DNA microarrays on a dendron-modified surface improve significantly the detection of single nucleotide variations in the p53 gene.

Abstract

Selectivity and sensitivity in the detection of single nucleotide polymorphisms (SNPs) are among most important attributes to determine the performance of DNA microarrays. We previously reported the generation of a novel mesospaced surface prepared by applying dendron molecules on the solid surface. DNA microarrays that were fabricated on the dendron-modified surface exhibited outstanding performance for the detection of single nucleotide variation in the synthetic oligonucleotide DNA. DNA microarrays on the dendron-modified surface were subjected to the detection of single nucleotide variations in the exons 5-8 of the p53 gene in genomic DNAs from cancer cell lines. DNA microarrays on the dendron-modified surface clearly discriminated single nucleotide variations in hotspot codons with high selectivity and sensitivity. The ratio between the fluorescence intensity of perfectly matched duplexes and that of single nucleotide mismatched duplexes was >5-100 without sacrificing signal intensity. Our results showed that the outstanding performance of DNA microarrays fabricated on the dendron-modified surface is strongly related to novel properties of the dendron molecule, which has the conical structure allowing mesospacing between the capture probes. Our microarrays on the dendron-modified surface can reduce the steric hindrance not only between the solid surface and target DNA, but also among immobilized capture probes enabling the hybridization process on the surface to be very effective. Our DNA microarrays on the dendron-modified surface could be applied to various analyses that require accurate detection of SNPs.

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DNA microarrays on a dendron-modified surface
improve significantly the detection of single
nucleotide variations in the p53 gene
Soon Jin Oh, Jimin Ju, Byung Chul Kim, Eunsil Ko, Bong Jin Hong,
Jae-Gahb Park1, Joon Won Park and Kwan Yong Choi*
Division of Molecular and Life Sciences, Pohang University of Science and Technology, Pohang 790-784, Korea and
1Research Institute and Hospital, National Cancer Center, Goyang, 411-764, Korea
Received April 9, 2005; Revised and Accepted May 16, 2005
ABSTRACT
Selectivity and sensitivity in the detection of single
nucleotide polymorphisms (SNPs) are among most
important attributes to determine the performance
of DNA microarrays. We previously reported the gen-
eration of a novel mesospaced surface prepared by
applyingdendronmoleculesonthesolidsurface.DNA
microarrays that were fabricated on the dendron-
modified surface exhibited outstanding performance
for the detection of single nucleotide variation in the
synthetic oligonucleotide DNA. DNA microarrays on
the dendron-modified surface were subjected to the
detection of single nucleotide variations in the exons
5–8 of the p53 gene in genomic DNAs from cancer
cell lines. DNA microarrays on the dendron-modified
surface clearly discriminated single nucleotide vari-
ations in hotspot codons with high selectivity and
sensitivity.Theratiobetweenthefluorescenceintens-
ity of perfectly matched duplexes and that of single
nucleotidemismatchedduplexeswas>5–100without
sacrificing signal intensity. Our results showed that
theoutstandingperformanceofDNAmicroarraysfab-
ricated on the dendron-modified surface is strongly
related to novel properties of the dendron molecule,
which has the conical structure allowing mesospa-
cing between the capture probes. Our microarrays
onthedendron-modifiedsurfacecanreducethesteric
hindrance not only between the solid surface and tar-
getDNA,butalsoamongimmobilized captureprobes
enabling the hybridization process on the surface
to be very effective. Our DNA microarrays on the
dendron-modifiedsurfacecouldbeappliedtovarious
analyses that require accurate detection of SNPs.
INTRODUCTION
Single nucleotide polymorphisms (SNPs) are distributed
throughout the human genome and implicated in genetic
disorders and disease susceptibility (1). DNA microarray tech-
niques allow parallel analysis of multiple DNA target samples
and can be applicable to gene expression profiling and gene
mutation analysis. Even if DNA microarray technology is
relatively well established for gene expression profiling
(2–4), accurate analyses of genetic mutation by DNA micro-
arrays (5) are still in an early stage because even SNPs that
are the most suitable targets for DNA microarray analysis are
detected with relatively poor accuracy.
