U.S. patent application number 09/978365 was filed with the patent office on 2002-03-21 for biological assays for analyte detection.
This patent application is currently assigned to Vysis, Inc., a Delaware corporation. Invention is credited to Bao, Yijia, Che, Diping, Muller, Uwe Richard.
Application Number | 20020034762 09/978365 |
Document ID | / |
Family ID | 22192864 |
Filed Date | 2002-03-21 |
United States Patent
Application |
20020034762 |
Kind Code |
A1 |
Muller, Uwe Richard ; et
al. |
March 21, 2002 |
Biological assays for analyte detection
Abstract
Fluorescence-based assay methods for detecting biological
analytes in a sample. The fluorescence background in these methods
is significantly lower than in conventional assay methods. Also
provided are methods of attaching nucleic acids to a metallic or
metalloid surface.
Inventors: |
Muller, Uwe Richard; (Plano,
IL) ; Che, Diping; (Westmont, IL) ; Bao,
Yijia; (Naperville, IL) |
Correspondence
Address: |
Norval B. Galloway, Esq.
Vysis, Inc.
3100 Woodcreek Drive
Downers Grove
IL
60515
US
|
Assignee: |
Vysis, Inc., a Delaware
corporation
|
Family ID: |
22192864 |
Appl. No.: |
09/978365 |
Filed: |
October 16, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09978365 |
Oct 16, 2001 |
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09085625 |
May 27, 1998 |
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6306589 |
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Current U.S.
Class: |
435/6.12 ;
435/243; 435/6.1; 435/91.2; 536/25.4 |
Current CPC
Class: |
C12Q 1/6834 20130101;
G01N 33/552 20130101 |
Class at
Publication: |
435/6 ; 435/91.2;
435/243; 536/25.4 |
International
Class: |
C12Q 001/68; C07H
021/00; C07H 021/02; C07H 021/04; C12P 019/34; C12N 001/00 |
Claims
What is claimed is:
1. A method of detecting an analyte in a sample, the method
comprising: providing an opaque glass support with a surface that
is in contact with the sample, wherein the analyte, if present in
the sample, is labeled with a fluorochrome; illuminating the
surface with light at a wavelength that excites the fluorochrome;
and detecting fluorescent emission from the surface as a indication
of the presence of the analyte in the sample.
2. The method of claim 1, wherein the analyte is bound to the
surface via a capture probe that (i) binds specifically to the
analyte and (ii) is immobilized on the surface.
3. The method of claim 1, wherein the analyte is a nucleic
acid.
4. A method of detecting an analyte in a sample, the method
comprising: providing a reflective surface that is in contact with
the sample, wherein the analyte, if present in the sample, is
labeled with a fluorochrome; illuminating the surface with light at
a wavelength that excites the fluorochrome; and detecting
fluorescent emission from the surface as an indication of the
presence of the analyte in the sample.
5. The method of claim 4, wherein the analyte is bound to the
reflective surface via a capture probe that binds specifically to
the analyte and is immobilized on the surface.
6. The method of claim 4, wherein the analyte is a nucleic
acid.
7. The method of claim 4, wherein the reflective surface is a
metallic or metalloid surface.
8. The method of claim 7, wherein the metallic surface is a
chromium surface.
9. The method of claim 7, wherein the metallic surface is an
aluminum surface.
10. The method of claim 7, wherein the metalloid surface is a
silicon surface.
11. A method of attaching a nucleic acid to a metallic or metalloid
surface, the method comprising: providing a solution that contains
the nucleic acid; denaturing the nucleic acid in the solution;
applying the solution to the metallic or metalloid surface; and
allowing the solution to dry on the surface, thereby attaching the
nucleic acid to the surface.
12. The method of claim 11, wherein the solution has a pH of at
least about 11.
13. The method of claim 12, wherein the solution comprises sodium
hydroxide.
14. The method of claim 11, wherein the nucleic acid is denatured
by heating the solution to a temperature and for a time sufficient
to denature the nucleic acid.
15. The method of claim 11, wherein the metallic surface is a
chromium surface.
