Room-Temperature Single-Nucleotide Polymorphism and Multiallele DNA Detection Using Fluorescent Nanocrystals and Microarrays
Identifieur interne : 000B18 ( Istex/Corpus ); précédent : 000B17; suivant : 000B19Room-Temperature Single-Nucleotide Polymorphism and Multiallele DNA Detection Using Fluorescent Nanocrystals and Microarrays
Auteurs : Daniele Gerion ; Fanqing Chen ; Balaji Kannan ; Aihua Fu ; Wolfgang J. Parak ; David J. Chen ; Arunava Majumdar ; A. Paul AlivisatosSource :
- Analytical Chemistry [ 0003-2700 ] ; 2003.
Abstract
We report two cDNA microarray-based applications of DNA−nanocrystal conjugates, single-nucleotide polymorphism (SNP) and multiallele detections, using a commercial scanner and two sets of nanocrystals with orthogonal emissions. We focus on SNP mutation detection in the human p53 tumor suppressor gene, which has been found to be mutated in more than 50% of the known human cancers. DNA−nanocrystal conjugates are able to detect both SNP and single-base deletion at room temperature within minutes, with true-to-false signal ratios above 10. We also demonstrate microarray-based multiallele detection, using hybridization of multicolor nanocrystals conjugated to two sequences specific for the hepatitis B and hepatitis C virus, two common viral pathogens that inflict more than 10% of the population in the developing countries worldwide. The simultaneous detection of multiple genetic markers with microarrays and DNA−nanocrystal conjugates has no precedent and suggests the possibility of detecting an even greater number of bacterial or viral pathogens simultaneously.
Url:
DOI: 10.1021/ac034482j
Links to Exploration step
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<front><div type="abstract">We report two cDNA microarray-based applications of DNA−nanocrystal conjugates, single-nucleotide polymorphism (SNP) and multiallele detections, using a commercial scanner and two sets of nanocrystals with orthogonal emissions. We focus on SNP mutation detection in the human p53 tumor suppressor gene, which has been found to be mutated in more than 50% of the known human cancers. DNA−nanocrystal conjugates are able to detect both SNP and single-base deletion at room temperature within minutes, with true-to-false signal ratios above 10. We also demonstrate microarray-based multiallele detection, using hybridization of multicolor nanocrystals conjugated to two sequences specific for the hepatitis B and hepatitis C virus, two common viral pathogens that inflict more than 10% of the population in the developing countries worldwide. The simultaneous detection of multiple genetic markers with microarrays and DNA−nanocrystal conjugates has no precedent and suggests the possibility of detecting an even greater number of bacterial or viral pathogens simultaneously.</div>
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Single-Nucleotide
Polymorphism and Multiallele DNA Detection Using
Fluorescent Nanocrystals and Microarrays</title>
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Chemical Society</publisher>
<availability><licence>Copyright © 2003 American Chemical Society</licence>
<p>American Chemical Society</p>
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<date>2003</date>
<date type="Copyright" when="2003">2003</date>
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<sourceDesc><biblStruct type="article"><analytic><title level="a" type="main">Room-Temperature
Single-Nucleotide
Polymorphism and Multiallele DNA Detection Using
Fluorescent Nanocrystals and Microarrays</title>
<author xml:id="author-0000" role="corresp"><persName><surname>Gerion</surname>
<forename type="first">Daniele</forename>
</persName>
<note place="foot" n="ac034482jAF2"><ref>†</ref>
<p>
Department of Chemistry, University of California.</p>
</note>
<note place="foot" n="ac034482jAF3"><ref>‡</ref>
<p>
Materials Sciences Division, Lawrence Berkeley National Laboratory.</p>
</note>
<note place="foot" n="ac034482jAF4"><ref>§</ref>
<p>
Present address: Lawrence Livermore National Laboratory,
P.O. Box 808,
L-415, Livermore, CA 94551.</p>
</note>
<affiliation role="corresp"> To whom correspondence should be sent. E-mails: gerion1@llnl.gov (D.G.); f_chen@lbl.gov (F.C.).</affiliation>
</author>
<author xml:id="author-0001" role="corresp"><persName><surname>Chen</surname>
<forename type="first">Fanqing</forename>
</persName>
<note place="foot" n="ac034482jAF5"><ref>‖</ref>
<p>
Life Sciences Division, Lawrence Berkeley National Laboratory.</p>
</note>
<affiliation role="corresp"> To whom correspondence should be sent. E-mails: gerion1@llnl.gov (D.G.); f_chen@lbl.gov (F.C.).</affiliation>
</author>
<author xml:id="author-0002"><persName><surname>Kannan</surname>
<forename type="first">Balaji</forename>
</persName>
<affiliation><orgName type="institution">Department of Chemistry and Department of Mechanical Engineering</orgName>
<orgName type="institution">University of California</orgName>
<address><addrLine>Berkeley</addrLine>
<addrLine>California 94720</addrLine>
<addrLine>and Materials Sciences Division and Life Sciences Division</addrLine>
<addrLine>Lawrence Berkeley National Laboratory</addrLine>
<addrLine>Berkeley</addrLine>
<addrLine>California 94720</addrLine>
</address>
</affiliation>
<note place="foot" n="ac034482jAF6"><ref>⊥</ref>
<p>
Department of Mechanical Engineering, University of California.</p>
</note>
</author>
<author xml:id="author-0003"><persName><surname>Fu</surname>
<forename type="first">Aihua</forename>
</persName>
<affiliation><orgName type="institution">Department of Chemistry and Department of Mechanical Engineering</orgName>
<orgName type="institution">University of California</orgName>
<address><addrLine>Berkeley</addrLine>
<addrLine>California 94720</addrLine>
<addrLine>and Materials Sciences Division and Life Sciences Division</addrLine>
<addrLine>Lawrence Berkeley National Laboratory</addrLine>
<addrLine>Berkeley</addrLine>
<addrLine>California 94720</addrLine>
</address>
</affiliation>
<note place="foot" n="ac034482jAF2"><ref>†</ref>
<p>
Department of Chemistry, University of California.</p>
</note>
</author>
<author xml:id="author-0004"><persName><surname>Parak</surname>
<forename type="first">Wolfgang J.</forename>
</persName>
<note place="foot" n="ac034482jAF2"><ref>†</ref>
<p>
Department of Chemistry, University of California.</p>
</note>
<note place="foot" n="ac034482jAF3"><ref>‡</ref>
<p>
Materials Sciences Division, Lawrence Berkeley National Laboratory.</p>
</note>
</author>
<author xml:id="author-0005"><persName><surname>Chen</surname>
<forename type="first">David J.</forename>
</persName>
<affiliation><orgName type="institution">Department of Chemistry and Department of Mechanical Engineering</orgName>
<orgName type="institution">University of California</orgName>
<address><addrLine>Berkeley</addrLine>
<addrLine>California 94720</addrLine>
<addrLine>and Materials Sciences Division and Life Sciences Division</addrLine>
<addrLine>Lawrence Berkeley National Laboratory</addrLine>
<addrLine>Berkeley</addrLine>
<addrLine>California 94720</addrLine>
</address>
</affiliation>
<note place="foot" n="ac034482jAF5"><ref>‖</ref>
<p>
Life Sciences Division, Lawrence Berkeley National Laboratory.</p>
</note>
</author>
<author xml:id="author-0006"><persName><surname>Majumdar</surname>
<forename type="first">Arunava</forename>
</persName>
<note place="foot" n="ac034482jAF3"><ref>‡</ref>
<p>
Materials Sciences Division, Lawrence Berkeley National Laboratory.</p>
</note>
<note place="foot" n="ac034482jAF6"><ref>⊥</ref>
<p>
Department of Mechanical Engineering, University of California.</p>
</note>
</author>
<author xml:id="author-0007"><persName><surname>Alivisatos</surname>
<forename type="first">A. Paul</forename>
</persName>
<note place="foot" n="ac034482jAF2"><ref>†</ref>
<p>
Department of Chemistry, University of California.</p>
</note>
<note place="foot" n="ac034482jAF3"><ref>‡</ref>
<p>
Materials Sciences Division, Lawrence Berkeley National Laboratory.</p>
</note>
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<profileDesc><abstract><p>We report two cDNA microarray-based
applications of
DNA−nanocrystal conjugates, single-nucleotide polymorphism
(SNP) and multiallele detections, using a commercial scanner and two
sets of nanocrystals with orthogonal emissions. We focus on SNP mutation
detection
in the human p53 tumor suppressor gene, which has
been found to be mutated in more than 50% of the known
human cancers. DNA−nanocrystal conjugates are able to
detect both SNP and single-base deletion at room temperature within
minutes, with true-to-false signal ratios
above 10. We also demonstrate microarray-based multiallele detection,
using hybridization of multicolor nanocrystals conjugated to two sequences
specific for the
hepatitis B and hepatitis C virus, two common viral
pathogens that inflict more than 10% of the population in
the developing countries worldwide. The simultaneous
detection of multiple genetic markers with microarrays
and DNA−nanocrystal conjugates has no precedent and
suggests the possibility of detecting an even greater
number of bacterial or viral pathogens simultaneously.