Poor accuracy of DNA microarray for the detection of
SNPs or single base mutation is considered to originate
from inherent properties of the surface and molecular inter-
layer structures that are not well characterized. A mixed
monolayer self-assembled on gold surface increased the hybri-
dization efficiency significantly (6) and a space-controlled
gold surface improved the efficiency of an alpha-helix forma-
tion for immobilized oligopeptides (7). Therefore, surface
characteristics is one of the critical major elements to deter-
mine the performance of microarray. Fabricating solid surface
that can provide excellent SNPs discrimination efficiency
could be a breakthrough in gene mutation analysis by DNA
microarray technology.
Previously, we reported the preparation of surface materials
allowing the mesospacing between capture probes by intro-
ducing a conical-shaped dendron that could provide enough
space for unhindered interactions between biomolecules (8).
We also observed that a glass substrate modified with the
dendron could significantly reduce non-specific binding
and enhance selectivity of DNA microarrays: The observed
selectivity was equivalent to that observed in solution for
detecting single point mutation in synthetic oligonucleotide
target DNA (9). This high SNPs discrimination efficiency
was found to be largely related to the characteristics of the
*To whom correspondence should be addressed. Tel: +82 54 279 2295; Fax: +82 54 279 8290; Email: kchoi@postech.ac.kr
? The Author 2005. Published by Oxford University Press. All rights reserved.
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Nucleic Acids Research, 2005, Vol. 33, No. 10 e90
doi:10.1093/nar/gni087

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dendron-modifiedsurfacethatguaranteedmesospacing among
the probe DNAs.
Inbiological system,the p53 gene playskeyrolesasatumor
suppressor in various biological events, such as cell cycle
regulation, gene transcription, DNA repair, genomic stability,
chromosomal segregation and apoptosis (10–12). The loss of
wild-type function of the p53 gene could lead to cancers and
was the most frequently mutated gene in a variety of human
cancers (12–14). For example, 70 and 60% of lung and colon
cancers were found to be related to p53 mutations. The p53
GeneChip?, which was developed by Affymetrix, showed
reasonable accuracy for detecting certain mutations in the
p53 gene in various cancers (15–19). However, the p53 Gene
Chip?still needs improvement in order to be widely adopted
for the accurate and reliable analysis of single nucleotide
mutations in the p53 gene. Creating new surface is essential
to improve the detection accuracy because the accuracy seems
to be strongly related to the properties of surface on which
DNA microarrays are fabricated.
In order to investigate the performance of the dendron-
modified surface characteristics for the detection capability
of DNA microarrays in real biological samples, we applied
our dendron-modified surface methodology for the detection
of single nucleotide variations in the p53 gene from cancer cell
lines. DNA microarrays on the dendron-modified surface
showed high selectivity and sensitivity, confirming that optim-
ization of surface characteristics could be very important to
fabricate DNA microarrays with enhanced performance.
MATERIALS AND METHODS
All chemicals were reagent grade or of better quality than
reagent grade and used as received unless mentioned. Ultra-
pure water (18 MW/cm) was obtained from a Milli-Q purifica-
tion system (Millipore, USA). Aldehyde-coated glass slides
were purchased from Telechem International (USA). Bare
glass slides were obtained from Corning (USA).
Capture probe oligonucleotide
The capture probe sequences were designed by the use of the
Array Designer software (PREMIER Biosoft International,
USA). Amine-modified capture probe oligonucleotides were
purchased from MWG-Biotech (Ebersberg, Germany) and
Bionics (Korea). Capture oligonucleotides used in this study
are listed in Table 1.