16. The method of claim 11, wherein the metallic surface is an
aluminum surface.
17. The method of claim 11, wherein the metalloid surface is a
silicon surface.
18. The method of claim 11, further comprising applying a
microscopy mounting medium to the metallic or metalloid surface so
as to enhance the attachment of the nucleic acid to the
surface.
19. The method of claim 18, wherein the microscopy mounting medium
is GEL/MOUNT.TM..
20. The method of claim 4, wherein the excitation light is directed
to the surface at an angle.
Description
FIELD OF THE INVENTION
[0001] The invention relates to biological methods of assaying
analytes.
BACKGROUND OF THE INVENTION
[0002] Biological assays for analyte detection generally involve
attaching to the analyte (e.g., nucleic acids, proteins, hormones,
lipids, or cells) a signal-generating moiety. Fluorescence-based
bioassays require the detection of weak fluorescence signals. In a
typical assay, the analyte is deposited onto a solid substrate such
as a microscope slide or a glass chip. After undergoing biochemical
treatment and fluorescent staining, the slide is examined with an
optical instrument such as a fluorescence microscope. Light of
certain wavelengths is applied to the slide, and the fluorescent
emission from the deposited biomaterial is collected as a
signal.
[0003] Transparent soda-lime and borosilicate glasses are commonly
used as substrates to support fluorescently labeled samples.
However, many of these materials exhibit significant
autofluorescence, have finite absorbance, and can produce
fluorescent emission throughout the visible region. A typical
soda-lime glass slide can produce background fluorescence
equivalent to a layer of a commonly used fluorescent dye with a
surface density of more than 1.times.10.sup.9 fluorophors/cm.sup.2.
This background fluorescence along with noise from other sources,
such as stray light and Rayleigh and Raman scattering, can obscure
the detection of weak fluorescent signals from the analyte,
limiting the sensitivity of the assay.
[0004] Further, in many fluorescent assays for nucleic acid
detection, nucleic acids are attached to a solid support via
chemical linkers. Such linkers often are autofluorescent and can
introduce background fluorescence.
SUMMARY OF THE INVENTION
[0005] The invention features improvements in biological assays. In
one aspect, the invention features fluorescence-based assays that
have a significantly reduced signal background compared to
conventional assays. These assays include the steps of: (i)
providing an opaque glass support with a surface that is in contact
with a sample containing an analyte (e.g., a protein, a nucleic
acid, a polysaccharide, a lipid, or a cell), where the analyte, in
present in the sample, is labeled with a fluorochrome; (ii)
illuminating the surface with light at a wavelength that excites
the fluorochrome; and (iii) detecting fluorescent emission from the
surface as an indication for the presence of the compound in-the
sample. As used herein, "an opaque glass support" refers to a glass
support that is impervious to the excitation and emission lights of
the fluorochrome used in an assay.
[0006] In the above assays, a reflective surface can be used in
lieu of an opaque glass support. By "a reflective surface" is meant
that, when incoming light is directed to the surface
perpendicularly, the surface reflects at least about 15% (e.g., at
least 25%, 50%, 75%, or 90%) of the incoming light, while
transmitting no more than 20% (e.g., no more than 10%, 5%, or 1%)
of the light. In assays using a reflective surface, the excitation
light can be directed to the surface at an angle, i.e.,
non-perpendicularly. A reflective surface can be, for instance,
metallic (e.g., chromium or aluminum) surface or metalloid (e.g.,
silicon) surface.
[0007] In the fluorescence assays of the invention, the analyte can
be bound to the surface via a capture probe that binds specifically
to the analyte and is immobilized on the surface.
