Patent Application
20050170383
The present invention relates generally to biological and chemical assay systems, and more specifically to microarray hybridization assemblies for use in conducting hybridization assays.
Biological and biochemical analysis often involves the use of glass slides that support hybridization reactions between bound probes and target molecules in a solution. A microarray commonly means a substrate (such as a glass slide, silicon wafer, and nylon or polymer-based substrate) that contains a plurality of different reagents immobilized on the surface. The substrate may have a shape of a rectangle, a square, a circle, a triangle, or any other convenient substantially planar shape. These reagents (known as probes) are usually selected for their high specificity in binding affinity or reactivity toward their counterparts (known as targets) in biological samples. After applying a biological sample onto a microarray under an experimentally-controlled condition, the interactions between each probe on a microarray and its corresponding target in the biological sample can be observed through various target labeling techniques and appropriate detection instrumentation, thus providing the microarray user with qualitative and quantitative information about the target in the tested biological sample.
A key consideration in conducting hybridization assays for a microarray is to provide a reaction region for hybridization reactions between immobilized probe molecules and target molecules in a hybridization solution. Hybridization reactions can often require time periods of up to several to tens of hours and are typically performed at high temperatures for DNA microarrays. Thus, the reaction region must be configured to prevent leaking or drying out of the solution. Several different systems have been developed to accommodate microarray slides for conducting hybridization reactions. For example, U.S. Pat. No. 6,258,593 discloses a slide system in which the slide cover is attached to a substrate through the use of screw fasteners. Such a system is labor intensive and requires the use of special materials and/or production tooling, thus adding a significant complication and expense to the microarray hybridization assay.
It is therefore desirable to provide a simple and economical apparatus and method for assembling slides used in microarray hybridization assay analysis.
A microarray hybridization assembly for conducting hybridization assay analysis is disclosed. The microarray assembly comprises a slide, a slide cover, and a spacer disposed between the slide and slide cover. The slide cover is placed over the slide and the spacer maintains a gap between the parallel surfaces of the slide and slide cover to create a reaction region for hybridization reactions between attached probe molecules and target molecules of a hybridization solution. The slide assembly is placed into a heat shrinkage bag. The bag and slide assembly are then heated in a heat cycle sufficient to seal the bag around at least three sides of the slide assembly. A hybridization solution can be introduced through an open end of the slide assembly or an open portion in the gap into the reaction region of the slide assembly. A second heat shrinkage bag, configured as a cap or cover can be fitted over the open end of the assembly and heated in a second heat cycle to seal the fourth side of the slide assembly after a hybridization solution is introduced into the reaction region of the slide assembly.
Other objects, features, and advantages of the present invention will be apparent from the accompanying drawings and from the detailed description that follows below.
The present invention is illustrated by way of example and not limitation in the figures of the accompanying drawings, in which like references indicate similar elements, and in which:
The present invention discloses a microarray hybridization assembly that uses a heat shrinkage bag to fit and seal the hybridization assembly for use in hybridization reaction processes. The microarrays include, but are not limited to, gene chips, DNA chips, oligonucleotide microarrays, polynucleotide microarrays, protein microarrays, antibody microarrays. For purposes of the following description the terms “hybridization assembly” or “slide assembly” refer to a structure comprising a slide cover mounted on a slide substrate with a spacer placed between them to form a reaction region that can hold a hybridization solution as a heat shrinkage bag is fitted around the slide assembly and then shrunk to form a hermetically sealed reaction region. In the following description, for purposes of explanation, numerous specific details are set forth in order to provide a thorough understanding of the present invention. It will be evident, however, to one of ordinary skill in the art, that the present invention may be practiced without these specific details. In other instances, well-known structures and devices are shown in block diagram form to facilitate explanation. The description of preferred embodiments is not intended to limit the scope of the claims appended hereto.
A reaction region enclosing the attached probes on the surface of slide 208 is created by mounting the slide cover 206 onto the slide 208 and separating the two mating surfaces by a small gap so that two parallel upper and lower surfaces are created.