</p>
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<title-group><article-title>Room-Temperature
Single-Nucleotide
Polymorphism and Multiallele DNA Detection Using
Fluorescent Nanocrystals and Microarrays</article-title>
</title-group>
<contrib-group><contrib contrib-type="author" corresp="yes" xlink:type="simple"><name name-style="western"><surname>Gerion</surname>
<given-names>Daniele</given-names>
</name>
<xref rid="ac034482jAF1">*</xref>
<xref rid="ac034482jAF2"><sup>†</sup>
</xref>
<xref rid="ac034482jAF3"><sup>‡</sup>
</xref>
<xref rid="ac034482jAF4"><sup>§</sup>
</xref>
</contrib>
<contrib contrib-type="author" corresp="yes" xlink:type="simple"><name name-style="western"><surname>Chen</surname>
<given-names>Fanqing</given-names>
</name>
<xref rid="ac034482jAF1">*</xref>
<xref rid="ac034482jAF5"><sup>‖</sup>
</xref>
</contrib>
<contrib contrib-type="author" xlink:type="simple"><name name-style="western"><surname>Kannan</surname>
<given-names>Balaji</given-names>
</name>
<xref rid="ac034482jAF6"><sup>⊥</sup>
</xref>
</contrib>
<contrib contrib-type="author" xlink:type="simple"><name name-style="western"><surname>Fu</surname>
<given-names>Aihua</given-names>
</name>
<xref rid="ac034482jAF2"><sup>†</sup>
</xref>
</contrib>
<contrib contrib-type="author" xlink:type="simple"><name name-style="western"><surname>Parak</surname>
<given-names>Wolfgang J.</given-names>
</name>
<xref rid="ac034482jAF2"><sup>†</sup>
</xref>
<xref rid="ac034482jAF3"><sup>‡</sup>
</xref>
</contrib>
<contrib contrib-type="author" xlink:type="simple"><name name-style="western"><surname>Chen</surname>
<given-names>David J.</given-names>
</name>
<xref rid="ac034482jAF5"><sup>‖</sup>
</xref>
</contrib>
<contrib contrib-type="author" xlink:type="simple"><name name-style="western"><surname>Majumdar</surname>
<given-names>Arunava</given-names>
</name>
<xref rid="ac034482jAF3"><sup>‡</sup>
</xref>
<xref rid="ac034482jAF6"><sup>⊥</sup>
</xref>
</contrib>
<contrib contrib-type="author" xlink:type="simple"><name name-style="western"><surname>Alivisatos</surname>
<given-names>A. Paul</given-names>
</name>
<xref rid="ac034482jAF2"><sup>†</sup>
</xref>
<xref rid="ac034482jAF3"><sup>‡</sup>
</xref>
</contrib>
<aff>Department
of Chemistry and Department of Mechanical Engineering, University
of California, Berkeley, California 94720,
and Materials Sciences Division and Life Sciences Division, Lawrence
Berkeley National Laboratory,
Berkeley, California 94720
</aff>
</contrib-group>
<author-notes><corresp id="ac034482jAF1">
To whom correspondence should be sent. E-mails: gerion1@llnl.gov
(D.G.);
f_chen@lbl.gov (F.C.).</corresp>
<fn id="ac034482jAF2"><label>†</label>
<p>
Department of Chemistry, University of California.</p>
</fn>
<fn id="ac034482jAF3"><label>‡</label>
<p>
Materials Sciences Division, Lawrence Berkeley National Laboratory.</p>
</fn>
<fn id="ac034482jAF4"><label>§</label>
<p>
Present address: Lawrence Livermore National Laboratory,
P.O. Box 808,
L-415, Livermore, CA 94551.</p>
</fn>
<fn id="ac034482jAF5"><label>‖</label>
<p>
Life Sciences Division, Lawrence Berkeley National Laboratory.</p>
</fn>
<fn id="ac034482jAF6"><label>⊥</label>
<p>
Department of Mechanical Engineering, University of California.</p>
</fn>
</author-notes>
<pub-date date-type="pub" publication-format="electronic"><day>20</day>
<month>08</month>
<year>2003</year>
</pub-date>
<pub-date date-type="issue-pub"><day>15</day>
<month>09</month>
<year>2003</year>
</pub-date>
<volume>75</volume>
<issue>18</issue>
<fpage>4766</fpage>
<lpage>4772</lpage>
<supplementary-material xlink:href="ac034482jsi20030702_060229.pdf" position="float" xlink:type="simple" orientation="portrait"></supplementary-material>
<history><date date-type="received"><day>07</day>
<month>05</month>
<year>2003</year>
</date>
<date date-type="asap"><day>20</day>
<month>08</month>
<year>2003</year>
</date>
</history>
<permissions><copyright-statement>Copyright © 2003 American Chemical Society</copyright-statement>
<copyright-year>2003</copyright-year>
<copyright-holder>American Chemical Society</copyright-holder>
</permissions>
<abstract><p>We report two cDNA microarray-based
applications of
DNA−nanocrystal conjugates, single-nucleotide polymorphism
(SNP) and multiallele detections, using a commercial scanner and two
sets of nanocrystals with orthogonal emissions. We focus on SNP mutation
detection
in the human p53 tumor suppressor gene, which has
been found to be mutated in more than 50% of the known
human cancers. DNA−nanocrystal conjugates are able to
detect both SNP and single-base deletion at room temperature within
minutes, with true-to-false signal ratios
above 10. We also demonstrate microarray-based multiallele detection,
using hybridization of multicolor nanocrystals conjugated to two sequences
specific for the
hepatitis B and hepatitis C virus, two common viral
pathogens that inflict more than 10% of the population in
the developing countries worldwide. The simultaneous
detection of multiple genetic markers with microarrays
and DNA−nanocrystal conjugates has no precedent and
suggests the possibility of detecting an even greater
number of bacterial or viral pathogens simultaneously.