Preparation of the dendron-modified surface
The di(N-succinimidyl)carbonate (DSC)-activated dendron-
modified surface was prepared according to the procedure
as described previously (9).
Preparing DNA microarrays
Amine-tethered capture probe oligonucleotides were immob-
ilized on the DSC-activated dendron-modified surface of the
glass slide by spotting the solution in a buffer containing
25 mM sodium bicarbonate, 5 mM MgCl2and 10% (v/v)
dimethyl sulfoxide at pH 8.5 using a microarrayer (Cartesian
Technologies, Microsys 5100) in a clean room (class 10 000).
After spotting the probe oligonucleotides side by side in a
10 · 1 format, the microarray was incubated in a chamber
maintained at ?85% humidity for overnight to give the amine-
tethered DNA sufficient reaction time. Slides were then stirred
in a buffer solution containing 2· SSPE (0.30 M sodium
chloride, 0.020 M sodium hydrogen phosphate and 2.0 mM
EDTA), pH 7.4 and 7.0 mM SDS at 37?C for 1 h and then in
boiling water for 5 min to remove non-specifically bound
oligonucleotides. Finally, the DNA-functionalized microarray
was dried under a stream of nitrogen for the subsequent
hybridization. Different kinds of probes were spotted in
a single plate. DNA microarrays on the aldehyde plates
(Telechem International, SMA) were prepared according to
the supplier’s protocol.
Genomic DNA samples
Genomic DNAs of SNU-cell lines (SNU-475 and 761) were
kindly provided by Prof. Ja-Lok Ku at Korean Hereditary
TumorRegistry,Seoul National University.SNU-celllines are
humancarcinomacelllinesdepositedatthe Korean Hereditary
Tumor Registry.
Subcloning and sequencing
The DNA segment from the exon 5 to the exon 8 in the p53
gene of each cell line was amplified by PCR with two kinds
of primer pairs: a forward primer, Fwd I (50-CTG ACT TTC
AAC TCT GTC TCC T-30) or Fwd II (50-TAC TCC CCT GCC
CTC AAC AA-30) and a reverse primer, Rev I (50-TGC ACC
CTT GGT CTC CTC CAC-30) or Rev II (50-CTC GCT TAG
TGC TCC CGG G-30). Each genomic DNA was amplified in
the 20 ml solution containing 10 pmol of the primer pair, Fwd I
and Rev I, 250 mM dNTP mixture, 2.5 U Taq polymerase
Table 1. Capture oligonucleotides used in this study
NoProbe name ExonSequencea(50!30)
GTTGTGAGGCNCTGCCCC N = G (wt), A, T, C (mt)
TTTCGACATANTGTGGTGGTG N = G (wt), A, T, C (mt)
TCGACATAGTNTGGTGGTGCC N = G (wt), A, T, C (mt)
CATGTGTNACAGTTCCTGCA N = A (wt), G, T, C (mt)
CATGAACNGGAGGCCCATC N = C (wt), A, T, G (mt)
TTGAGGTGCNTGTTTGTGC N = G (wt), A, T, C (mt)
GAGAGACNGGCGCACAG N = C (wt), A, T, G (mt)
(T)30-GTTGTGAGGCNCTGCCCC N = G (wt), A, T, C (mt)
(T)30-CATGTGTNACAGTTCCTGCA N = A (wt), G, T, C (mt)
Nucleotide
1
2
3
4
5
6
7
8
9
175
215
216
239
248
273
282
175-T30
239-T30
5
6
6
7
7
8
8
5
7
18
21
21
20
19
19
17
48
50
The sequences underlined represent the codons as numbered under ‘Probe name’. wt, wild type; mt, mutant type.
aThe oligonucleotides have an amino group at the 50end.