It should be noted that the spacer 204 can be of various different forms from that shown in
Generally the spacer is placed around the outer edge of the slide 208 of the microarray, the spacer can be affixed around the outer edge of the slide 208, to create a reaction region for hybridization on the substrate surface when the slide cover 206 is mounted on the slide 208. As illustrated in
The region of the spacer contained within its three walls defines the reaction area of the hybridization assembly. The spacer height, H5, is typically on the order of 3 to 10 mm, and the spacer width, W5, is typically on the order of 3 to 5 mm. In general, the spacer may have any suitable shape or dimension as long as the spacer does not cover any portion of the probe area of the microarray and a suitable volume of the reaction region can be created. In addition, the spacer may be configured to be placed proximately around an inside portion of at least two outer edges of the slide and the slide cover. For most typical oligonucleotide hybridization reactions, the volume of the reaction region is on the order of about 100 μl to about 500 μl. This volume should be carefully calibrated, depending upon the application. If the reaction volume is too small, due to too thin a spacer, it may complicate the hybridization treatment. If the reaction region volume is too big, the concentration of target sample molecule is decreased, and so therefore is the sensitivity of hybridization assays.
As illustrated in
Upon shrinking, the slide assembly can be sealed within the bag as shown in
In one embodiment, the heat shrinkage bag 202 can be sized so that sufficient material is available at the top (open end) of the bag when the bag is immersed in water, such that adequate surface area is available to grip the bag using fastener 302 and such that water does not enter the bag during the sealing process. The bag should be placed sufficiently low in the water so that the whole height H2 of the two sides of slide 208 is sealed during the heating cycle. The excess top portion of the bag above the top (open end) of the slide assembly can then be cut away.
The slide assembly described with reference to
For the embodiment illustrated in
The heat shrinkage cap embodiment is typically used in applications in which the hybridization solution is introduced into the slide assembly after the first heat shrinkage obag is sealed around the slide assembly to form the reaction chamber. This application is shown in
Once the slide assembly is filled with the target hybridization solution and sealed with the first heat shrinkage bag and the heat shrinkage cap, it can be incubated under hybridization conditions in accordance with the requirements of the interaction of probe molecules and target molecules. After incubation, the array can be washed and read through suitable means.
In typical applications, the assembly may be disassembled after the hybridization reaction is complete, so that the slide with the hybridized target molecules on its surface is analyzed directly. For these applications, the bag material is removed from the hybridization assembly after incubation.
As described with reference to the embodiment illustrated in
The following example illustrates the steps taken to prepare a hybridization assembly and conduct a hybridization assay with the method and techniques described above.
(A) Preparation of Hybridization Solution:
HepG2 (ATCC No. HB-8065)cell was grown on a laboratory dish in ATCC (American Type Culture Collection) complete growth medium at 37° C. to a cell number of 6×106. The cell was then harvested for total RNA extraction. The total RNA was extracted using Qiagen RNeasy Midi Kit (Catalogue No. 75144). After extraction, 20 kg of the total RNA was taken and converted to Cy5 labeled cDNA according to Agilent Fluorescent Direct label Kit (Catalogue No.G2557A) in each run of a total of ten runs. In each run, a Perkin-Elmer Cy5-dCTP reagent was used for direct labeling, and a Qiagen PCR Purification Kit (Catalogue No. 28106) was applied to provide purified fluorescent labeled cDNA elution. The elution was concentrated using Millipore YM30 filter (Catalogue No. 42410) so that one μl of the final cDNA solution corresponded to 2.5 μg of total RNA converted in the run. In this manner, 200 μg of total RNA was converted to 80 μl of fluorescent labeled cDNA solution. The following steps were taken to prepare a target hybridization mix, target sample solution:
(1) A water bath was preheated to about 90° C.
(2) The 1.5× hybridization buffer (7.5×SSC, 45%Foramide, 0.15%BSA, 1.5 mM EDTA, 0.75%SDS) was agitated in a 65° C. water bath for 10 minutes.
(3) 25 μl of the fluorescent labeled cDNA and 173 μl of the 1.5× hybridization buffer were mixed, and nuclease-free ddH2O was added to bring up a total volume of 260 μl of target hybridization mix comprising the fluorescent labeled cDNA hybridization mix.
(4) A denature program was set in a PCR machine to 95° C. for 5 minutes, then to a steady temperature of 60° C. The target hybridization mix was placed in the PCR machine and the Denature program was started. While the hybridization mix was denaturing, a hybridization assembly was prepared according to step B.
