Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving viable micro-organism
Reexamination Certificate
2001-05-14
2004-01-13
Redding, David A. (Department: 1744)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving viable micro-organism
C435S091200, C435S286500, C435S287200, C435S288300
Reexamination Certificate
active
06677131
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to devices, kits and methods for fluid processing of biological material, particularly processing biological material with controlled and minimal fluid quantities. The devices, kits and methods of the invention are particularly useful for hybridization of biomolecules, which may be bound to a substrate.
BACKGROUND OF THE INVENTION
High-throughput processing of biological material using fluids containing biomolecules is widely used in several fields. The processing of biological material surface mounted on planar substrates such as slides is known and used in various procedures. For example, biotechnology, biochemistry, molecular biology, molecular genetics, cytogenetics, cell biology, pharmacology, and immunology, are examples of fields in which such procedures have been used for analytical and diagnostic purposes. In a typical procedure, a slide containing immobilized biological material is contacted with a variety of fluids, and usually involves a number of processing steps. It is essential that the processing steps are carried out properly to obtain repeatable and comparable results.
Two examples of such procedures include hybridization of slide-bound nucleic acid probes with labeled targets that incorporate complementary nucleotide sequences and staining of biological material. In a procedure involving the staining of slide mounted specimens of biological materials, the prepared slides may be dipped successively into a series of small vessels or jars, each about 0.2 to about 2 liters in volumetric capacity, and each containing a particular liquid treating composition, such as washing agents, buffers, dehydrating agents, dyes and other solutions. Dye materials may be used to highlight different cells, structures of cells, intracellular structures, and cellular products. Resulting dyed slides may be washed and dried, possibly stored, and then microscopically examined. Selective staining procedures using specific antibody or gene probes have been developed and are advantageously highly specific and sensitive. However, such procedures require many successive steps to be carried out.
Another example of a procedure that utilizes slide-mounted biological materials is hybridization of biomolecules. Screening of biomolecules such as nucleic acids, protein sequences of amino acids, carbohydrates, lipids and living cells containing biomolecules provides information about changes in physiological, biochemical and molecular interactions of biological samples at the cellular and/or subcellular level. Techniques such as hybridization utilize an analyte and a complementary binding entity that form a bound pair of the analyte and the binding entity. Such hybridization can be performed in solution, or alternatively, the analyte can be immobilized on a support such as a glass slide and contacted with a solution containing a binding entity. Typically a pattern or an array of different analytes (usually called probes) are immobilized on a glass slide, and a solution containing a binding entity (usually called the target) contacts the array. Unbound binding entities can be washed from the slide, and various types of analytical technique involving, for example, phosphorescence, fluorescence, and radioactivity, can be performed to determine which specific sites or probes were bound to the target or targets.
In a more specific example, the high throughput screening of nucleic acids is typically performed by attaching base pairs of nucleic acid sequences in an array of locations on a glass plate or slide. Each spot location provides an address for later reference to each spot of nucleic acid. Hybridization techniques utilize markers such as radioactive or fluorescent compounds to label particular nucleic acid sequences that are complementary to the nucleic acid sequences on the glass slide. Signal measurement equipment is then utilized to measure each address on the array to determine if the labeled sequences have attached to the complementary sequence on the glass slide. The resulting slide is examined using an evaluation procedure such as, for example, microscopy, autoradiography, fluorescence measurement, photon emission, or the like. A single hybridization procedure may involve as many as thirty or more controlled step sequences.
Since multi-step processing of slide mounted biological materials typically involves numerous slides and a variety of process liquids and steps, it may be difficult to control the identical treatment of all slides. An additional concern in such processes is that some processing liquids, such as liquids containing nucleotides and other biomolecules, are very costly and must be used in small volumes.
Hybridization reactions are usually carried out in fluid-containing structures such as wells, bottles and other structures designed to contain the target-containing hybridization solution and a substrate containing an array of biomolecular probes. Conventional hybridization chamber devices, particularly hybridization chambers used with microarrays printed on a glass or plastic slide have several shortcomings.
For example, referring to
FIGS. 1-7
, which shows a typical and commercially available hybridization chamber assembly that includes three layers and a glass slide
10
, which is shown in FIG.
1
. The glass slide is provided with an array of biomolecules
12
arranged thereon, or an array of biomolecules may be printed on the surface of the glass slide
10
. An example of a suitable slide for printing microarrays of biomolecules such as DNA is a CMT-GAPS™ coated slide available from the assignee of the present invention. Referring now to
FIG. 3
, a frame
14
for a commercially available hybridization chamber frame typically includes three layers. The first layer is a cover sheet
16
, which may be made from glass or plastic and may include an inlet opening
18
and an outlet opening
20
. The frame includes a second frame layer
22
, which typically includes an adhesive backing and is attached to a disposable backing sheet
24
.
According to known hybridization procedures, the slide
10
containing an array of biomolecules
12
immobilized on the surface of the slide is typically washed separately before attachment of the hybridization chamber frame. Referring to
FIG. 4
, after the slide
10
has been washed and dried, the adhesive backing sheet
24
is peeled from the frame layer
22
and the frame layer
22
and cover sheet
16
are positioned over the microarray of biomolecules
12
on the slide
10
. Referring to
FIG. 5
, the frame layer
22
and cover sheet
10
are placed over the microarray of biomolecules
12
to surround the biomolecules and form a well structure
26
with the slide
10
.
As shown in
FIG. 6
, a mixture of hybridization solution containing target molecules is injected in the inlet opening
18
with a pipette
28
or other suitable fluid injection device. Excess fluid may flow out of the outlet opening
20
. After injection of the fluid, the hybridization chamber including the frame layer
22
, the cover sheet
16
and the slide
10
are left for a time and under conditions to allow hybridization of the target molecules in the hybridization solution and probe biomolecules
12
on the slide
10
. Referring now to
FIG. 7
, the frame layer
22
and the cover sheet
16
are removed from the slide
10
, and additional processing steps such as post hybridization washing of the slide
10
containing the biomolecules are performed. Thereafter, the slide
10
is scanned and analyzed.
As evidenced by the above discussion, the delivery and removal of fluids to a hybridization chamber as shown in
FIGS. 1-7
is not designed for rapid processing of slide-mounted material. It would be advantageous to provide a chamber frame having connectors that could be readily attached to standard laboratory tubing to facilitate the supply and removal of fluids from the well. Such a chamber would facilitate processing such slide-mounted material in a replicable manner. It would also be useful if the delivery and removal
Corning Incorporated
Kung Vincent T.
Redding David A.
Servilla Scott S.
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