[0008] In another aspect, the invention features methods of
efficiently attaching nucleic acid to a metallic or metalloid
surface. These methods include the steps of: (i) providing a
solution that contains the desired nucleic acid; (ii) denaturing
the nucleic acid in the solution; (iii) applying the solution to
the metallic (e.g., chromium or aluminum) or metalloid (e.g.,
silicon) surface; and (iv) allowing the solution to dry on the
surface, thereby attaching the nucleic acid to the surface. In
these methods, the nucleic acid can be denatured in an alkaline
solution (e.g., a NaOH solution) that has a pH of at least about
11, or by being heated to a temperature and for a time sufficient
to denature the nucleic acid. A microscopy mounting medium, e.g.,
Gel/Mount.TM. (Biomeda Corp., Foster City, Calif.), can be
optionally applied to the metal or metalloid surface to enhance the
attachment of the nucleic acid to the surface.
[0009] Unless otherwise defined, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs.
Exemplary methods and materials are described below, although
methods and materials similar or equivalent to those described
herein can be used in the practice or testing of the present
invention. All publications and any other references mentioned
herein are incorporated by reference in their entirety. In case of
conflict, the present specification, including definitions, will
control. The materials, methods, and examples are illustrative only
and not intended to be limiting.
[0010] Other features and advantages of the invention will be
apparent from the following detailed description, and from the
claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0011] FIG. 1 is a diagram illustrating a fluorescence-based assay
in which a chromium-coated soda-lime glass slide is used as a
support for analyte DNA.
[0012] FIG. 2 is a graph showing that treatment with GEL/MOUNT.TM.
(a permanent aqueous mounting medium available from Biomeda Corp.,
Foster City, Calif.) improves the attachment of DNA, especially
short DNA, to a chromium chip. The Y axis represents the ratio of
the signal from a DNA spot treated with GEL/MOUNT.TM. to the signal
from a control DNA spot treated with 2.times. SSC.
DETAILED DESCRIPTION
[0013] The invention features (i) improved fluorescence-based
bioassay methods in which background fluorescence is significantly
reduced as compared to conventional methods, and (ii) improved
methods for attaching nucleic acids to solid supports.
[0014] Methods of Reducing Fluorescence Background
[0015] Fluorescently labeled analytes are detected on a substrate
(e.g., black glass) that is opaque in the wavelength regions where
the fluorescent label absorbs and emits. Due to opaqueness of the
substrate, penetration of the excitation light into the substrate
and any returning fluorescence is substantially reduced. Rayleigh
and Raman scattering from the interior of the substrate will also
be prevented. Further, contamination and dust particles on the side
and back surfaces of the substrate, which can generate background
of much higher intensity than the real signal, will not be
detected. Background originating stray light from below the
substrate is also reduced or eliminated. Consequently, the
background noise from this type of substrate is significantly lower
than that from a conventional substrate (e.g., soda-lime
glass).
[0016] The substrate is preferably non-fluorescent or has low
autofluorescence. The chemical and physical properties of the
substrate material should also be compatible with the assay.
Suitable materials include, but are not limited to, colored or
opaque glasses and opaque, plastic-based materials. Exemplary
colored glasses are Schott M-UG-2, M-UG-6, UG-1, UG-11, ND, RG715,
RG9, RG780, ND-1, and ND-10 (Germany); and Corning 2030, 2540,
2550, 2600, 5840, 5860, 5874, and 9863. The substrate can be
fabricated into forms such as slides, wafers, or chips.
[0017] Alternatively, the analyte-receiving surface of the
substrate is reflective, and can be, for example, a surface of a
solid support coated with a metalloid or metallic thin film, or a
polished surface of a metal or metalloid plate. The reflective
surface eliminates the transmission of excitation and fluorescent
emission through the substrate if the substrate is otherwise
transparent to the excitation and emission light. By illuminating
the surface at an appropriate angle, excitation light is reflected
away from the collection optics, eliminating autofluorescence from
the collection optics; consequently, less efficient filters can be
used to absorb autofluorescence.
[0018] A variety of reflective coatings can be used, as long as the
chemical and physical properties of the coating material is
compatible with the assay and efficient attachment of an analyte
can be achieved. Suitable substrates include, but are not limited
to, coated glass materials used in glass lithography, e.g.,
chromium-coated glass available from Nanofilm (Westlake Village,
Calif.).