</p>
</abstract>
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<body><sec id="d7e221"><title></title>
<p>Genotyping of human diseases and detection of pathological
microorganisms, such as infectious virus and biowarfare reagents,
depend mainly on the molecular detection of nucleic acids or
proteins. The advent of microarray and microanalytical device
analysis has provided unprecedented high throughput, convenience,
capability, and opportunities for genotyping and microorganism detection.<named-content content-type="bibref-group" xlink:type="simple"><xref rid="ac034482jb00001" ref-type="bibr"></xref>
,<xref rid="ac034482jb00002" ref-type="bibr"></xref>
</named-content>
Many detection assays are performed with
reaction volumes in the microliter or nanoliter range. Detection
of biological samples at such extremely low amounts is a major
challenge for clinical diagnosis and detection. Most biochemical
assays require secondary detection of a label, because biomolecules
lack intrinsic properties that are useful for direct high-sensitivity
detection. Most assays are based on hybridization or
specific interaction, which exploit the high binding specificity of
nucleic acids and polypeptides. Label detection is a key determinant
of sensitivity. The continuing quest for improved labels has
produced a series of markers, including new fluorophores such
as cyanine dyes, enzymes, and particles such as phosphors and
nanocrystals<named-content content-type="bibref-group" xlink:type="simple"><xref rid="ac034482jb00003" ref-type="bibr"></xref>
,<xref rid="ac034482jb00004" ref-type="bibr"></xref>
</named-content>
(for further reviews,
see also ref <xref specific-use="ref-style=base-text" rid="ac034482jb00005" ref-type="bibr"></xref>
).
</p>
<p>Among the most commonly used labels in biological diagnostics
are organic fluorescent dyes. But while new types of fluorescent microscopes
and techniques have pushed the resolution
well below the diffraction limit of light,<xref rid="ac034482jb00006" ref-type="bibr"></xref>
fluorescent probes have
not followed the same impressive evolution trend. Organic dyes
still suffer from notorious limitations such as photobleaching and
discrete excitation bands that preclude their use in many applications.
An alternative technology, based on inorganic semiconductor nanocrystals,
might overcome some of these limitations. These
materials are crystals made of semiconductor CdSe/ZnS core/shell structures
and have a diameter of a few nanometers.<xref rid="ac034482jb00007" ref-type="bibr"></xref>
Nanocrystals possess many properties that make them attractive
for biolabeling: broad excitation spectrum but narrow and
precise
tunable emission<sup>6</sup>
by simply varying the size of the nanoparticles
(see Figure <xref rid="ac034482jf00001"></xref>
), negligible photobleaching,<xref rid="ac034482jb00008" ref-type="bibr"></xref>
fairly high quantum
yields, stability, and negligible phototoxicity.<xref rid="ac034482jb00009" ref-type="bibr"></xref>
Recent synthetic
progress has allowed the conjugation of nanocrystals to a wide
variety of biomolecules such as DNA,<named-content content-type="bibref-group" xlink:type="simple"><xref rid="ac034482jb00010" ref-type="bibr"></xref>
−<xref rid="ac034482jb00011" specific-use="suppress-in-print" ref-type="bibr"></xref>
<xref rid="ac034482jb00012" ref-type="bibr"></xref>
</named-content>
proteins,<named-content content-type="bibref-group" xlink:type="simple"><xref rid="ac034482jb00013" ref-type="bibr"></xref>
,<xref rid="ac034482jb00014" ref-type="bibr"></xref>
</named-content>
antibodies,<named-content content-type="bibref-group" xlink:type="simple"><xref rid="ac034482jb00015" ref-type="bibr"></xref>
,<xref rid="ac034482jb00016" ref-type="bibr"></xref>
</named-content>
short peptides,<xref rid="ac034482jb00017" ref-type="bibr"></xref>
or neurotransmitters<xref rid="ac034482jb00018" ref-type="bibr"></xref>
and their targeting
to their receptor sites. Until now though, biological applications
of these biomodified nanocrystals are still in their infancy because
of the delicate process of synthesizing and manipulating them.
Ongoing improvements however have already led to in vivo
targeting, and there is great hope of using nanocrystal technology
in more complex biological problems.
<fig id="ac034482jf00001" position="float" orientation="portrait"><label>1</label>
<caption><p>Comparison of the excitation and fluorescence of Cy3−oligos,
Cy5−oligos (19-mer), and yellow and red DNA−nanocrystals
(not to scale). Note that yellow and red nanocrystals are the same
material but differ only in the size of the nanocrystal core. The
dyes spectra are adapted from the Amersham Bioscience website. The
asterisks in the excitation spectra indicate the wavelengths of excitation
used in this study (532 and 634 nm). Emission of Cy3−oligos
and Cy5−oligos are 566 and 668 nm, respectively. Yellow C5−nanocrystals
(566 nm, fwhm ∼32 nm) and red B3−nanocrystals (644 nm,
fwhm ∼34 nm) are used in this study. Excitation at 408 nm with
a blue laser diode (blue arrow) is a better choice to excite nanocrystals.</p>
</caption>
<graphic xlink:href="ac034482jf00001.tif" position="float" xlink:type="simple" orientation="portrait"></graphic>
</fig>
</p>
<p>One of these applications is DNA microarrays. Advances in
this technology have allowed high-throughput gene expression
profiling and nucleotide polymorphism detection, which have
profound implications in medical sciences. Indeed, small variations
in the genome influence our predisposition to diseases, such as
cancer, and congenital genetic diseases.<named-content content-type="bibref-group" xlink:type="simple"><xref rid="ac034482jb00019" ref-type="bibr"></xref>
−<xref rid="ac034482jb00020" specific-use="suppress-in-print" ref-type="bibr"></xref>
<xref rid="ac034482jb00021" ref-type="bibr"></xref>
</named-content>
The identification of
these variations has become a major issue in genetics since they
are believed to be major determinants to disease onset, progression,
and clinical prognosis. Also, such small variations are
extremely useful in categorization of infectious disease pathogens,
such as the highly mutable HIV,<xref rid="ac034482jb00022" ref-type="bibr"></xref>
and the classification of
biowarfare reagents, such as anthrax.<named-content content-type="bibref-group" xlink:type="simple"><xref rid="ac034482jb00023" ref-type="bibr"></xref>
,<xref rid="ac034482jb00024" ref-type="bibr"></xref>
</named-content>
These variations include
insertions, deletions, rearrangements of the sequences, or alterations
of a single base pair also called single-nucleotide polymorphism (SNP).
Although DNA microarray technology using conventional organic fluorescent
dyes is extremely powerful in
detecting multiple genetic markers with high throughput, it
presents a certain number of drawbacks, such as high incubation
temperature requiring special ovens, overnight incubation time,
and possible leakage of a peculiar fluorescent color in another
channel caused by overlapping spectra.<xref rid="ac034482jb00025" ref-type="bibr"></xref>
Each of these shortcomings limits the potential of DNA
microarrays.
</p>
<p>In recent years, DNA−nanocrystal conjugates have emerged
as efficient probes, capable of hybridizing to the complement
strands and generating up to five perfectly orthogonal channels
of detection within the visible range.<xref rid="ac034482jb00006" ref-type="bibr"></xref>
Combined with their high
sensitivity, DNA−nanocrystal probes are ideal candidates for
DNA
microarrays with an even higher throughput. So far however,
DNA−nanocrystal probes have been tested only in proof-of-concept
experiments where they were exposed to either their fully
complement sequence or a fully random one as a control.<named-content content-type="bibref-group" xlink:type="simple"><xref rid="ac034482jb00011" ref-type="bibr"></xref>
,<xref rid="ac034482jb00012" ref-type="bibr"></xref>
,<xref rid="ac034482jb00026" ref-type="bibr"></xref>
</named-content>
The possibility to pinpoint some genetic variations in the genome
remained purely theoretical.