(B) Preparation of Hybridization Assembly:
(1) A glass microarray slide and glass slide cover each having dimensions on the order of of 2.5 cm×7.6 cm (approximately 1 inch×3 inch) were obtained. The slide comprised an oligonucleotide microarray slide containing a plurality of oligonucleotides attached to the surface of the slide. A spacer made of silicon rubber was cut to a three sided shape for placement between the slide and slide cover as shown in
(2) The microarray probe side of the slide was positioned upwards and the spacer was placed on the upper surface of the slide with its corners aligned with two corners of the microarray. The blank slide cover was placed on the top of the spacer and the edges of the two slides were aligned to form a hybridization assembly, such as that shown in
(3) The hybridization assembly was fitted into a polyvinylchloride heat shrinkage bag, and the thickness of the bag material was on the order of 0.04 mm. The bag was cut to a size of about 30 mm×100 mm. The sample loading side of the hybridization assembly faced the opening side of the bag. The assembly was lowered to the end of the bag, then the bag opening side was clipped with a binder clip.
(4) The clipped assembly of the slide assembly in the heat shrinkage bag was immersed into a beaker of water heated to about 90° C. for about 10-20 seconds to shrink and wrap the slides inside the bag. After shrinkage, the bag was lifted out of the water, and the clips were removed. The portion of the excess bag was trimmed off the sample loading side of the assembly.
(C) Addition of Target Sample Solution to the Hybridization Assembly:
(1) After production of the hybridization assembly with the open bag side, the assembly was preheated in a 50° C. oven for more than 10 minutes. About 260 μl of the fluorescent labeled cDNA hybridization mix prepared in step (A) was added to the reaction region through the open side of the hybridization assembly.
(2) The sample loading side of the hybridization assembly was covered with a polyvinylchloride heat shrinkage cap, which was cut to a size of about 30 mm×7 mm. The thickness of the cap material was on the order of 0.04 mm. The cap was held in place through a friction fit, and the capped side of the assembly was immersed in 90° C. hot water to seal the sample loading opening of the assembly. After sealing of the assembly end, the entire assembly was immersed in the hot water bath to completely seal the assembly in the polyvinylchloride heat shrinkage film.
(D) Hybridization Reaction and Analysis:
(1) After sealing, the hybridization assembly was placed in a 50° C. oven with rotation (3 rpm) for 14-16 hours for incubation of the hybridization reaction. The oven was kept humidified with 2×SSC.
(2) After incubation, an incision was made along the edge of the glass slides with a craft knife to open the wrapped hybridization assembly. The wrapped film was removed and the hybridization assembly was entirely submerged in 42° C. 2×SSC, 0.2% SDS solution. The assembly was disassembled in the solution.
(3) The microarray was washed with excess amount of pre-warmed 2×SSC, 0.2% SDS for ten minutes at 42° C.
(4) The microarray was then washed with excess amount of pre-warmed 2×SSC for ten minutes at 42° C.
(5) The microarray was finally washed with excess amount of 0.2×SSC for ten minutes at room temperature, and then dried in a spin dry centrifuge.
(6) The microarray was scanned by Axon Genepix 4000B scanner and analyzed by fluorescence detection to quantify the amount of cDNA hybridized with each oligonucleotide probe of the microarray.
Further, a polyethyleneterephthalate heat shrinkage bag with dimensions on the order of 30 mm×100 mm and a thickness of the bag material on the order of 0.06 mm worked similarly in the first heating cycle as the polyvinylchloride heat shrinkage bag of the Example 1. A polyethyleneterephthalate heat shrinkage cap with dimensions on the order of 30 mm×7 mm and a thickness of the bag material on the order of 0.06 mm also worked similarly in the second heating cycle as the polyvinylchloride heat shrinkage cap of the Example 1.
In the foregoing, a slide system for microarrays has been described. Although the present invention has been described with reference to specific exemplary embodiments, it will be evident that various modifications and changes may be made to these embodiments without departing from the broader spirit and scope of the invention as set forth in the claims. For example, although embodiments of the invention were described with reference to use with oligonucleotide or polynucleotide arrays, it should be noted that the use of a heat shrinkage bag and a spacer to produce a sealed reaction region between two laboratory slides can be used for analysis of many other different biological or chemical substances, such as protein arrays, antibody arrays, and the like. Accordingly, the specification and drawings are to be regarded in an illustrative rather than a restrictive sense.
The present application is a Continuation-In-Part of U.S. patent application Ser. No. 10/769,960, filed Feb. 2, 2004, entitled “Method of Preparing Reaction Regions for Biochips,” which is currently pending, and which is assigned to the assignee of the present application.
| Number | Date | Country | |
|---|---|---|---|
| Parent | 10769960 | Feb 2004 | US |
| Child | 10996975 | Nov 2004 | US |