[0019] FIG. 1 shows a substrate 1 formed in the shape of a
conventional microscope slide with a smooth surface coated with a
thin chromium film 2. Fluorescently labeled DNA fragments 3 are
deposited onto the surface dither randomly or addressably in an
array. After biochemical treatments of the deposited molecules, a
liquid media 5 that provides proper pH is optionally applied onto
the slide and covered with a thin coverslip 6. The liquid media can
optionally contain an antifade reagent, which is a composition that
prevents oxidation of a fluorochrome. An exemplary antifade reagent
is p-phenylenediamine, available from Aldrich Chem., Co.
(Milwaukee, Wis.). The liquid media can also contain a
counterstain, e.g., 4',6-diamidino-2-phylindole ("DAPI"). The slide
is then examined with a fluorescence imaging system, in which
excitation light illuminates the chromium-coated side of the slide.
The collection optics for fluorescent emission 4 is positioned
directly above the illuminated surface, with the optical axis
perpendicular to the coated surface. Such a position allows for
reflection of fluorescent light into the collection optics,
approximately doubling the intensity of the fluorescent signal
detected by the optics. The excitation light is applied in an angle
such that the reflected beam does not enter the collection optics.
A mirror 7 can also be used to enhance the intensity of the
excitation light.
[0020] The new methods can be used in a variety of fluorescence
assays such as fluorescence immunoassays, fluorescence in situ
hybridization, comparative genomic hybridization ("CGH"),
genosensor-based CGH ("gCGH"; see, e.g., Kallioniemi et al.,
Science, 258:818-821, 1992), molecular lawn (see U.S. patent
application Ser. Nos. 08/768,177 and 08/991,675), or general DNA
chip based assays (see, e.g., U.S. Pat. Nos. 5,445,934, 5,510,270,
and 5,556,752).
[0021] Well established methods can be used to attach analyte
nucleic acids, or capture probes specific for analyte nucleic
acids, to glass or metallic coated surfaces. See, e.g., Joos et
al., Analytical Biochemistry, 247: 96-101, 1997; Maskos et al.,
Nucleic Acids Research, 20: 1679-1684, 1992; Fodor et al., Science,
251: 767-773, 1991; Lowe, Chemical Society Reviews, 24: 309-317,
1995; Guo et al., Nucleic Acids Research, 22: 5646-5465, 1994; and
Bischoff, Analytical Biochemistry, 164: 336-344, 1987. Attachment
methods that work for glass surfaces also work well for silicon
substrates. To do this, the silicon substrate is heated to oxidize
the surface layer so that the surface has the same chemical
properties as a glass slide.
[0022] To attach a nucleic acid to a metallic surface, the surface
can be treated with a first silane compound (e.g., Gelest's WAS
7021 (Tullytown, Pa.)) and then with a second silane compound
(e.g., (3-Glycidoxypropyl)-Trimethoxysilane). The first silane
coating binds to the metallic surface, and the second silane
coating provides a reactive group (e.g., an epoxy group) for
attachment of an appropriately modified nucleic acid (e.g., an
aminated nucleic acid). Preferably, these two silane coatings are
transparent in the wavelength regions that are most often used.
[0023] Analyte proteins (e.g., cell surface proteins) can be
attached to a solid support by any of a number of standard methods,
including direct adsorption or chemical coupling to reactive groups
on the surface. For example, a solid surface can be derivatized to
generate active amine groups; then an amine- and
sulfhydryl-reactive heterobifunctional crosslinker (e.g.,
succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carbox- ylate or
other DOUBLE-AGENT.TM. crosslinkers available from Pierce,
Rockford, Ill.) is used to link a free cysteine group in a
polypeptide to the amine group on the solid surface.
Homobifunctional crosslinkers can be used as well.
[0024] An analyte can be linked covalently or noncovalently
(directly or indirectly) to a detectable label moiety such as a
fluorochrome (e.g., fluorescein, phycoerythrin, Texas Red, or
Allophycocyanin), or an enzyme that catalyzes a fluorescence
reaction (e.g., horseradish peroxidase). By way of example, an
analyte nucleic acid may be labeled with a probe that is attached
to a fluorochrome; and an antigen can be labeled with a specific
antibody conjugated to horseradish peroxidase.