</p>
<p>We report, in this article, the first study of DNA−nanocrystal
conjugates as efficient probes in SNP human genotyping and
pathogen detection using cDNA arrays. Specifically, we focus on
mutation detection in human oncogene p53, a tumor suppressor
gene, which has been found to be mutated in 50% of the known
cancers.<named-content content-type="bibref-group" xlink:type="simple"><xref rid="ac034482jb00027" ref-type="bibr"></xref>
−<xref rid="ac034482jb00028" specific-use="suppress-in-print" ref-type="bibr"></xref>
<xref rid="ac034482jb00029" ref-type="bibr"></xref>
</named-content>
Our model experiments
have used a SNP located at
amino acid residue 248, on exon 7 of p53, which is one of the
most common mutation hot spots in p53.<xref rid="ac034482jb00030" ref-type="bibr"></xref>
In addition, we also
demonstrate the feasibility of simultaneous detection of multiple
biological pathogens, by using hepatitis B virus (HBV) and
hepatitis C virus (HCV) as a widespread model system. The anti-HCV
and anti-HBV positive rate in the general population or
healthy blood donors is 2−10% worldwide.<named-content content-type="bibref-group" xlink:type="simple"><xref rid="ac034482jb00031" ref-type="bibr"></xref>
,<xref rid="ac034482jb00032" ref-type="bibr"></xref>
</named-content>
With our detection
system based on nanocrystal-conjugated probe DNA and microarray, we
have been successful in identifying multiplexed HBV and
HCV targets. The potential single-molecule detection sensitivity
of nanocrystals could enable the direct detection of large numbers
(hundreds or thousands) of pathogens at the same time on a
microarray, thus serving as a replacement technology for PCR.
</p>
<p>In all our experiments, hybridization of DNA−nanocrystal
probes is extremely fast (∼10 min) and is performed at room
temperature. Signal-to-background ratios of >100 can be readily
obtained in conditions that discriminate down to three base
mismatches in a 25-mer sequence. No false positive signals are
observed when formamide is used in the hybridization solution.
We also pinpoint more stringent conditions for hybridization that
lead to SNP detection with true-to-false signal ratios over 10. All
this is achieved with a commercial scanner that is not optimized
for nanocrystal detection. Overall, our results might be a first step
toward the use of nanocrystal probes for an ultrafast detection of
a great number of viral or bacterial pathogens simultaneously.
</p>
</sec>
<sec id="d7e330"><title>Experimental Section</title>
<p><bold>Array Layout.</bold>
Two sets of arrays are produced for this study.
</p>
<p><bold>Set 1.</bold>
10 rows of 6 identical spots each row.
</p>
<p>Row 1. cDp53a: 5‘-aggatgggcctcAggttcatgccgc-3‘.
</p>
<p>Row 2. cDp53c: 5‘-aggatgggcctcCggttcatgccgc-3‘.
</p>
<p>Row 3. cDp53g: 5‘-aggatgggcctcGggttcatgccgc-3‘.
</p>
<p>Row 4. cDp53t: 5‘-aggatgggcctcTggttcatgccgc-3‘.
</p>
<p>Row 5. cDp53x: 5‘-aggatgggcctc-ggttcatgccgc-3‘.
</p>
<p>Row 6. Dp53g, which is the reverse complement of cDp53c
with a G in the middle place: 5‘gcggcatgaaccGgaggcccatcct-3‘.
</p>
<p>Row 7. cB3 is the reverse complement to portion of the HBV
genomic sequence (hepatitis B virus sequence, Genebank accession number <ext-link ext-link-type="gen" xlink:href="AF461043" xlink:type="simple">AF461043</ext-link>
):5‘-cgagattgagatcttctgcgacgcggcgattgagaccttcgtctgcgaggcgagggagttcttcttctag-3‘.
</p>
<p>Row 8. cC5 is the reverse complement to portion of the HCV
genomic sequence (hepatitis C virus sequence, Genebank accession number <ext-link ext-link-type="gen" xlink:href="D90208" xlink:type="simple">D90208</ext-link>
): 5‘-ctagacgctttctgcgtgaagacagtagttcctcacaggggagtgatctatggtggagtgtcgcccccaa-3‘.
</p>
<p>Row 9. B3, which is the reverse complement of cB3:
5‘-ctagaagaagaactccctcgcctcgcagacgaaggtctcaatcgccgcgtcgcagaagatctcaatctcg-3‘.
</p>
<p>Row 10. C5, which is the reverse complement of cC5:
5‘-ttgggggcga cactccacca tagatcactc ccctgtgagg aactactgtc ttcacgcaga
aagcgtctag ccatggcgtt-3‘.
</p>
<p><bold>Set 2.</bold>
DNA with mismatches for SNP detection.
10 rows, 3
spots for each DNA, 2 types of DNA per row.
</p>
<p>Row 1. cDp53t and 5bpm@end.
</p>
<p>Row 2. 10 bpm@end and 15 bpm@end.
</p>
<p>Row 3. 20bpm@end and 3bpm@center.
</p>
<p>Row 4. 7bpm@center and 11bpm@center.
</p>
<p>Row 5. 15 bpm@center and blank.
</p>
<p>Row 6. cDp53a and cDp53c.
</p>
<p>Row 7. cDp53g and cDp53t.
</p>
<p>Row 8. cDp53x and Dp53t.
</p>
<p>Row 9. cC5 (HCV capture probe) and cB3 (HBV capture
probe).
</p>
<p>Row 10. C5 and B3.
</p>
<p>Sequences from 5‘ to 3‘ end.
</p>
<p>cDp53a: aggatgggcctcaggttcatgcgcgc.
</p>
<p>5bpm@end: aggatgggcctcaggttcatATATA.
</p>
<p>10bpm@end: aggatgggcctcaggCATCCATATA.
</p>
<p>15bpm@end: aggatgggccCACATCATCCATATA.
</p>
<p>20bpm@end: aggatAATACCACATCATCCATATA.
</p>
<p>3bpm@center: aggatgggcctATAgttcatgcgcgc.
</p>
<p>7bpm@center: aggatgggccAATACCAcatgcgcgc.
</p>
<p>11bpm@center: aggatgggCTAATACCAACtgcgcgc.
</p>
<p>15bpm@center: aggatgCACTAATACCAACCAcgcgc.
</p>
<p><bold>Oligonucleotide Synthesis.</bold>
The following sequences
were
custom-synthesized at Operon. The wild-type 25-mer encompassing the
amino acid residue 248 on exon 7 of the human p53
oncogene is called cDp53a. The nucleotide in the middle of the
cDp53a sequence is mutated into either A, C, G, or T or deleted
to generate oligonucleotides cDp53a, cDp53c, cDp53g, cDp53t,
and cDp53x. The 5‘s of the above sequences are modified with
a
C-12 amino linker (Glen Research). cDp53t (5‘-gcggcatgaaccTgaggcccatcct-3‘)
sequence, which is from the wild-type p53 and
complements to Dp53a sequences, is conjugated to nanocrystals.
</p>
<p>We also synthesized four sequences corresponding to genomic
sequences in hepatitis virus B and hepatitis virus C. (1) cB3 is
reverse complement to a portion of the HBV genomic sequence
(hepatitis B virus sequence, Genebank accession number
<ext-link ext-link-type="gen" xlink:href="AF461043" xlink:type="simple">AF461043</ext-link>
). cB3 will capture HBV genomic DNA. (2) cC5 is
reverse complement to a portion of the HCV genomic sequence
(hepatitis C virus sequence, Genebank accession number <ext-link ext-link-type="gen" xlink:href="D90208" xlink:type="simple">D90208</ext-link>
).
cC5 will capture the HCV genomic DNA. (3) B3, which is a reverse
complement of cB3, is conjugated to nanocrystals and can be
specifically captured by cB3 on the microarray. (4) C5, which is
a reverse complement of cC5 is conjugated to the nanocrystals
and can be specifically captured by cC5 on the microarray. The
four hepatitis B/C virus related sequences are also amino-modified
at the 5‘ end of the oligonucleotides.
</p>
<p><bold>Fabrication of Microarray.</bold>
The amino-modified
cDp53a,c,g,t,x,
Dp53t, and other sequences were diluted to 25 μM in a 1:1 mixture
of 3× SSC buffer and microprinting buffer from Telechem. The
printing of the microarray has been carried out by a state-of-art
high-speed microarrayer OmniGrid II from GeneMachine, with
SMP3 Stealth split pins from Telechem. Pin speed is 2 cm/s, and
dwell time on slide is 25 ms. Humidity is maintained at 45%.