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(Takara, Japan) in 1· buffer (supplemented with Taq
polymerase) in a thermocycler (Hybaid, Multiblock System,
UK).Thereactioncycleswereprogrammedasfollows:aninitia-
tionactivationofthepolymerase at95?Cfor1min,subsequent
20 cycles of 95?C for 30 s, 58?C for 30 s and 72?C for 90 s,
followed by a final elongation step at 72?C for 5 min. The
amplified PCR products of genomic DNA were diluted20-fold
and subjected to the second nested PCR with the same reaction
conditions using the second primer pair, Fwd II and Rev II.
After separated on agarose gel and purified by a gel extraction
kit (Qiagen, USA), the amplified DNAs were subcloned
into pGEM T-easy vector (Promega) and used to transform
Escherichia coli DH5a. The subcloned plasmid was purified
by the use of a Plasmid Mini kit (Qiagen) for the deter-
mination of nucleotide sequences. Bidirectional sequencing
was performed using pUC/M13 sequencing primers with the
sequences, 50-GTT TTC CCA GTC ACG ACG TTG-30and 50-
TGA GCG GAT AAC AAT TTC ACA CAG-30, respectively.
Preparation of target DNA
The amplified plasmid DNA was digested with EcoRI to
release the exons 5–8 region of the p53 gene. The insert
DNA was separated on agarose gel and purified by the use
of a gel extraction kit (Qiagen). Target DNAs spanning SNPs
sites were random primed and labeled in the 20 ml solution
containing 50 ng of template DNA with 50 U Klenow enzyme
(NEB), 1· EcoPol buffer supplemented with Klenow enzyme,
6 mg of random octamer (Bionics, Korea), low dT dNTP
mixture (100 mM dATP, 100 mM dGTP, 100 mM dCTP and
50 mM dTTP) and 50 mM Cyanine3-dUTP (NEN) at 37?C
for 2 h. Unincorporated nucleotides were removed using a
QIAquick PCR purification kit (Qiagen). After assessing the
specific activity and the number of nucleotides per incorpor-
ated fluorescent dye by UV spectrophotometry, aliquots of the
labeled DNA were subjected to hybridization.
Hybridization
Hybridization was performed in the hybridization buffer con-
taining 30 nM target DNA tagged with Cy3 fluorescent dye
for 1 h at 50?C, 1 h at 47?C and 2 h at 45?C and then washed
at room temperature in a buffer containing 1· SSC and 0.1%
SDS, 0.1· SSC and 0.1% SDS, and 1· SSC respectively, in a
hybridization station (Genomic solutions, GeneTAC).
Fluorescence scanning and signal quantitation
The image acquisition and the fluorescence intensity analysis
were carried out by the use of a confocal laser scanner (GSI
Lumonics, ScanArray Lite) and a quantitative microarray
analysis software (BioDiscovery, ImaGene).
RESULTS AND DISCUSSION
Design of capture probes and preparation of
target DNAs
Codons, 175, 215, 216, 239, 248, 273 and 282 in the p53 gene,
which have been known as missense mutational hotspots
among cancer patients, were selected for this study. Codons
175, 248, 273 and 282 were chosen from the international
IARC TP53 mutation database (20), and other codons 215,
216 and 239 found very frequently among Korean cancer
patients were selected from the p53 mutational hotspot data-
base in the Korean Hereditary Tumor Registry. The amino
acid encoded by the codon 175 occurs in the L2 loop in the
vicinity of the zinc binding site and plays a critical role in
stabilizing the L2 and L3 loops. The two amino acids encoded
by the codons 248 and 273 interact directly with the minor
groove and the backbone phosphate of DNA, respectively.
The codon 282 encodes a residue that plays a structural
role in the loop–sheet–helix motif (21).
The capture probe sequences designed for the detection of
seven codons were 18–21 nt long depending on the sequences
around the respective codon and their calculated Tmvalues
were ?55?C (Table 1). Four capture probes, one perfectly
matched and three single-nucleotide-mismatched probes were
prepared to investigate the selective detection of each codon.