[0025] The experiments described below illustrate several
embodiments of the new fluorescence-based bioassays methods.
[0026] Quantitative determinations of signals emanating from
fluorescently labeled compounds were carried out with a large field
fluorescent imaging system developed at Vysis, Inc. (Downers Grove,
Ill.). The imaging system consisted of a 450 W Xenon arc lamp (SLM
Instruments Inc., Champaign Urbana, Ill.), a charge coupled device
detector (CH200, Photometrics, Tucson, Ark.), and a filter set
(Chroma Technology, Brattleboro, Vt.) for three commonly used
fluorescent dyes--DAPI for blue, fluorescein isothiocyanate
("FITC") for green, and Texas red ("TRED") for red. The imaging
system was controlled by a Power Macintosh 7100/80 with an imaging
acquisition/analysis software (IpLab, Signal Analytics, Corp.,
Vienna, Va.). The results are shown in Table 1.
1TABLE 1 Relative Background Fluorescence Slide Blue Green Red
Source Soda-lime glass 1 1 1 VWR Scientific products Fused Silica
0.43 0.38 0.59 Haraeus Amersil (Duluth, GA) M-UG-2 black 0.11 0.08
0.16 Schott Mainz glass (Germany) M-UG-6 black 0.10 0.08 0.18
Schott Mainz glass (Germany) Soda-lime with 0.04 0.03 0.09 Nanofilm
Cr coating Soda-lime with 0.05 0.03 0.09 Nanofilm Al coating
Silicon 0.26 0.06 0.13 Nova Electronic Materials (Richardson,
TX)
[0027] Table 1 shows the relative background fluorescence of
untreated materials scaled to the commonly used soda-lime
microscope slide (VWR Scientific Products, West Chester, Pa.). The
relative background fluorescence includes contributions from light
scattering, stray light and filter imperfections, but all
electronic noise has been subtracted. The results show that black
glass slides and slides with metallic coatings offer 4 and 10 fold
background reductions, respectively, over fused silica.
[0028] In another experiment, fluorescent intensity was measured on
fluorescein-conjugated beads (Flow cytometry Standards Corp., Hata
Rey, PR) mixed with a commonly used antifade medium (Catalogue
#824-28, Flow Cytometry Standards Corp.) and sandwiched between a
slide and a glass coverslip. Quantitative analysis of the images
revealed that the overall background for the Cr-coated slide was
only 21% of that for regular glass. The overall background included
not only contributions from light scattering, stray light, and
filter imperfections, but also autofluorescence from the mounting
medium and the coverslip. Notably, the net signal intensity was
doubled, due to reflection of incident light, passing through the
fluorophors twice, and reflection of the fluorescent light, which
would otherwise not enter the detection optics.
[0029] Another experiment demonstrates that the new
fluorescence-based assay methods can be used to detect analyte DNA
complexes. In this experiment, the slide materials listed in Table
1 were used as array supports (i.e., chips) in genosensor-based
comparative genomic hybridization ("gCGH"). The gCGH technology was
developed to improve on standard CGH, where DNA from a sample
tissue is labeled with one fluorophor (e.g., TRED), mixed with an
equal amount of reference DNA labeled with a different fluorophor
(e.g., FITC), and then co-hybridized to metaphase chromosomes that
are affixed to microscope slides. In gCGH, cloned DNA fragments
immobilized on the chip surface in an array format are used in lieu
of metaphase chromosomes. After hybridization, the target spots are
washed and counter-stained with DAPI, which stains all DNA blue.
The slide is then analyzed with a multi-color fluorescence imaging
system. Image analysis software determines the presence of a target
spot by the DAPI fluorescence, and then determines the relative
amount of sample and reference DNA hybridization by measuring the
red to green fluorescence ratio.