Sonication and washing are carried out between samples to avoid
sample carryover. Glass slides with an aldehyde-activated surface
(Superaldehyde slide from Telechem) were used for the microprinting
of the microarrays. After printing, the DNA with amino
end modification is allowed to react to the aldehyde group
overnight to form a covalent link between the DNA probe and
the microarray substrate surface before being subjected to further
processing.
</p>
<p><bold>Processing of the DNA Array Slide. </bold>
The arrays
are hydrated
for ∼15 min in a humid atmosphere. They are washed twice for
2 min in 0.1% Tween 20 in water, twice 1 min in ddH<sub>2</sub>
O.
Then the
Schiff base is reduced in (25% methanol in 50 mM phosphate
buffer) and 5 g/L sodium borohydride. The slides are washed
again twice for 1 min in 0.1% Tween 20 in water and 1-min final
wash in ddH<sub>2</sub>
O. The slides are dried by centrifugation for
1 min
at 500<italic toggle="yes">g</italic>
. A rubber seal defines eight reaction compartments
of ∼40
μL each.
</p>
<p><bold>DNA</bold>
<bold>−</bold>
<bold>Nanocrystal Synthesis
and Purification.</bold>
Core/shell
CdSe/ZnS nanocrystals, with core size of ∼2.5 (yellow emission)
and ∼4 nm (red emission), are embedded in a silica shell, which
confers them high stability in high-salt buffers and in a wide
ranges of pHs. The polymerized shell can be tailored to have any
electric charge and to contain a wide array of functional groups
(thiols, amines, carboxyls). The resulting nanoparticles have ∼10
nm in diameter and resemble very much globular proteins. Single-stranded
DNA of any length can be covalently linked to nanoparticles by standard
bioconjugation techniques. Each nanoparticle
is likely to carry a few DNA. The synthesis of these biolabels has
been extensively described elsewhere.<named-content content-type="bibref-group" xlink:type="simple"><xref rid="ac034482jb00008" ref-type="bibr"></xref>
,<xref rid="ac034482jb00010" ref-type="bibr"></xref>
</named-content>
The bioconjugates are
purified from excess of free DNA by electrodialysis in 5−10
mM
phosphate buffer, pH ∼7.3, in a 200-μL Teflon chamber.
The
chamber is closed by two regenerated cellulose membrane of
100 000 MWCO. We apply a voltage of 3−5 V/cm, <italic toggle="yes">I</italic>
∼10−15 mA
for a period of 45 min to ensure a complete removal of free DNA.
The DNA−nanocrystal conjugates are collected from the dialysis
chamber, diluted to 500 μL, and concentrated in a Microcon 100
down to ∼50 μL. This procedure is repeated twice. The
concentration of the DNA−nanocrystal solution is checked by
UV−visible
spectroscopy. We consider extinction coefficients of 3 × 10<sup>5</sup>
mol<sup>-1</sup>
cm<sup>-1</sup>
at 555 nm for yellow- (emission 565 nm) and 9 ×
10<sup>5</sup>
mol<sup>-1</sup>
cm<sup>-1</sup>
at 630 nm for red-emitting (emission 644 nm) nanocrystals,
respectively. The quantum yields are estimated at ∼20 and 5%
for yellow and red samples, respectively. Purified samples are
stable for months. However, hybridization efficiency was observed
to drop after a few days, a fact attributed to wrapping of the oligo
around the particle. We recover the initial hybridization efficiency
by applying a small voltage (∼3−5 V/cm) to the DNA−nanocrystal
conjugates. This step is performed in the electrodialysis
chamber with the same conditions described above.
</p>
<p><bold>DNA</bold>
<bold>−</bold>
<bold>Nanocrystal Hybridization
and Washing. </bold>
DNA−nanocrystal conjugates are diluted
to the desired concentration
for hybridization. All reactions, including the incubation, are
performed at room temperature, on a rocking platform. Typically,
we use 40 μL of nanocrystal solution in 10 mM potassium
phosphate buffer, 30 mM NaCl, 0.1% Tween 20, and 30−70%
formamide. Concentrations of nanocrystals vary from a few
nanomolar to 1 μM. Incubation time is typically 1 h but can
be as
low as ∼10 min. The arrays are then washed for 30 s in 10 mM
phosphate buffer, 300 mM NaCl followed by 1 min in pure water.
The slides are then dried by a brief centrifugation, after which
they are ready for imaging.
</p>
<p><bold>Imaging and Analysis. </bold>
Fluorescence from the arrays
is
measured with a commercial objective-scanning confocal Axon
3000i scanner (Axon Instruments), which has two excitation laser
diodes (532 and 635 nm). Two orthogonal detection channels, for
yellow and red fluorescence, respectively, are detected with two
distinct photomultipliers. Both yellow and red channels are saved
as 8-bit jpg images independently. A homemade Matlab routine
allows the processing of the images to calculate the histogram of
each blots. The average fluorescence intensity for each oligonucleotide
printed on the slide is determined and then normalized
with respect to the intensity of the spots corresponding to the
perfect complement sequence.
</p>
</sec>
<sec id="d7e488"><title>Results</title>
<p><bold>Multicolor
DNA Array with Mild Hybridization Conditions.
</bold>
The first set of experiments intends to probe the possibility
of
using DNA−nanocrystal conjugates and microarrays for hepatitis
genotyping and detection in the presence of a background of
human genes. For this purpose, we choose two particular
sequences specific to HBV and HCV and a third sequence specific
to human oncogene p53. We call them B3, C5, and Dp53,
respectively. B3 and C5 are 75-mers corresponding to portions of
the HBV and HCV, respectively. Dp53 is a 25-mer, covering the
25 nucleotides flanking codon 248 in gene p53; its 13th (central)
base is a T, and hence, we will call it Dp53t. These three sequences
are independently conjugated to the nanocrystals and their
complements (cB3, cC5, cDp53a, respectivelycDp53a means the
oligonucleotide sequence is complement to Dp53 and that its 13th
base is an “A”) are printed on the DNA array.
</p>
<p>These sequences allow us to monitor the specificity of
hybridization and the possibility of recording nonoverlapping
(orthogonal) channels using an Axon microarray scanner (Axon
Instruments). Previous experiments using gold patterns suggest
that 10 mM phosphate buffer, 50 mM NaCl, and 0.1% Tween 20,
pH ∼7.3, is a reasonable incubation buffer.<xref rid="ac034482jb00026" ref-type="bibr"></xref>
Using these conditions, we target
yellow Dp53t−nanocrystal conjugates to cDp53a
(Figure <xref rid="ac034482jf00002"></xref>
, panel a). In panel b,
red B3-nanocrystals are targeted
to cB3, while in panel c, yellow C5−nanocrystals are targeted
to
cC5. Panel d is an overlay of the previous panels.
<fig id="ac034482jf00002" position="float" orientation="portrait"><label>2</label>
<caption><p>Multicolor targeting of SNP mutations in human oncogene p53 (rows
1−6) and of human hepatitis B and C viruses (rows 7−10)
with different DNA−nanocrystal probes. The arrays are printed
in 10 rows of 6 identical blots. The five top rows are 25-mers related
to the sequence encompassing the amino acid residue 248 on exon 7
of the human oncogene p53 called Dp53. The nucleotide in the middle
of the cDp53 is mutated in either A, C, G, T or deleted X and generates
cDp53a, cDp53c, cDp53g, cDp53t, or cDp53x, respectively. Row 6 contains
Dp53g, the complement sequence of cDp53c. Row 7 is cB3, the reverse
complement to a portion of the 3‘ end of HBV. Row 8 is cC5,
the reverse complement to a portion of the 3‘ end of HCV. Row
9 is B3 the complement of the sequence cB3, and row 10 is C5, the
complement of the sequence cC5.a<bold>)
</bold>
In panel a, yellow Dp53g−nanocrystals are targeted only
toward mutations in human oncogene p53, but SNP detection is not achieved.
