Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1998-11-18
2001-10-30
Jones, W. Gary (Department: 1655)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091100, C435S287800, C435S288300, C435S288400, C435S288500, C435S288600, C536S023100, C536S025300
Reexamination Certificate
active
06309828
ABSTRACT:
TECHNICAL FIELD
The invention relates generally to the chemical, biological, medical and diagnostic applications of nucleic acid molecules. In particular, the present invention relates to a device for the synthesis and placement of a plurality of discrete nucleic acids in an array on a substrate.
BACKGROUND OF THE INVENTION
An array is a collection of nucleic acid molecules of a chosen length, arranged in a spatially defined and physically addressable manner. Arrays of nucleic acid molecules are used in a variety of screening techniques such as diagnostics, scanning, sequencing and analysis of probes or target molecules, An array of probes such as nucleic acid molecules can be fabricated by depositing the preformed nucleic acid molecules on a substrate, or by forming nucleic acids using in situ synthesis techniques. In nucleic acid sequencing and analysis, there is a growing emphasis on the use of high density arrays of immobilized nucleic acid probes. The arrays can be used to assay for activity against a particular receptor, for screening in drug discovery, for sequencing, and as diagnostics.
Such arrays can be prepared by massive parallel schemes, e.g., using the selective photomask techniques described in U.S. Pat. No. 5,445,934. Arrays constructed in this manner are typically formed on a planar area of between about 4-100 mm
2
, and can have densities of up to several hundred thousand or more distinct array members/cm
2
. However, this method is expensive and requires sophisticated equipment. Further, this method requires photomask cells which are four times the nucleic acid probe length. Additionally, the photo-deprotection step is not as efficient as chemical deprotection, thus resulting in lower quality probes.
Conventional in situ synthesis using nanoliter drops can also be used to fabricate arrays, as described in, for example, U.S. Pat. No. 5,474,796 issued to Brennan. However, the number of arrays that can be produced in parallel using this technique is limited. Thus, fabrication of a large number of replicates of the same array is both labor- and time-intensive.
In order to avoid problems inherent in in situ synthesis techniques, polynucleotides can be synthesized prior to attachment to an appropriate substrate. U.S. Pat. Nos. 5,529,756 and 5,472,672, both issued to Brennan, describe an apparatus and a method for polymer synthesis. Preformed nucleic acid molecules can then be deposited on a substrate using several techniques, such as the method described in WO 95/35505 and U.S. Pat. No. 5,807,522. However, synthesis of such polynucleotides is laborious and expensive. Moreover, commercially available DNA synthesizers, such as the ABI 394 DNA Synthesizer, are limited to producing only 4 different oligonucleotides at a time.
Further, the transfer of nucleic acid molecule solutions from the original containers/chambers to the substrate requires a spatial translation from the original format to the final format. This translation is time-intensive and limits the number of parallel transfers possible. For example, WO 95/35505 and U.S. Pat. No. 5,807,522 describe a system to deposit presynthesized materials, wherein a pen-like capillary is used to transfer DNA or c-DNA solutions from a 96 well plate to glass microscope slides. The pen is dipped in the stock solution, and touched on the substrate to spot the liquid on the slide. However, this method has several disadvantages. For instance, the tips must be cleaned before contacting a different probe, and the tips dull easily. Further, the stock solutions are in a different spatial format from that of the actual array. This difference in formats limits the number of parallel transfers, making this process very time-consuming. For example, it would take approximately 50 hours to fabricate 100 arrays of 6000 different probes.
The above problems can be solved in part using robotics. However, current robotic technology can handle only a limited number of samples simultaneously.
Thus, there is a need for an improved apparatus and method to achieve more efficient fabrication of high density arrays, wherein the spatial format of the source of the nucleic acid molecules is similar to the array format, especially when the spatial format of the nucleic acid source is integral to the spatial formation of the deposition system and that of the final array.
SUMMARY OF THE INVENTION
The present method provides an efficient means for arranging an array of nucleic acid molecules onto a substrate. Unlike conventional methods, the present method does not rely on intricate masking/lithographic, protecting, and selective activation techniques to form an array, or labor-intensive methods for applying discrete aliquots of preformed molecules to the substrate. Accordingly, the present invention provides an improved and cost-effective method and device for the fabrication and placement of an array of nucleic acid molecules onto a substrate.
In one aspect, the invention relates to a device for applying an array of nucleic acid molecules onto a substrate, comprising a synthesis unit, and optionally a purification unit and a printing unit. The synthesis unit comprises a plurality of synthesis chambers with corresponding synthesis outlet means, wherein the synthesis chambers are spatially arranged relative to each other to provide an array suitable for conducting parallel nucleic acid syntheses, and wherein the spatial format of the synthesis chambers is integral to the spacial format of the array of nucleic acid molecules on the substrate. Each synthesis chamber may comprise a reactive surface on which nucleic acid molecules are synthesized. For example, the reactive surface can comprise controlled pore glass (CPG) in solution, such as CPG beads in a microtiter well, or in a flow-through format, such as a CPG column or a membrane. Examples of outlet means include, but are not limited to, capillary tubing, any microscaled outlet, pipettes, micropipette tips, inkjet components, pens and the like. The nucleic acid molecules can be directly transferred from the outlet means to a substrate. The outlet means may optionally include partitioning means for separating and/or purifying nucleic acid molecules of different lengths as they pass through the outlet means from the chambers. Further, the synthesis chambers can comprise inlet means which interface with printers or dispensers such as inkjet components or flow-through methodologies, for the introduction of reagents, samples, solutions and the like.
In an alternative embodiment, the invention device further comprises a printing unit wherein the printing unit comprises a plurality of printing chambers with corresponding printing outlet means, and further wherein the spatial format of the printing chambers is correspond to the spacial format of the synthesis unit. The nucleic acid solution is transferred from the synthesis unit into the printing chambers. The nucleic acid solution is then deposited onto the substrate by contacting the printing unit with the substrate. In one embodiment, the printing unit is a rubber stamp or a capillary deposition unit. In a preferred embodiment, the printing unit is an inkjet device.
In another alternative embodiment, the device further comprises a purification unit comprising a plurality of purification chambers with corresponding purification outlet means, wherein the spatial format of the purification chambers corresponds to the spacial format of the synthesis unit. The nucleic acid molecule solution is transferred from the synthesis unit into the purification unit for separation and/or purification of the solution. The purified solution is then transferred into the printing unit and deposited on the substrate as described above.
In another embodiment, the device for applying an array of nucleic acid molecules onto a substrate comprises a synthesis unit, a purification unit, and a printing unit; wherein each of the units is configured as described above.
In another aspect, the invention relates to a method for applying an array of nucleic acid molecules onto a substrate
Caren Michael P.
Hotz Charles Z.
Leonard Leslie A.
Perbost Michel G. M.
Schleifer Arthur
Agilent Technologie,s Inc.
Forman B J
Jones W. Gary
LandOfFree
Method and apparatus for fabricating replicate arrays of... does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Method and apparatus for fabricating replicate arrays of..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Method and apparatus for fabricating replicate arrays of... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2594504