[0030] Cr-coated glass slides were made from Cr-coated glass
obtained from Nanofilm. The following steps were carried out to
prepare the metallic surface for DNA attachment. These steps are
also applicable to other metallic surfaces such as Al-coated
surfaces. Briefly, the metallic surface was treated with a 2%
water-based solution of silesquioxane oligomers (Gelest, Inc.) for
10 minutes at room temperature, and washed with water. Silanization
was then carried out with a 5% solution of
glycidoxypropyl-trimethoxysilane ("GPTS"; Gelest, Inc.) in water at
room temperature for 2 hours. Aminated DNA was then attached to the
treated surface by reaction of the primary amine with the epoxy
group.
[0031] DNA extracted from COLO 320 (American Type Culture
Collection ("ATCC") #CCL-220) or HTB-18 (ATCC #HTB-18) cells under
standard conditions were used in the experiment. The hybridization
mixture contained, in 20 .mu.l, 200 ng of human reference DNA
(i.e., human blood DNA) probes labeled with Spectrum Red, 200 ng of
test DNA probes (derived from COLO 320 or HTB-18 cells) labeled
with Spectrum Green, 2.times. SSC, 10% dextran sulfate, 1
.mu.g/.mu.l Cot1 DNA, 1 .mu.g/.mu.l salmon sperm DNA, and 5.times.
Denhardts solution. The mixture was incubated for 4 hours at
37.degree. C. before being added to the chips onto which the target
DNA had been immobilized.
[0032] Hybridization on the chips was carried out overnight at
37.degree. C. The chips were washed with 2.times. SSC at room
temperature for 5 minutes, with 2.times. SSC and 50% formamide at
40.degree. C. for 30 minutes, and then with 2.times. SSC at room
temperature for 10 minutes. Subsequently, the chips were dried at
room temperature in the dark. Before imaging, 10 .mu.l of
GEL/MOUNT.TM. was placed onto the chip in the area of the array,
which was then covered with a coverslip. For imaging, the chromium
chip was imaged with an integration time of 20 seconds. For
comparison, DNA was attached to a soda-lime glass chip (i.e., a
microscope slide) via standard epoxysilane chemistry and hybridized
under identical conditions; and the glass chip was imaged for 10
seconds. The results showed that more than 4-fold reduction in
overall background was achieved with Cr-coated slides. Similar
results were obtained with dark glass slides, silicon slides and
Al-coated slides.
[0033] An advantage of using the Cr-coated surface is that if an
appropriate hybridization fluid, it is very easy to remove unbound
probe from the chip surface due to the hydrophobic properties of
chromium. An appropriate hybridization fluid can be one that does
not contain a detergent capable of altering the hybrophobic
property of the chromium surface. As a result, the fluorescent
background due to Non-specific binding of probe is lessened or even
eliminated. The hybridization efficiency may also be increased,
since less non-specific binding allows for increased probe
availability for specific binding.
[0034] Methods of Attaching Nucleotide Acids to Substrates
[0035] The invention provides a fast and surprisingly simple and
convenient method for attaching nucleic acids to a solid surface.
In the new attachment methods, nucleic acids are bound
noncovalently to a substrate surface, e.g., a metallic or metalloid
surface. To accomplish this, a solution containing denatured
nucleic acid is applied to a substrate and allowed to dry at room
temperature or in an oven. The nucleic acid can be denatured by
raising the pH of the solution to a level of about 11.0 or higher.
High pH in a solution can be achieved by use of a variety of
alkaline materials, e.g., an alkali metal or alkaline earth metal
hydroxide such as NaOH, KOH and the like.
[0036] Alternatively, the nucleic acids can be denatured by heat,
e.g., by heating a solution containing the nucleic acids at
95.degree. C. or higher for 2 to 5 minutes. The solution containing
the denatured nucleic acids is then applied to a metallic or
metalloid surface and allowed to dry.
[0037] It is believed that the electrostatic forces present in the
denatured, single-stranded nucleic acids are typically adequate;
for effective attachment to the substrate. The above processes work
particularly well for long polynucleotides (e.g., more than about
550 nucleotides ("nt") in length).