All the probe oligonucleotides have a terminal amine group
required for the covalent attachment to the DSC-activated
dendron surface or the aldehyde surface. Cy3-incorporated
target DNAs were prepared by the random priming (22) of
the exons 5–8 region in the p53 gene, which had been amp-
lified by PCR of the genomic DNA obtained from cancer cell
lines, SNU 761 and 475. The size of random primed target
DNAs was found to be 100–200 nt and the incorporation ratio
was ?80 bases per dye molecule. SNU 761 has no mutation in
exons 5–8, and SNU 475 has a single point mutation in the
codon 239 (AAC!GAC). The nucleotide sequence of each
PCR product was confirmed before random priming.
Preparation of the dendron-modified surface
The structure of the dendron, which is a second generation
dendrimer with reactive functional groups at the branch ter-
mini, is shown in Figure 1. The dendron-modified surface was
prepared by self-assembling dendron molecules on a slide (9).
Each reaction step was confirmed by both UV spectrophoto-
metry and atomic force microscopy. High-resolution scanning
Figure 1. Structure of the dendron molecule.
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electron microscope (HRSEM) image showed that the surface
manifests the mesospacing of 3.2 nm in average between the
dendrons. Tapping mode AFM image also showed that the
resulting layer was smooth and homogeneous without any
aggregates or holes (9).
Selectivity of the DNA microarrays on the
dendron-modified surface
The selectivity indicating the capability of discriminating
single nucleotide variation is one of the most important
factors to determine the performance of DNA microarrays.
In order to investigate the selectivity of DNA microarrays
on the dendron-modified surface, single nucleotide variations
in seven hotspot codons of the p53 gene were simultaneously
examined.
After 28 kinds of capture probes that have all the possible
nucleotide sequences at seven codons were spotted on a single
slide, 30 nM target DNA for SNU 761 was hybridized. The
matched sequence of each codon in SNU 761 was CGC, AGT,
GTG, AAC, CGG, CGT and CGG for the codons 175, 215,
216, 239, 248, 273 and 282, respectively; underlined: the mis-
matched nucleotides. The fluorescence images of all seven
codons after hybridization are shown in Figure 2. Although
the fluorescence intensity was different from codon to codon,
all matched sequences of seven codons exhibited invariably
strong fluorescence intensity. There is a drastic difference
in the intensity between the matched and mismatched pairs.
The normalized fluorescence signal ratio, i.e. intensity for one
base mismatched pair to that for the perfectly matched pair,
MM/PM, ranged from 0.007 to 0.16 when the relative intens-
ities were assessed from 10 independent measurements of
the fluorescence intensities (Table 2). We also observed
that the DNA microarrays on the dendron-modified surface
discriminated singlenucleotidevariationswithhighselectivity
factor of <0.01 in synthetic oligonucleotide target DNAs (9).
These discriminating efficiencies obtained in both synthetic
and real target DNAs are unprecedentedly high because select-
ivity factors of 0.19–0.57 were observed previously for
microarrays fabricated on various amine surfaces, including
a mixed self-assembled monolayer for the detection of single
nucleotide variations even in short synthetic oligonucleotide
target DNAs (23).
We also investigated the dependence of discrimination effi-
ciency for the detection of single nucleotide variations on the
concentrations of target DNAs. Interestingly, it was observed
that high discrimination efficiency (<0.10) as judged by the
signal intensity ratio (MM/PM) of codon 273 was maintained
in the broad range of target concentration from 3 to 100 nM
(Figure 3). For other codons, similar results were obtained
Figure 2. Fluorescence images after hybridization for the detection of single nucleotide variations in seven hotspot codons of p53 gene on the dendron-modified
surface. All the capture probes were spotted in a 10 · 1 format.
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(data not shown). The fluorescence signal intensity of the
matched duplex was increased with the concentration of the
target DNAs and saturated at ?30 nM target DNA as shown in
Figure 3a. These results demonstrated that DNA microarrays
fabricated on the dendron-modified surface could detect target
DNA of nanomolar concentration with high discriminating
ability in the broad range of target DNA concentration.