In
panel b, red B3−nanocrystals are targeted toward the hepatitis
B virus sequence. In panel c, yellow C5−nanocrystals are targeted
only toward
the hepatitis C virus sequence. Panel d is an overlay of panels a−c.
Note that in both red and yellow channels, signal-to-noise ratio is
>100,
and no cross-talk is observed. Incubation time is ∼1 h. Each
spot is ∼100 μm.</p>
</caption>
<graphic xlink:href="ac034482jf00002.tif" position="float" xlink:type="simple" orientation="portrait"></graphic>
</fig>
</p>
<p>Due to their relatively narrow emission (Figure <xref rid="ac034482jf00001"></xref>
), yellow
nanocrystals are never seen in the red channel and red nanocrystals
do not appear in the yellow channel of the Axon scanner.
It is therefore clear from Figure <xref rid="ac034482jf00002"></xref>
that DNA−nanocrystals are
targeted to their complement strands without cross-talks. These
perfectly orthogonal channels are detected with signal to surrounding
background ratios, also called signal-to-noise ratio, as
high as 150. For a given set of hybridization conditions but for
different slides, the intensities of the spot signals are constant
within 15−20%.
</p>
<p>Although C5 and B3 are quite long (75-mers), they do not bear
any homologies longer than seven bases with themselves or with
cDp53a. On the other hand, cDp53a and its single-base mutants
show up with about the same intensity, and therefore, SNP
detection is not reached with such mild hybridization conditions.
Different washing stringencies, including heat, do not yield to SNP
detection either. This first set of experiments tells us that DNA−nanocrystal
probes can easily discriminate genotypes with low
homologies, such as the three we used here. However, a wider
application of these probes in multiplexed genotyping would
require the possibility to detect SNP.
</p>
<p><bold>Conditions To Reach SNP Selectivity: Effects
of Formamide and Salt </bold>
<bold>Concentrations. </bold>
To precisely
pinpoint the
conditions for SNP detections, we focus on the sequence specific
to human oncogene p53, Dp53t, and its complement cDp53a. We
print on the array 13 sequences of 25 bases homologous to cDp53a
but with an increasing number of base mismatches either at the
center or at the 3‘ end. We also print on the array the sequences
specific to HBV and HCV as background genes, but we will not
consider them hereafter since they never yield any signal. Figure
<xref rid="ac034482jf00003"></xref>
illustrates schematically the
structure of the sequences homologous to cDp53a printed on the slide
and indicates where the
mismatches are located. The right part of Figure <xref rid="ac034482jf00003"></xref>
also shows
that Dp53t−nanocrystal probes achieve SNP detection. The ratio
of the signal from the complement spot to the signal from the
SNP spots, also called the true-to-false signal ratio, is ∼12.
Notice
here that all noncomplement sequences give a signal that reaches
the noise floor. The key strategy to achieve such selectivity is to
use unfavorable conditions for hybridization so that only DNA−nanoparticle
conjugates with a perfect match have a chance to
hybridize. As a result, although the overall signal is lower, the
discrimination between the perfect match sequence and the SNP
sequences is higher.
<fig id="ac034482jf00003" position="float" orientation="portrait"><label>3</label>
<caption><p>Geometry for probing SNP in human oncogene p53. On the left, the
schematic of the 25-mer sequences used to probe SNP detection. From
the perfect complement sequence, cDp53a, we introduce more and more
base mismatches either at the center or at the 3‘ end. The
sequences of all oligonucleotides are given in the Experimental Section.
On the right side, a composite image of the array after 1 h of hybridization
in 70% formamide, 30 mM NaCl, 0.1% Tween 20. The only signal visible
comes from the position of the perfect complement, cDp53a. The histogram
for each sequence shows that signal-to-background ratio is ∼12.
Incubation time is ∼1 h, but we also observe SNP detection
for incubation times as short as 10 min. Each spot is ∼100
μm.</p>
</caption>
<graphic xlink:href="ac034482jf00003.tif" position="float" xlink:type="simple" orientation="portrait"></graphic>
</fig>
</p>
<p>DNA arrays are ideal to explore these unfavorable conditions.
Low-salt concentrations only, i.e., 2−10 mM NaCl, do not yield
to
SNP detection. A combination of low-salt concentrations and the
presence of denaturing organic solvents such as formamide are
necessary, much like the case of dye-labeled oligos. We thus
performed a two-dimensional study varying independently the salt
concentration and the formamide content in the hybridization
process. We monitored the intensity of the signal of each cDp53a
homologues on the array.
</p>
<p>Figure <xref rid="ac034482jf00004"></xref>
a summarizes the
effect of formamide content while
keeping 30 mM NaCl in the incubation buffer. With no formamide,
the perfect match sequence (column 1, cDp53a) and the sequences with
SNP homologues (columns 2−5) have nearly the
same intensity, arbitrarily set to 1 for the perfect match. In this
peculiar case, we observe a prominent false-positive signal from
column 6, corresponding to a mutant sequence with a three-base
mismatch in the center. All other sequences show a negligible
contribution. The signal from the three-base mismatch mutant
can be reduced by increasing the content of formamide in solution.
At 40% formamide in the incubation buffer, the intensity of the
three-base mismatch mutant is similar to the intensity of cDp53a,
but SNP detection is not yet reached. At 60% formamide, the false-positive
signal is totally suppressed and we observe SNP detection
with a true-to-false ratio of ∼2. At slightly higher formamide
content, we reach the peak of selectivity. Only the perfect match
sequence cDp53a gives a signal. All other mutant sequences are
totally black. The signal-to-noise ratio as well as the true-to-false
ratio is ∼12. A further increase in formamide content to 80%
does
not produce any hybridization at all.
<fig id="ac034482jf00004" position="float" orientation="portrait"><label>4</label>
<caption><p>Evolution of SNP detection in human p53 versus hybridization conditions.
We report the intensity of the signal on the spots of the DNA sequences
of Figure <xref rid="ac034482jf00002"></xref>
as a function of hybridization
conditions. In all these graphs, lane 1 is the perfect match sequence,
cDp53a. Its intensity is the reference and is set to 1. Lanes 2−13
correspond to the sequences described in Figure <xref rid="ac034482jf00002"></xref>
(i.e., 2 = cDp53c, ..., 13 = 20bpm@end).
Reducing stringencies, by either increasing the formamide concentration
or reducing the NaCl concentration, leads to SNP with a true-to-false
signal ratio of ∼12. Note how, with formamide in solution,
SNP is progressively lost as [NaCl] is increased. In this case, at
75 mM NaCl, only sequences with a single-base difference give a signal.
At 100 mM, the sequence with a three-base mismatch at the center (lane
6) give a signal ∼<sup>1</sup>
/<sub>3</sub>
of the complement
sequence.</p>
</caption>
<graphic xlink:href="ac034482jf00004.tif" position="float" xlink:type="simple" orientation="portrait"></graphic>
</fig>
</p>
<p>Similarly, for a fixed amount of formamide in solution (Figure
<xref rid="ac034482jf00004"></xref>
b), the hybridization gets less
and less selective as the salt
concentration is increased. At 70% formamide, 10 mM NaCl, no
hybridization occurs, while at 30 mM NaCl we observe the highest
selectivity. With increasing concentrations of NaCl, the signal-to-noise
ratio gets bigger, but SNP detection becomes weaker. Also,
there are no false positive signals in this case. At relatively high
salt concentration, i.e., 100 mM NaCl, SNP detection is almost
lost. A signal from the three-base mismatch mutant sequence
(column 6) appears with intensity ∼3 times lower than that
of
cDp53a.
</p>
<p><bold>Sensitivity of the Probes. </bold>
The high selectivity
of DNA−nanoparticle conjugates constitutes only one step for
their use in
cDNA microarray technology. A second aspect is related to the
sensitivity of the probe. We have performed titration experiments
by targeting DNA−nanoparticles to their complement sequence.
