Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1998-07-21
2001-06-19
Fredman, Jeffrey (Department: 1639)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091200, C435S071100, C427S333000
Reexamination Certificate
active
06248521
ABSTRACT:
TECHNICAL FIELD
This invention relates generally to enzymatic reactions performed on nucleic acids that are arrayed on a solid substrate, and in particular, to amplification of nucleic acids that are arrayed.
BACKGROUND OF THE INVENTION
Replicate arrays of biological agents have been used to facilitate parallel testing of many samples. For example, sterile velvet cloths and a piston-ring apparatus has long been used to make replicates of bacterial and yeast colonies to agar plates each containing a different growth medium, as a means of rapidly screening a large number of independent colonies for different growth phenotypes (Lederberg and Lederberg,
J. Bacteriol
. 63 :399, 1952). Likewise, 96-well microtiter plates are used to organize and store in an easily accessed fashion large numbers of e.g. cell lines, virus isolates representing recombinant DNA libraries, or monoclonal antibody cell lines.
The advent of large scale genomic projects and the increasing use of molecular diagnostics has necessitated the development of large volume throughput methods for screening nucleic acids. Recently, methods have been developed to synthesize large arrays of short oligodeoxynucleotides (ODNs) bound to a glass or silicon surface that represent all, or a subset of all, possible nucleotide sequences (Maskos and Southern,
Nucl. Acids Res
. 20: 1675, 1992). These ODN arrays have been made used to perform DNA sequence analysis by hybridization (Southern et al.,
Genomics
13: 1008, 1992; Drmanac et al.,
Science
260: 1649, 1993), determine expression profiles, screen for mutations and the like. In all these uses, the ODNs are covalently attached to the surface of the substrate. However, some useful screening techniques and assays are not readily adaptable to a format in which ODNs are immobilized.
In particular, amplification of nucleic acids, notably the polymerase chain reaction (PCR) and its many variations, has found wide application to many different many biological problems and is not easily moved to a format where the ODNs are immobilized. In its standard format, PCR has two major limitations to its commercial utilization: the cost of reagents and the ability to automate the process. Reagent costs, especially DNA polymerase, can be lowered if the total volume of each reaction is decreased. An accurate and reliable means to array small volumes of reagents using a robotically controlled pin tool would miniaturize the reactions. Additional hurdles to moving amplification to an array format include preventing evaporation during heating and cooling cycles and preventing spreading and merging of the reactions on the array.
The present invention discloses methods and compositions for performing amplification and other enzymatic reactions in an array format without the need to immobilize the components, and further provides other related advantages.
SUMMARY OF THE INVENTION
Within one aspect of the present invention, methods of amplifying nucleic acid molecules from a template are provided comprising (a) mixing single-stranded nucleic acid templates on a solid substrate with a solution comprising an oligonucleotide primer that hybridizes to the templates and a DNA polymerase, wherein the mixing occurs in discrete areas on the substrate, and wherein the solution remains in the discrete areas; (b) synthesizing a complementary strand to the template to form a duplex; (c) denaturing the duplex; and (d) synthesizing complementary strands to the template, therefrom amplifying nucleic acid molecules; wherein mixing, synthesizing, and denaturing are conducted at dew point. The solid substrate may be a silicon wafer or glass slide. The templates may be covalently attached to the solid substrate or deposited on the surface of the substrate. The template may be uniformly applied to the entire array prior to mixing or applied individually to each discrete area on the substrate. When applied individually, preferably the applying is performed using spring probes. In a most preferred embodiment, an apparatus is used to control the dew point.
Within a related aspect, the method of amplifying uses a first oligonucleotide primer that hybridizes to the templates, a second oligonucleotide primer that hybridizes to a complementary strand of the template, and after sythesizing, denaturing the duplex; and synthesizing complementary strands to the template and the complementary strand of the template, therefrom amplifying nucleic acid molecules.
In preferred embodiments, the denaturing and synthesizing steps are performed multiple times. In other preferred embodiments, the solution contains a compound that confers viscosity, such as glycerol or a sugar. In other preferred embodiments, the DNA polymerase is a thermostable polymerase and synthesis and denaturation are performed at different temperatures.
In yet other preferred embodiments, the method further comprises detecting the duplexes. Most preferably, the oligonucleotide primers are labeled with a tag that is detectable by non-fluorescent spectrometry or potentiometry, and preferably by mass spectrometry, infrared spectrometry, ultraviolet spectrometry, or poteniostatic amperometry.
In another aspect, a method of synthesizing a nucleic acid molecule from a template is provided, comprising (a) mixing single-stranded nucleic acid templates on a solid substrate with a solution comprising an oligonucleotide primer that hybridizes to the templates and a DNA polymerase, wherein the mixing occurs in a discrete area of an array, and wherein the solution remains in discrete areas; and (b) synthesizing a complementary strand to the template to form a duplex, wherein mixing and synthesis are performed at dew point, wherein dew point is maintained or achieved by an apparatus, comprising: a container capable of being pressurized; a heating device; a means for generating pressure; and a means for generating saturated steam; wherein the heating device, pressure generating means, and steam generating means are controllable.
In yet another aspect, a method of detecting a single base alteration in a nucleic acid molecule, is provided comprising (a) mixing single-stranded nucleic acid molecules on a solid substrate with a solution comprising a first and a second oligonucleotides that hybridize to the nucleic acid molecules and a DNA ligase, wherein the mixing occurs in a discrete area of an array, and wherein the solution remains in the discrete areas; and (b) detecting a ligation product; wherein the first and second oligonucleotides will not ligate when there is a single base alteration at the junction base on the nucleic acid molecule, wherein mixing is performed at dew point.
In yet another aspect, a method of performing single nucleotide extension assay is provided, comprising (a) mixing oligonucleotides on a solid substrate with a solution comprising single-stranded nucleic acid molecules that hybridize to the oligonucleotides, a single nucleotide, and a DNA polymerase, wherein the mixing occurs in discrete areas of the substrate, and wherein the solution remains in discrete areas; and (b) detecting an extension product of the oligonucleotide; wherein the oligonucleotide will be extended only when the single nucleotide is complementary to the nucleotide adjacent to the hybridized oligonucleotide, wherein mixing is performed at dew point.
In other aspects, the invention provides a kit for genotyping, comprising a solid substrate containing an array of labeled oligonucleotide primer pairs. In preferred embodiments, the kit further comprises nucleic acid templates and a viscous solution.
These and other aspects of the present invention will become evident upon reference to the following detailed description and attached drawings. In addition, various references are set forth below which describe in more detail certain procedures or compositions (e.g., plasmids, etc.), and are therefore incorporated by reference in their entirety.
The methods and kits of the present invention may include tagged biomolecules, for example, oligonucleotides covalently bonded to cleavable tags. E
Moynihan Kristen
Ness Jeffrey Van
Tabone John C.
Chakrabarti Arun
Fredman Jeffrey
QIAGEN Genomics, Inc.
Seed Intellectual Property Law Group PLLC
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