[0038] To improve attachment of polynucleotides that are less than
about 550 nt in length), the nucleic acids spotted to a substrate
surface can be treated with a microscopy mounting medium such as
GEL/MOUNT.TM. or an equivalent of GEL/MOUNT.TM.. While not wishing
to be bound by any specific theory, it is believed that
GEL/MOUNT.TM., which contains polymeric molecules, acts as a volume
displacement reagent, bringing nucleic acids to closer proximity to
the substrate surface. Alternatively, this mounting reagent may
allow the nucleic acids to be in closer contact with each other,
thereby promoting formation of a nucleic acid network that traps
nucleic acids not directly attached to the substrate surface. Any
reagent that has similar effects on nucleic acid can be used.
[0039] The conventional nucleic acid attachment methods are known
to introduce background fluorescence due to the fact that most
chemical linkers are autofluorescent. By eliminating the use of
such linkers, the new attachment methods circumvent this
problem.
[0040] In one example, 0.9 .mu.g/.mu.l unmodified and undigested
plasmid DNA (6 kb) in water or 100 mM NaOH was manually spotted
onto untreated Cr-coated chips and allowed to dry. After a wash
with 2.times. SSC, the DNA was stained with GEL/MOUNT.TM./DAPI and
examined for DAPI fluorescent signals. The results showed that the
spots of undenatured DNA, i.e., DNA in water, were washed out; in
contrast, the spots of denatured DNA, i.e., NaOH-treated DNA, bound
well, with similar DAPI intensity to 49 kb long lambda DNA spots
generated under NaOH denaturation conditions. Specific
hybridization to the attached DNA was observed using nick
translated plasmid probe under standard hybridization and wash
conditions.
[0041] To test whether the alkaline method described above works
well for attachment of short DNA molecules, NaOH-treated, sonicated
lambda DNA, which was about 500 nt long on average, were spotted to
Cr-coated chips as described herein. Subsequent to washing with
2.times. SSC, the spotted DNA was stained with GEL/MOUNT.TM./DAPI
and examined for fluorescent signal. The results showed that the
DNA remained attached to the chips even after the wash.
[0042] These results demonstrate that binding of DNA to a Cr- or
Al-coated surface achieved by the new method endures the harsh
conditions of hybridization and wash steps as well as the binding
achieved by the conventional covalent methods.
[0043] In another example, the effect of GEL/MOUNT.TM. treatment on
DNA attachment and hybridization was tested. Sonicated lambda DNA
of various lengths (i.e., 48 kb, 2.5 kb, 900 nt, 550 nt, 400 nt,
and 300 nt, respectively) were used in the experiment.
[0044] The sonicated DNA was suspended in 100 mM NaOH and attached
onto an untreated chromium chip as described above. Fifteen .mu.l
of GEL/MOUNT.TM. were then added to the DNA chips, and incubated at
room temperature for 1 hour. The chips were washed with 2.times.
SSC and stained with DAPI. The data shown in FIG. 2 demonstrates
that the GEL/MOUNT. treatment significantly improves the attachment
of DNA, especially short DNA (e.g., about 300 to 400 nt in length),
to the chromium chip. For instance, the blue DAPI signal from the
GEL/MOUNT.TM.-treated 300 nt spots was found to be 7 fold higher
than that from the corresponding SSC-treated spots.
[0045] To examine the effect of GEL/MOUNT.TM. on hybridization, the
DNA chips were incubated with 20 ng of Spectrum green labeled
lambda DNA and 20 ng of Spectrum red labeled lambda DNA. For the
spots containing 300 nt or 400 nt long target DNA, the fluorescent
signals of the GEL/MOUNT.TM.-treated chips were 4 to 7 fold higher
than those of untreated chips
Other Embodiments
[0046] It is to be understood that while the invention has been
described in conjunction with the detailed description thereof, the
foregoing description is intended to illustrate and not to limit
the scope of the invention, which is defined by the scope of the
appended claims.
[0047] Other aspects, advantages, and modifications are within the
scope of the following claims.
* * * * *