70.38, 114.09, 123.85, 129.89,
130.04, 135.58, 148.19, 166.48, 168.90; ESI-MS m/z 529 (M
+ H); ESI-HRMS calcd for C21H28N4O12 (M + NH4) 546.20475,
found 546.20655.
Synthesis of N4-Aminodeoxycytidine 5′-Triphosphate-
dPEG4-DNP (4). Active ester 3 (124 mg, 0.234 mmol) in 2
mL of dry dimethyl sulfoxide (DMSO) was added to a solution
of N4-aminodeoxycytidine 5′-triphosphate triethylammonium salt
(173 mg, 0.195 mmol) in 1.5 mL of anhydrous DMSO. The
solution was blanketed with dry argon and vortexed until all
solids had dissolved. After being stirred for 16 h at room
temperature, the reaction mixture was fractionated by HPLC
utilizing a Waters Delta 600 HPLC system fitted with a model
2996 photodiode array detector and a Sunfire Prep 10 µ, C18(2),
100A, 250 mm × 50 mm column. Elution with 125 mM
triethylammonium carbonate and 5-50% ACN over 60 min at
pH 8.5 and a flow rate of 20 mL/min provided the triethylam-
monium salt of N4-amino-dCTP-dPEG4-DNP. Cation ex-
change was performed on a 50 mL column packed with Sp
Sephadex C-25 equilibrated with 0.2 N NaOH and washed with
water until the pH of the eluent buffer was 7.0. The lyophilized
triethylammonium salt was taken up in 1 mL (4 × 0.250 mL)
of DI water loaded on top of the cation exchange column and
eluted with water. The UV-vis spectra of the collected fractions
were recorded on an Agilent 8453 UV-vis spectrophotometer,
and the N4-amino-dCTP-dPEG4-DNP fractions were pooled
and lyophilized to give 145 mg of the tetrasodium salt of N4-
Synthesis of 3-[2-(2-{2-[2-(2,4-Dinitrophenylamino)ethoxy]-
ethoxy}ethoxy)ethoxy]propanoic Acid (2). To a solution of
3-[2-(2-{2-[2-(2-aminoethoxy)ethoxy]ethoxy}ethoxy)ethoxy]-
propanoic acid (0.5 g, 1.89 mmol) and Na2CO3 (0.317 g, 3.77
mmol) in 5 mL of water was added 1-fluoro-2,4-dinitrobenzene
(227 mL, 1.89 mmol), and the solution was irradiated in a CEM
Discovery microwave synthesizer set at 80 °C (30 W maxi-
mum), with cooling for 30 min. After cooling, the solution was
acidified with 0.25 M HCl and extracted three times with 20
mL of ethyl acetate. Concentration of the combined organic
fractions under vacuum provided a thick yellow oil that was
purified utilizing an Isco CombiFlash automated chromatography
1
amino-dCTP-dPEG4-DNP as a yellow crystalline solid: H
NMR (250 MHz, D2O) δ 2.27 (m, 1 H), 2.36 (br m, 1 H), 2.63
(t, J ) 4.8 Hz, 2 H), 3.85-3.63 (m, 18 H), 4.18 (d, J ) 4 Hz,
3 H), 4.58 (br s, 1 H), 6.19 (br s, 2 H), 7.15 (d, J ) 8 Hz), 7.94
(br s, 1 H), 8.27 (d, J ) 8.3 Hz, 1 H), 9.04 (s, 1 H); 13C NMR
(126 MHz, D2O) δ 34.4, 39.8, 42.8, 65.47, 65.51, 66.62, 68.82,
69.94, 70.02, 70.06, 70.10, 70.72, 71.94, 85.98, 86.37, 92.7,
115.4, 124.9, 130.3, 130.8, 135.7, 141.7, 143.6, 149.3, 173.2,
173.6; 31P NMR (121 MHz, D2O) δ -8.39 (d, J ) 41 Hz, 1 P),
10.58 (d, J ) 48 Hz, 1 P), -22.00 (t, J ) 48 Hz, 1 P); ESI-MS

Microwave-Mediated Synthesis of Labeled Nucleotides
Bioconjugate Chem., Vol. 21, No. 10, 2010 1775
m/z 984.1 (M + H); FAB-HRMS calcd for C26H37N7Na4O22P3
(M + H) 984.0795, found 984.0790.
issue, ion pair chromatography was explored. Under the
appropriate ion pair conditions, N4-amino-dCTP was easily
fractionated with negligible degradation.
Labeling DNA with DNP-PEG4-dCTP. Double-stranded
DNA was labeled with DNP-dPEG4-dCTP by nick translation
(34). No unmodified dCTP was added to the nick translation
reaction mixture. The reaction was stopped when the size of
the DNA was reduced to ∼300 bp as judged by agarose gel
electrophoresis. The resulting DNA was purified by column
chromatography (Qiagen) and precipitation by 2-propanol. The
extent of labeling was estimated from optical absorption
measurements at 260 and 365 nm. Typically, from ∼1 to 5%
of the nucleotides in the resulting DNA was labeled with DNP.