Sensitivity of the DNA microarrays on the
dendron-modified surface
The sensitivity of DNA microarrays is another important
factor to determine the performance of DNA microarrays.
Previously, we reported that the signal intensity was strongly
dependent on the density of functional groups on the surface
(23). In order to compare the signal intensity of our DNA
microarrays on the dendron-modified surface with that on
the aldehyde surface, we also fabricated the DNA microarray
on the aldehydesurface forthe detectionofthe codons 175 and
239. The signal intensity on the dendron-modified surface
(Figure 4a, left panel) was found to be similar to or higher
than that on the aldehyde surface (Figure 4b, left panel).
This is an interesting result since it was reported that the
surface density of reactive aldehyde groups was 5 · 1012/mm2
(www.arrayit.com), whereas that of reactive amine groups of
the dendron-modified surface was 1 · 1011/mm2(9). This
result suggests that the hybridization efficiency of DNA
microarrays on the dendron-modified surface is much higher
than that on the aldehyde surface.
To investigate the effect of the linker in the capture probe on
hybridized signal intensity and/or discrimination efficiency,
a T30 spacer composed of 30 thymidine molecules was placed
between the 50ends of capture probes and terminal amine
groups. Spacers were reported to increase the hybridization
efficiency in the detection of single nucleotide variations in
synthetic oligonucleotide target DNAs because spacers kept
capture probes away from the solid surface minimizing the
influence of the surface (24,25). As shown in Figure 4, DNA
microarrays on the dendron-modified and aldehyde surfaces
showed different effect of the T30 spacer on the signal
intensity and the discrimination efficiency of SNPs after the
hybridization of DNA. In the case of DNA microarrays on the
dendron-modified surface, the T30 spacer could enhance
the hybridization signal intensity by a factor of ?3 for both
codons 175 and 239 without any loss of SNPs discrimination
efficiency (Figure 4a). Therefore, synergistic effects were
observed when the vertical separation from the surface was
combined with optimum lateral spacing between the probes.
In contrast, T30 spacer on the aldehyde surface could not
enhance the signal intensity (Figure 4b), implying that T30
spacer alone was not effective in enhancing the hybridization
of long target DNAs. These results again support that precise
control of the optimum spacing of capture probes is utmost
important to give high selectivity and sensitivity of DNA
microarrays.
Table 2. Relative fluorescence intensity of seven hotspot codons in p53 gene on dendron-modified surface
CodonRelative intensitya(%)
GATC
175
215
216
239
248
273
282
100 (–3.4)
100 (–7.1)
100 (–1.7)
15.9 (–2.4)
2.1 (–0.4)
100 (–8.4)
8.1 (–0.3)
10.1 (–0.9)
4.9 (–0.7)
8.2 (–0.9)
100 (–3.4)
4.9 (–1.0)
5.5 (–0.7)
13.4 (–1.0)
3.1 (–0.3)
3.3 (–0.4)
7.4 (–0.6)
9.4 (–1.1)
5.9 (–0.5)
4.5 (–0.2)
6.8 (–0.5)
1.7 (–0.1)
2.1 (–0.2)
3.4 (–0.2)
9.2 (–0.8)
100 (–6.9)
1.3 (–0.1)
100 (–4.9)
aThe fluorescence intensity of perfectly matched sequence of each codon was set to 100 and shown in bold. Numbers in parenthesis represent relative standard
deviation for each capture probe based on 10 independent measurements.
Figure 3. (a) Dependence of fluorescence signal intensity and (b) relative
fluorescence intensity depending on the concentration of target DNAs for
the detection of codon 273. PM, perfectly matched; MM, mismatched. The
fluorescence intensity of perfectly matched sequence of each concentration of
target DNA was set to 100.
