Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2001-07-02
2002-11-26
Chin, Christopher L. (Department: 1641)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S283100, C435S286700, C435S303300, C435S305300, C435S305400, C435S287100, C422S051000, C422S105000, C422S105000, C422S940000, C422S942000, C436S518000, C436S524000, C366S267000
Reexamination Certificate
active
06485918
ABSTRACT:
FIELD
The present invention relates generally to methods and systems for hybridizing and/or incubating microarrays.
BACKGROUND OF THE INVENTION
Microarrays are arrays of very small samples of purified DNA, protein, antibody, or small molecule target material arranged as a grid of up to hundreds or thousands of small spots immobilized onto a solid substrate.
FIG. 1A
is a top view of a typical microarray. The microarray substrate
10
is typically coated or derivatized uniformly over its top surface
12
to afford chemical or electrostatic binding of small droplets of the target material in solution. The droplets of target solution dry and bind to the top surface
12
of the substrate
10
, forming target spots
5
that are is generally from tens to hundreds of microns in diameter. The target spots
5
form a spotted area
7
on the top surface
12
of the substrate
10
. The spotted area
7
in the substrate
10
of
FIG. 1A
is rectangular and has a broken line drawn around it.
A microarray can be used to detect complementary probes. The immobilized target spots on the microarray substrate are exposed to complementary DNA, protein, antigen, or chemical probe samples in liquid solution. The probe materials in solution, which are generally derived from cells, bodily fluids, or combinatorial chemistry libraries, are labeled with fluorescent dyes. The probe materials bind at complementary target spots on the microarray, and the dyes allow for subsequent detection and measurement of the relative concentration of each species of complementary probe material at each target spot. Other detection schemes may be used aside from fluorescence, such as the use of radioactive markers, chemiluminescence, and surface plasmon resonance (SPR).
In some references relating to microarrays, the nomenclature for the immobilized spot material on the microarray substrate (called “target” material here) and the solution applied to the spots for selective binding assays (called “probe” material here) is reversed.
Through a process called hybridization, DNA probe material in solution selectively binds to target spots on the microarray substrate only where complementary bonding sites occur. Similarly, labeled protein probe material only binds selectively to target spots with specific complementary bonding sites; this process is called affinity binding and incubation in protein and antibody assays. Selective reactions of smaller organic or inorganic chemicals (small molecules) to one another or to proteins or DNA can occur in the same way. DNA hybridization and the terminology associated with DNA microarrays will be used throughout this specification, but it is to be understood that the same processes and effects apply to these other types of microarrays.
After the reaction between the probe material and the target material is allowed to occur, quantitative scanning in a fluorescent microarray scanner produces a pixel map of fluorescent intensities. This fluorescent pixel map can be analyzed by special purpose quantitation algorithms to reveal the relative concentrations of the fluorescent probe materials at each target spot on the microarray, thus indicating the level of gene expression, protein concentration, or the like present in the cells from which the probe materials were extracted.
The microarray substrate is generally made of glass that has been treated chemically to provide for molecular attachment of the target spot samples of microarray target material. The substrate
10
can also be made of plastic, silicon, ceramic, metal, or other rigid material. The microarray substrate
10
is also generally of the same size and shape as a standard microscope slide, about 25 mm×75 mm×1 mm thick. The array area of target spots can extend to within about 1.5 mm of the edges of the substrate, although this array area can also be smaller. Typically, the target spots are approximately round. The target spot diameter can vary from about 50 microns to about 500 microns, depending on the dispensing or spotting technique used to apply the target spots to the microarray substrate. The center-to-center spacing between the target spots on the microarray substrate usually falls into the range of about 1.5 to 2.5 target spot diameters. The target spots are typically printed or “spotted” on the top surface of the substrate by pin-type spotting instruments which deposit droplets by a stamping process, where a small (<1 nanoliter) amount of liquid from the wetted end of the pin is transferred to the top surface of the substrate. Alternately, piezo-electric dispensers can dispense drops onto a substrate's activated surface (called the spotted area of the top surface here) in a manner similar to an ink-jet printer.
The protocols for producing the fluorescently labeled probe solutions can be fairly complex. For differential gene-expression DNA microarrays, exemplary probe preparation steps are:
Tissue or cell isolation
RNA extraction
RNA purification
Reverse transcription of RNA to cDNA
Attachment of the fluorescent label to all species of DNA the solution
Dye teminator cleanup
Addition of buffer to attain desired volume, concentration, pH, etc.
A common type of microarray is used for analyzing differential gene expression. Labeled probe material is prepared from each of two or more tissues or cell types; the RNA/cDNA extracted from each tissue type is labeled with a different dye. Then, the two or more labeled probes are mixed together and applied in solution form to the microarray. The probe mixture is kept in intimate contact with the immobilized target spots on the microarray for some number of hours, typically at a temperature above ambient temperature, to allow the complementary strands of DNA to come into contact with one another and to bind. This process is generally called “incubation,” and “hybridization” is used to refer to single-stranded DNA segments binding into a double-helix. In contrast, antibody-antigen assay incubation is often carried out at room temperature for times on the order of 5-60 minutes.
FIG. 1B
shows a side view of a typical cover glass arrangement that has been used for reacting the probe material with target spots. In the arrangement of
FIG. 1B
, the microarray substrate
10
is placed on a work surface
14
with the top surface
12
of the microarray substrate
10
having the spotted area
7
facing up. A selected volume of liquid probe solution
16
is then placed as a thin layer on the top surface
12
where the spotted area
7
(not shown) is located, and a cover glass
18
is placed over the liquid probe solution
16
. A typical volume of the liquid probe solution
16
is about 15-25 microliters. This small volume of liquid probe solution
16
is deposited on the spotted area
7
of the top surface
12
as a drop. The cover glass
18
placed on top of the drop of liquid probe solution
16
spreads the drop into a thin layer over the spotted area
7
of the top surface
12
with about the same dimensions as the cover glass
18
. The layer of liquid probe solution
16
can be about 10-60 microns thick and is kept in place by the capillary effect of being sandwiched between two planar pieces of glass. The dimensions of the spotted area
7
on the top surface
12
and the cover glass
18
are usually smaller than the dimensions of the microarray substrate
10
, but in some cases the spots, the cover glass
18
and the liquid probe solution
16
can cover the entire top surface
12
of the microarray substrate
10
.
The microarray substrate
10
with liquid probe solution
16
and the cover glass
18
is then placed in a sealed chamber of some sort to prevent the probe from evaporating or drying during incubation. Specially designed hybridization chambers are available for this (a Telechem Hybridization Cassette, for example), but many researchers use common labware such as 50 ml centrifuge tubes or Copeland jars. Often, a laboratory wipe or other absorbent object soaked with water is placed into the hybridization chamber with the microarray and probe liquid to keep th
Lombardo C. Todd
Milkowski Robert
Regan Donald
Schermer Mack J.
St. Cyr Paul
Chin Christopher L.
Counts Gary
Hale and Dorr LLP
Packard Bioscience Corporation
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