Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2000-01-04
2004-11-16
Celsa, Bennett (Department: 1639)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S089000, C435S091100, C435S174000, C435S177000, C436S501000
Reexamination Certificate
active
06818394
ABSTRACT:
BACKGROUND OF THE INVENTION
In the fields of molecular biology and biochemistry, as well as in the diagnosis of diseases, nucleic acid hybridization has become a powerful tool for the detection, isolation and analysis of specific oligonucleotide sequences. Typically, such hybridization assays utilize an oligodeoxynucleotide probe that has been immobilized on a solid support; as for example in the reverse dot blot procedure (Saiki, R. K., Walsh, P. S., Levenson, C. H., and Erlich, H. A. (1989)
Proc. Natl. Acad. Sci. USA
86, 6230). More recently, arrays of immobilized DNA probes attached to a solid surface have been developed for sequencing by hybridization (SBH) (Drmanac, R., Labat, I., Brukner, I., and Crkvenjakov, R. (1989)
Genomics
, 4, 114-128), (Strezoska, Z., Pauneska, T., Radosavljevic, D., Labat, I., Drmanac, R., and Crkvenjakov, R. (1991)
Proc. Natl. Acad. Sci. USA
, 88, 10089-10093). SBH uses an ordered array of immobilized oligodeoxynucleotides on a solid support. A sample of unknown DNA is applied to the array, and the hybridization pattern is observed and analyzed to produce many short bits of sequence information simultaneously. An enhanced version of SBH, termed positional SBH (PSBH), has been developed which uses duplex probes containing single-stranded 3′-overhangs. (Broude, N. E., Sano, T., Smith, C. L., and Cantor, C. R. (1994)
Proc. Natl. Acad. Sci. USA
, 91, 3072-3076). It is now possible to combine a PSBH capture approach with conventional Sanger sequencing to produce sequencing ladders detectable, for example by gel electrophoresis (Fu, D., Broude, N. E., Köster, H., Smith, C. L. and Cantor, C. R. (1995)
Proc. Natl. Acad. Sci. USA
92, 10162-10166).
For the arrays utilized in these schemes, there are a number of criteria which must be met for successful performance. For example, the immobilized DNA must be stable and not desorb during hybridization, washing or analysis. The density of the immobilized oligodeoxynucleotide must be sufficient for the ensuing analyses. There must be minimal non-specific binding of the DNA to the surface. In addition, the immobilization process should not interfere with the ability of the immobilized probes to hybridize and to be substrates for enzymatic solid phase synthesis. For the majority of applications, it is best for only one point of the DNA to be immobilized, ideally a terminus.
In recent years, a number of methods for the covalent immobilization of DNA to solid supports have been developed which attempt to meet all the criteria listed above. For example, appropriately modified DNA has been covalently attached to flat surfaces functionalized with amino acids (Running, J. A., and Urdea, M. S. (1990)
Biotechniques
, 8, 276-277), (Newton, C. R., et al., (1993)
Nucl. Acids. Res
., 21, 1155-1162.), (Nikiforov, T. T., and Rogers, Y. H. (1995)
Anal. Biochem
., 227, 201-209), carboxyl groups, (Zhang, Y., et al., (1991)
Nucl. Acids. Res
., 19 3929-3933), epoxy groups (Lamture, J. B. et al., (1994)
Nucl. Acids. Res
., 22, 2121-2125), (Eggers, M. D., et al., (1994)
BioTechniques
, 17, 516-524) or amino groups (Rasmussen, S. R., et al., (1991)
Anal. Biochem
., 198, 138-142). Although many of these methods were quite successful for their respective applications, the density of oligonucleotide bound (maximum of approximately 20 fmol of DNA per square millimeter of surface) (Lamture, J. B., et al., (1994)
Nucl. Acids. Res
. 22, 2121-2125), (Eggers, M. D., et al., (1994)
BioTechniques
, 17, 516-524), was far less than the theoretical packing limit of DNA.
Therefore, a method for achieving higher densities of immobilized nucleic acids on a surface is needed. In particular, a method for achieving higher densities of surface immobilized nucleic acids which permits use, manipulation and further reaction of the immobilized nucleic acids, as well as analysis of the reactions, is needed.
In connection with the need for improved nucleic acid immobilization methods for use, for example, in analytical and diagnostic systems, is the need to develop sophisticated laboratory tools that will automate and expedite the testing and analysis of biological samples. At the forefront of recent efforts to develop better analytical tools is the goal of expediting the analysis of complex biochemical structures. This is particularly true for human genomic DNA, which is comprised of at least about one hundred thousand genes located on twenty four chromosomes. Each gene codes for a specific protein, which fulfills a specific biochemical function within a living cell. Changes in a DNA sequence are known as mutations and can result in proteins with altered or in some cases even lost biochemical activities; this in turn can cause a genetic disease. More than 3,000 genetic diseases are currently known. In addition, growing evidence indicates that certain DNA sequences may predispose an individual to any of a number of genetic diseases, such as diabetes, arteriosclerosis, obesity, certain autoimmune diseases and cancer. Accordingly, the analysis of DNA is a difficult but worthy pursuit that promises to yield information fundamental to the treatment of many life threatening diseases.
Unfortunately, the analysis of DNA is made particularly cumbersome due to size and the fact that genomic DNA includes both coding and non-coding sequences (e.g., exons and introns). As such, traditional techniques for analyzing chemical structures, such as the manual pipeting of source material to create samples for analysis, are of minimal value. To address the scale of the necessary analysis, scientists have developed parallel processing protocols for DNA diagnostics.
For example, scientists have developed robotic devices that eliminate the need for manual pipeting and spotting by providing a robotic arm that carries at its proximal end a pin tool device that consists of a matrix of pin elements. The individual pins of the matrix are spaced apart from each other to allow each pin to be dipped within a well of a microtiter plate. The robotic arm dips the pins into the wells of the microtiter plate thereby wetting each of the pin elements with sample material. The robotic arm then moves the pin tool device to a position above a target surface and lowers the pin tool to the surface contacting the pins against the target to form a matrix of spots thereon. Accordingly, the pin tool expedites the production of samples by dispensing sample material in parallel.
Although this pin tool technique works well to expedite the production of sample arrays, it suffers from several drawbacks. First during the spotting operation, the pin tool actually contacts the surface of the substrate. Given that each pin tool requires a fine point in order that a small spot size is printed onto the target, the continuous contact of the pin tool against the target surface will wear and deform the fine and delicate points of the pin tool. This leads to errors which reduce accuracy and productivity.
An alternative technique developed by scientists employs chemical attachment of sample material to the substrate surface. In one particular process, DNA is synthesized in situ on a substrate surface to produce a set of spatially distinct and diverse chemical products. Such techniques are essentially photolithographic in that they combine solid phase chemistry, photolabile protecting groups and photo activated lithography. Although these systems work well to generate arrays of sample material, they are chemically intensive, time consuming, and expensive.
It is further troubling that neither of the above techniques provide sufficient control over the volume of sample material that is dispensed onto the surface of the substrate. Consequently, error can arise from the failure of these techniques to provide sample arrays with well controlled and accurately reproduced sample volumes. In an attempt to circumvent this problem, the preparation process will often dispense generous amounts of reagent materials. Although this can ensure sufficient sample volumes, it is wasteful of sample materials, which are of
Cantor Charles R.
Köster Hubert
O'Donnell-Maloney Maryanne J.
Celsa Bennett
Heller Ehrman White & McAuliffe LLP
Seidman Stephanie L.
Sequenom Inc.
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