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Detection of heterozygous mutation
In order to identify the heterozygous mutations, we analyzed
a 1:1 mixture of the target DNA from SNU 761 and 475 that
have wild (AAC) and mutant (GAC) sequence at the codon
239, respectively. The 1:1 mixture (in terms of concentration)
of target DNA was prepared by the random priming of the
1:1 mixture of genomic DNA templates that were extracted
from both cancer cell lines. A representative fluorescence
image and quantitative results are shown in Figure 5a and b,
respectively. When 100% of DNA template from SNU
761 was used as a target DNA, the ratio between the fluores-
cence intensity of wild-type AAC and that of GAC, TAC or
CAC mutant was <0.2, while 100% of DNA template from
SNU 475 as a target DNA also exhibited the same ratio
of <0.2 between the fluorescence intensity of GAC mutant
and that of AAC wild-type, TAC or CAC mutant. For the
1:1 mixture of target DNA (50% DNA template from
SNU-761 + 50%DNAtemplatefromSNU-475),the observed
ratio was 1.0:1.0:<0.2 (wild-type AAC: GAC mutant: TAC or
CAC mutant). The quantitative signal intensity ratio of 1.0:1.0
between wild-type AAC and GAC mutant demonstrates again
high selectivity of DNA microarrays on the dendron-modified
surface. These results indicate that DNA microarrays on the
dendron-modified surface were capable of detecting hetero-
zygous mutations reliably.
We believe that the high performance of DNA microarrays
on the dendron-modified surface is attributed to the inherent
characteristics of the surface, which allows mesospacing
between functional groups resulting in the reduction of the
steric hindrance not only among immobilized capture probes,
but also between immobilized capture probes and target
DNAs. Because steric hindrance and surface effect could be
Figure4.Fluorescenceimageforthedetectionofcodons175and239onthe(a)dendron-modifiedsurfaceand(b)aldehydesurfaceusingcaptureprobeswithoutand
with T30 spacer.
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reduced significantly to mimic the solution phase phenomena,
enhanced DNA hybridization efficiency with high selectivity
and signal intensity was achieved for the DNA microarrays on
the nano-scale controlled surface. This is well coincident with
previous reports that the steric hindrance by the adjacent
DNA probe molecules plays an important role in governing
the amount of hybridization and hybridization efficiency
(6,25–28). For example, Georgiadis et al. (6,26) showed that
hybridization on the surface was strongly dependent on the
density of capture probes when the hybridization on two kinds
of slides with different probe density was analyzed by surface
plasmon resonance spectroscopy. Shchepinov et al. (25) also
showed that high hybridization yield could be achieved
by adjusting the capture probe density on the surface using
a combination of cleavable and stable linkers. Moreover,
enhanced hybridization efficiency in the detection of M13
phage DNA was achieved on a mixed self-assembled mono-
layer boasting reduced hindrance among capture probes (28).
Our observation again supports that the properties of surface
on which DNA microarrays fabricated play critical roles to
determine the performance of DNA microarrays.
In conclusion, we have shown that the DNA microarrays
fabricated on the dendron-modified surface could unam-
biguously detect single nucleotide variations in seven hotspot
codons of the p53 tumor-suppressor gene simultaneously with
high selectivity and sensitivity. The outstanding performance
ofDNAmicroarrays onthedendron-modifiedsurfaceseems to
be attributed to the inherent characteristics of the dendron
molecule on the surface, which has conical structure, render-
ing mesospacing between immobilized capture probes. It
remains to be investigated that our DNA microarrays on the
dendron-modified surface can be applied to various analyses
that require accurate and precise detection of SNPs.
ACKNOWLEDGEMENTS
This work was supported by the Korea Research Foundation
Grant(KRF-2002-005-C00010,
Funding to pay the Open Access publication charges for this
article was provided by POSTECH.
KRF-2002-005-C00011).
Conflict of interest statement. None declared.
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