Figure <xref rid="ac034482jf00005"></xref>
illustrates the case of
yellow B3−nanoparticles targeted
to the reverse complement to a portion of the 3‘ end of HBV
genomic sequence. As indicated in the inset, a positive identification
of a printed sequence requires ∼2 nM concentration of DNA−nanoparticle
probes. In comparison, we can detect the same
sequence using about 10−50 pM Cy5-DNA. This comparison is
only indicative here since the sensitivity of nanocrystal probes
cannot be fully assessed with our setup because the Axon scanner
is only optimized for Cy3 and Cy5 dyes and not for nanocrystal
photophysics.
<fig id="ac034482jf00005" position="float" orientation="portrait"><label>5</label>
<caption><p>Titration curve for sensitivity determination. Yellow C5−nanocrystals
are hybridized at different concentration. The intensity of the signal
is measured as a function of the concentration of C5−nanoparticle
conjugates. We consider an extinction coefficient of 3 × 10<sup>5</sup>
M<sup>-1</sup>
cm<sup>-1</sup>
at 555 nm, the exciton peak
of the yellow C5−nanocrystals. Inset: At ∼4
nM, the hybridization signal is still clearly
visible with a signal-to-noise ratio of ∼2. Present resolution
is ∼2 nM,
but the true sensitivity of the nanocrystal technology cannot be fully
assessed with our nonoptimized Axon scanner. Incubation time is
∼1 h. Each spot is ∼100 μm.</p>
</caption>
<graphic xlink:href="ac034482jf00005.tif" position="float" xlink:type="simple" orientation="portrait"></graphic>
</fig>
</p>
</sec>
<sec id="d7e577"><title>Discussion</title>
<p>Our results show
that DNA−nanocrystal probes of different
colors are able to target hepatitis B and C genotypes in the
presence of a background of human genes, such as p53. Such
simultaneous detection of multiple pathogens has never been
reported before for microarray-based nanocrystal labeling. We see
potential applications in clinical diagnosis with an expanded
number of pathogens. With improvements of sensitivity down
toward the single-molecule detection level, where nanocrystals
are regularly studied, our method could have the potential to be
a replacement technology for PCR<bold>. </bold>
However, for that to
be
realized, a number of modifications will have to be implemented
as will be discussed further on. Indeed, nanocrystal technology
is attractive in many aspects. Even if the sequences specific to
HBV and HCV are G−C rich (∼68%), we do not observe significant
false-positive artifacts. We believe the same conclusion holds true
for any genotype, since G−C base pairing is stronger than A−T
and G−C content can reach 25−75% in bacterial genomes
but only
41% in the human genome.<xref rid="ac034482jb00033" ref-type="bibr"></xref>
Therefore,
the use of DNA−nanocrystal probes in cDNA microarrays suppresses
the issue of
cross-talks between detection channels. This constitutes a marked
improvement compared to certain sets of organic dyes commonly
used, especially for high-sensitivity applications such as gene
expression profiling. In all our experiments, we used yellow- and
red-emitting nanocrystals with emissions at 566 (fwhm ∼32 nm)
and 644 nm (fwhm ∼34 nm), respectively, and no leakage on the
opposite channel is ever observed (Figure <xref rid="ac034482jf00001"></xref>
). Moreover, due to
their low adsorption on glass, DNA−nanocrystal conjugates yield
fairly large signal-to-noise ratios over 100 that are reproducible
with different microarray slides.
</p>
<p>A further interesting aspect is the selectivity of the DNA−nanocrystal
conjugates. In fact, cDNA sequences with more than
3 base mismatches with DNA−nanoparticle probes do not produce
any signal in an extremely wide range of hybridization conditions.
The cDNA homologues that yield a measurable signal are the
full complement sequence, its SNP mutants, and the three-base
pair mutant. If no formamide is present during the incubation,
the three-base pair mutant sometimes gives the largest signal, a
signature of false-positive detection. This strong false-positive
signal appears in many different slides but its origin is still unclear.
A simple speculation may attribute it to the peculiar interaction
of the DNA and the nanoparticle favoring locally a non-Watson−Crick
base pairing. Anyway, the false positive signal can be
suppressed if formamide is added to the hybridization solution.
Above ∼40% formamide, the three-base pair mutant is never
observed and SNP detection becomes unambiguous. In optimal
conditions, SNP detection gives a true-to-false signal ratio of 12
and compares favorably with allele-specific hybridization using
dyes where the ratio is between 1 and 5.<named-content content-type="bibref-group" xlink:type="simple"><xref rid="ac034482jb00034" ref-type="bibr"></xref>
,<xref rid="ac034482jb00035" ref-type="bibr"></xref>
</named-content>
The concentrations
of formamide and NaCl to get such a ratio vary for different
experiments, but the important parameter is their relative ratio.
We work with concentrated salt solutions (1 M NaCl), and we
pipet ∼1 μL of NaCl in an overall 50-μL incubation
volume, which
might lead to pipetting errors. In general, however, SNP detection
with true-to-false ratios of >10 appear for formamide contents
of
50−70% and NaCl concentrations of 25−40 mM. The conditions
to reach SNP detection with nanocrystals are similar albeit more
stringent than those used with dyes.
</p>
<p>Other striking aspects of our DNA−nanocrystal conjugates
are
their stability, their fast hybridization, and their room-temperature
incubation. DNA−nanocrystal conjugates stored at 4 °C
maintain
stable activity for more than 6 months. Also, the hybridization is
carried out at room temperature; thus, no heating is necessary
for the hybridization. We typically incubate the solution for 1 h
to get SNP detection, but we found that incubation times of 10
min also yield a SNP detection signal with true-to-false ratios of
>10. Such short incubation time was already noticed with CdSe/ZnS
nanocrystals encapsulated into phospholipids micelles and
modified by DNA.<xref rid="ac034482jb00012" ref-type="bibr"></xref>
Incubation
times longer than 1 h did not result
in higher signals.
</p>
<p>The sensitivity of the nanocrystals does not reach that of
the
dyes yet, and this constitutes the weakest point of this technology
for their use in DNA microarrays so far. There are essentially
two reasons for this lower sensitivity. First, the single-wavelength
laser diodes and the Axon scanner optics are optimized for Cy3
and Cy5 dyes. In this study, nanocrystals are excited at 532 nm
where their cross section is relatively modest (Figure <xref rid="ac034482jf00001"></xref>
). Second,
the density of the nanocrystals on the array may well be 1 order
of magnitude lower than that of the dyes, as was observed in the
case of DNA−gold conjugates.<named-content content-type="bibref-group" xlink:type="simple"><xref rid="ac034482jb00003" ref-type="bibr"></xref>
,<xref rid="ac034482jb00036" ref-type="bibr"></xref>
</named-content>
Despite the lower sensitivity of
our nanocrystals with respect to dyes in this study, simple
modifications in the optical setup could greatly enhance the
present sensitivity. For instance, nanocrystals have a continuous
excitation band and an absorption cross section in the UV
substantially higher than in the visible. A cross section 2 times
higher results in a ∼10-fold increase in the number of photons
absorbed. Thus, UV excitations would produce higher absorption
and consequently higher fluorescence emission. Such sources
include commercially available blue laser diodes with a 408-nm
excitation line, Ar<sup>+</sup>
lasers, and mercury or xenon lamps.