Tissue Staining. The BenchMark XT automated slide pro-
cessing system (Ventana Medical Systems, Inc., Tucson, AZ)
was used for to assess performance of the in situ hybridization
assay for HER2 and HPV DNA targets.
Pretreatments. Automated deparaffinization, pretreatment,
hybridization, stringency wash, signal detection, and counter-
staining were performed on the BenchMark XT system. For-
malin-fixed paraffin-embedded tissue sections on glass slides
were baked at 65 °C for 20 min prior to the deparaffinization
step with EZ Prep (Ventana) at 75 °C for 16 min. Deparaffinized
tissue sections were exposed to a combination of heat treatment
with reaction buffer [Tris-based solution (pH 7.6), Ventana] and
ISH Protease 2 or ISH Protease 3 (Ventana) to unmask DNA
targets.
Hybridization and Signal Development. For HER2 gene
detection, the INFORM HER2 DNA Probe (Ventana) was
applied to the glass slide and the probe and sample DNA were
codenatured by incubation at 95 °C for 10 min. Then, the
hybridization step was conducted at 52 °C for 2 h. After three
stringency wash steps were performed at 72 °C with 2× SCC
(Ventana), tissue sections were incubated with monoclonal rabbit
anti-DNP antibody (Ventana) for 20 min and then with HRP-
conjugated anti-rabbit antibody for 16 min at 37 °C. The
peroxidase catalyzed silver deposition was developed using the
ultraView SISH Detection Kit (Ventana) (36). HER2 probe
slides were counterstained with Hematoxylin II (Ventana) for
4 or 8 min and Bluing Reagent (Ventana) for 4 min.
For the HPV assays, the DNP-labeled HPV probe was
detected using the Ventana ISH iView Blue Plus detection kit
on the BenchMark XT system.
The synthesis of the electrophilic species, DNP-dPEG4-
NHS, also benefited from the use of microwave conditions.
Under aqueous microwave conditions, DNP-dPEG4-CO2H
was obtained in >90% yield after irradiation for only 30 min.
The corresponding reaction in organic solvent in the absence
of microwave irradiation never afforded a >60% yield and
required several hours to complete. The synthesis of
DNP-dPEG4-NHS was accomplished by conversion of the
acid intermediate to the NHS ester via treatment with NHS and
DCC in DCM overnight. The crude NHS ester obtained
following the removal of the urea byproduct was sufficiently
stable to allow for purification on the silica gel.
Overnight reaction of the nucleotide (1) and the NHS ester
(3) in anhydrous DMSO proceeded smoothly to give the final
nucleotide conjugate that was efficiently purified by ion pair
chromatography. Ion exchange provided the sodium salt of N4-
amino-dCTP-dPEG4-DNP that was used in the labeling of
DNA.
DNA Labeling with DNP-dPEG4-dCTP and Tissue
Staining. Double-stranded HER2 and HPV DNA were labeled
with DNP-dPEG4-dCTP by nick translation using Escherichia
coli DNA polymerase I. The resulting probes were typically
labeled at 1-5% of the nucleotide residues. The probes were
used to visualize gene amplification of HER2 DNA in HER+
breast cancer and to detect human papilloma virus DNA in
cervical tumors. In situ hybridization assays for HER2 and HPV
DNA signals utilizing the BenchMark XT automated slide
processing system (Ventana) were effective in visualizing the
HER2 and HPV DNA targets in tissue. HPV was visualized
utilizing a chromogenic ISH protocol, whereas HER2 was
visualized via SISH (Figure 1). The observed signals were strong
and easily discernible with little or no background (Figure 2).
DISCUSSION
Efforts to efficiently produce nucleotide conjugates suitable
for the labeling of double-stranded DNA via nick translation
have led to a novel methodology involving the bisulfite-
mediated, microwave-promoted hydrazinolysis of cytosine at
the C4 position. By utilizing microwave acceleration, the
hydrazine moiety was effectively installed while side reactions
were prevented and phosphate hydrolysis was minimized. HPLC
analysis of thermally promoted and microwave-promoted reac-
tions indicated that microwave energy accelerates the hydrazi-
nolysis reaction with no effect on the rate of phosphate
hydrolysis. This observation allowed the optimization of
4-amino-dCTP formation by employing the minimum effective
reaction time. The reaction was shown to be scalable within
the capacity limits of the microwave synthesizer. As the scale
was increased, slightly longer microwave reaction times were
required to affect complete hydrazinolysis. As such, larger scale
reactions produced slightly lower yields.
There are several examples of nucleotides that have been
synthetically modified to contain nucleophilic handles (37, 38).