On the
other hand, the use of a grated spectrometer with CCD camera
allows the unambiguous detection of at least four orthogonal
channels in the visible range.<named-content content-type="bibref-group" xlink:type="simple"><xref rid="ac034482jb00006" ref-type="bibr"></xref>
,<xref rid="ac034482jb00026" ref-type="bibr"></xref>
</named-content>
With such a setup, it has long
been observed that nanocrystals photobleach at a much lower rate
than organic dyes and that they can be detected at the single-molecule
level for extended periods of time. Finally, commercially
available quantum dots<sup>15</sup>
have larger quantum yields than
our
samples and may further increase the overall sensitivity of this
labeling technology. As a consequence, DNA−nanocrystals will
be superior probes at spot sizes where the detection of dyes is
limited by photobleaching. We view nanometer-size arrays as a
potential technology where nanocrystals can surpass dyes. A DNA
spot of ∼50 nm in diameter may contain only 100 probes. At
this
scale, the unambiguous detection of dyes with different colors is
challenging, while it has become routine to detect multicolor single
nanocrystals by stage-scanning confocal microscopy. <sup>37,38</sup>
</p>
<p>Overall, our experiments indicate that DNA−nanocrystal
conjugates have realistic potential for DNA microarray applications,
for both SNP detection and multimarker detection. The
stability and room-temperature hybridization condition make a
nanocrystal/microarray-based field test a possibility. So far, our
model system has used only synthetic targets, but we believe we
can extend our results to real samples such as blood after
appropriate extraction and purification of the genetic material. For
instance, one application that can be derived from this study is to
use nanocrystals and microarrays to do fast and full-panel viral
and bacterial pathogen screening for blood banks. The microarray
panel can include probes for hundreds of blood-borne pathogens
such as hepatitis B virus, hepatitis A, C, D, E, F, G viruses, HIV,
and sepsis-inducing bacteria. Another application is to use the SNP
detection capability of nanocrystal−DNA conjugates to detect
SNP
arrays for different genes, such as the p53 oncogene in this study.
Because of the superior true-to-false signal ratio and faster
hybridization of the nanocrystal−DNA conjugates over other
conventional labeling methods such as cyanine dyes, better
discrimination and faster throughput can be achieved.
</p>
</sec>
</body>
<back><ack><p>We thank Xavier Michalet for comments
on the manuscript.
This work was supported by NIH National Center for Research
Resources through the University of California, Los Angeles
subaward agreement 0980GCD709 through the U.S. Department
of Energy under Contract DE-AC03-76SF00098, by NIH
Grant 1R21CA95393-01, and by Basic Energy Sciences, Department of
Energy.
</p>
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<mods version="3.6"><titleInfo><title>Room-Temperature Single-Nucleotide Polymorphism and Multiallele DNA Detection Using Fluorescent Nanocrystals and Microarrays</title>
</titleInfo>
<titleInfo contentType="CDATA"><title>Room-Temperature Single-Nucleotide Polymorphism and Multiallele DNA Detection Using Fluorescent Nanocrystals and Microarrays</title>
</titleInfo>
<name type="personal" displayLabel="corresp"><namePart type="family">GERION</namePart>
<namePart type="given">Daniele</namePart>
<affiliation>Departmentof Chemistry and Department of Mechanical Engineering, Universityof California, Berkeley, California 94720,and Materials Sciences Division and Life Sciences Division, LawrenceBerkeley National Laboratory,Berkeley, California 94720</affiliation>
<affiliation> Department of Chemistry, University of California.</affiliation>
<affiliation> Materials Sciences Division, Lawrence Berkeley National Laboratory.</affiliation>
<affiliation> Present address: Lawrence Livermore National Laboratory,P.O. Box 808,L-415, Livermore, CA 94551.</affiliation>
<affiliation> To whom correspondence should be sent. E-mails: gerion1@llnl.gov(D.G.);f_chen@lbl.gov (F.C.).</affiliation>
<role><roleTerm type="text">author</roleTerm>
</role>
</name>
<name type="personal" displayLabel="corresp"><namePart type="family">CHEN</namePart>
<namePart type="given">Fanqing</namePart>
<affiliation>Departmentof Chemistry and Department of Mechanical Engineering, Universityof California, Berkeley, California 94720,and Materials Sciences Division and Life Sciences Division, LawrenceBerkeley National Laboratory,Berkeley, California 94720</affiliation>
<affiliation> Life Sciences Division, Lawrence Berkeley National Laboratory.</affiliation>
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<affiliation>Departmentof Chemistry and Department of Mechanical Engineering, Universityof California, Berkeley, California 94720,and Materials Sciences Division and Life Sciences Division, LawrenceBerkeley National Laboratory,Berkeley, California 94720</affiliation>
<affiliation> Department of Mechanical Engineering, University of California.</affiliation>
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<affiliation>Departmentof Chemistry and Department of Mechanical Engineering, Universityof California, Berkeley, California 94720,and Materials Sciences Division and Life Sciences Division, LawrenceBerkeley National Laboratory,Berkeley, California 94720</affiliation>
<affiliation> Department of Chemistry, University of California.</affiliation>
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<name type="personal"><namePart type="family">PARAK</namePart>
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<affiliation>Departmentof Chemistry and Department of Mechanical Engineering, Universityof California, Berkeley, California 94720,and Materials Sciences Division and Life Sciences Division, LawrenceBerkeley National Laboratory,Berkeley, California 94720</affiliation>
<affiliation> Department of Chemistry, University of California.</affiliation>
<affiliation> Materials Sciences Division, Lawrence Berkeley National Laboratory.</affiliation>
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</role>
</name>
<name type="personal"><namePart type="family">CHEN</namePart>
<namePart type="given">David J.</namePart>
<affiliation>Departmentof Chemistry and Department of Mechanical Engineering, Universityof California, Berkeley, California 94720,and Materials Sciences Division and Life Sciences Division, LawrenceBerkeley National Laboratory,Berkeley, California 94720</affiliation>
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</role>
</name>
<name type="personal"><namePart type="family">MAJUMDAR</namePart>
<namePart type="given">Arunava</namePart>
<affiliation>Departmentof Chemistry and Department of Mechanical Engineering, Universityof California, Berkeley, California 94720,and Materials Sciences Division and Life Sciences Division, LawrenceBerkeley National Laboratory,Berkeley, California 94720</affiliation>
<affiliation> Materials Sciences Division, Lawrence Berkeley National Laboratory.</affiliation>
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<role><roleTerm type="text">author</roleTerm>
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</name>
<name type="personal"><namePart type="family">ALIVISATOS</namePart>
<namePart type="given">A. Paul</namePart>
<affiliation>Departmentof Chemistry and Department of Mechanical Engineering, Universityof California, Berkeley, California 94720,and Materials Sciences Division and Life Sciences Division, LawrenceBerkeley National Laboratory,Berkeley, California 94720</affiliation>
<affiliation> Department of Chemistry, University of California.</affiliation>
<affiliation> Materials Sciences Division, Lawrence Berkeley National Laboratory.</affiliation>
<role><roleTerm type="text">author</roleTerm>
</role>
</name>
<typeOfResource>text</typeOfResource>
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<originInfo><publisher>AmericanChemical Society</publisher>
<dateIssued encoding="w3cdtf">2003-09-15</dateIssued>
<copyrightDate encoding="w3cdtf">2003</copyrightDate>
</originInfo>
<language><languageTerm type="code" authority="iso639-2b">eng</languageTerm>
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<abstract>We report two cDNA microarray-based applications of DNA−nanocrystal conjugates, single-nucleotide polymorphism (SNP) and multiallele detections, using a commercial scanner and two sets of nanocrystals with orthogonal emissions. We focus on SNP mutation detection in the human p53 tumor suppressor gene, which has been found to be mutated in more than 50% of the known human cancers. DNA−nanocrystal conjugates are able to detect both SNP and single-base deletion at room temperature within minutes, with true-to-false signal ratios above 10. We also demonstrate microarray-based multiallele detection, using hybridization of multicolor nanocrystals conjugated to two sequences specific for the hepatitis B and hepatitis C virus, two common viral pathogens that inflict more than 10% of the population in the developing countries worldwide. The simultaneous detection of multiple genetic markers with microarrays and DNA−nanocrystal conjugates has no precedent and suggests the possibility of detecting an even greater number of bacterial or viral pathogens simultaneously.</abstract>
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