To the best of our knowledge, the methodology that we reported
here represents a particularly efficient route for producing such
nucleotides.
The utility of 4-amino-dCTP as a reagent for the assembly
of hapten-labeled nucleotides that can be incorporated into DNA
via nick translation has been demonstrated via the synthesis of
DNP-dPEG4-dCTP. Nick translation of double-stranded HER2
and HPV DNA in the prescence of DNP-dPEG4-4-amino-
dCTP consistently produced probes labeled at 3-4% of the
nucleotide residues. Visualization of HER2 and HPV DNA
targets in tissue via in situ hybridization utilizing the BenchMark
RESULTS
Synthesis. A novel methodology involving the bisulfite-
mediated, microwave-promoted hydrazinolysis of cytosine was
utilized to efficiently produce nucleotide conjugates suitable for
the labeling of DNA via nick translation. The synthesis of one
such conjugate, N4-amino-dCTP-dPEG4-DNP, is outlined in
Scheme 1. A number of microwave-mediated transamination
reactions of deoxycytosine triphosphate (dCTP) with bidentate
nitrogen nucleophiles were explored. Of these, the reaction with
hydrazine to produce N4-aminodeoxycytidine 5′-triphosphate
(N4-amino-dCTP) proved to be the highest yielding and most
reproducible. To obtain optimal results, strict control of reaction
parameters, including applied microwave power, pH, and
temperature, had to be maintained. On a 300 mg scale, under
optimal microwave conditions, the hydrazinolysis of cytosine
proceeded nearly quantitatively within 30 min. The correspond-
ing thermal reaction produced a <20% yield of the hydrazi-
nolysis product after heating for 1 h. The microwave reaction
was shown to be scalable to 3.5 g, and the final product was
amenable to labeling with a wide variety of molecules such as
haptens or fluorophores.
Early attempts to purify N4-aminodeoxycytidine 5′-triphos-
phate under acidic HPLC conditions described in the literature
led to extensive product degradation (23). To circumvent this

1776 Bioconjugate Chem., Vol. 21, No. 10, 2010
Lefever et al.
Scheme 1. Synthesis of N4-Amino-dCTP-PEG4-DNP
XT automated slide processing system (Ventana) consistently
provided strong and easily discernible signals with little or no
background.
As the percent of labeled nucleotide incorporation increases
for a given probe, effects on the DNA melting temperature and
hybridization specificity became evident. These phenomena are
Figure 1. Alkaline phosphatase (AP)-mediated chromogenic detection (SISH) and horseradish peroxidase (HRP)-mediated silver deposition detection
(SISH) (5).

Microwave-Mediated Synthesis of Labeled Nucleotides
Bioconjugate Chem., Vol. 21, No. 10, 2010 1777
an individual patient sample. This last objective is of critical
importance in the fast developing area of individualized
medicine and diagnostics, which seeks to do away with the
cancer paradigms of “trial and error” and “one drug fits all”,
replacing them with targeted therapies tailored for the specific
genomic profiles of an individual patient’s disease. This has
become possible because of the recent advent of multiplexed
light microscopy-based in situ hybridization methods, which
allow for the visualization of individual copies of genes and
amplification states. The use of HER2 as a prognostic and
predictive biomarker for the determination of breast cancer
patient eligibility for Herceptin (Genentech) treatment exempli-
fies this emerging approach to personalized cancer diagnosis
and therapy.
ACKNOWLEDGMENT
We thank Dr. Lidija Pestic-Dragovich for biological tissue
staining data and many discussions and Mr. Philip Miller for
continuing support of this work. Ventana, BenchMark, IN-
FORM, and UltraView are trademarks of Roche. All other
trademarks are property of their respective owners.
Supporting Information Available: Characterization of
Figure 2. (a) Detection of HPV DNA in cervical cancer tissues by
in situ hybridization with a DNP-labeled HPV DNA probe cocktail
followed by immunohistochemical detection of the probe with a
rabbit anti-DNP monoclonal antibody followed by an alkaline
phosphatase-conjugated goat anti-rabbit antibody and Ventana’s
IViewBlue Plus Detection kit. The blue nuclear signal indicates the
presence of HPV DNA. (b) Detection of HER2 genes in breast cancer
cell lines. In situ hybridization with a DNP-labeled HER2 DNA
probe was followed by reaction with a rabbit anti-DNP monoclonal
antibody that was detected by a peroxidase-conjugated goat anti-
rabbit antibody. Peroxidase-catalyzed silver deposition generated
the black spots representing the HER2 genes (Ventana’s INFORM
HER2 SISH assay). MCF-7 cells contain the normal two copies of
the gene. In BT474 cells, the HER2 gene is amplified as indicated
by the many spots in each nucleus.
synthesized small molecules, including 1H NMR, 13C NMR, 31
P
NMR, and HRMS data. This material is available free of charge
via the Internet at http://pubs.acs.org.
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