Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound containing saccharide radical
Reexamination Certificate
2000-10-10
2003-10-14
Siew, Jeffrey (Department: 1637)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound containing saccharide radical
C435S006120, C435S007100, C435S091100, C435S287200, C530S333000, C530S333000, C530S333000, C530S333000, C530S333000, C530S333000
Reexamination Certificate
active
06632641
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to a method and apparatus for performing a large number of reactions using array assembly. In particular, the present invention features a method and apparatus for performing a large number of chemical and biological reactions by bringing two arrays into close apposition and allowing reactants on the surfaces of two arrays to come into contact. The present invention is exemplified by performing a large number of polynucleotide amplification reactions using array assembly. In addition, the present invention features a method and apparatus for coupling the amplification of polynucleotides and the detection of sequence variations, expression levels, and functions thereof.
BACKGROUND OF THE INVENTION
Intense efforts are under way to map and sequence the human genome and the genomes of many other species. In June 2000, the Human Genome Project and Celera Genomics announced that a rough draft of the human genome had been completed. This information, however, represents only a reference sequence of the 3-billion-base human genome. The remaining task lies in the determination of sequence variations (e.g., mutations, polymorphisms, haplotypes) and sequence functions, which are important for the study, diagnosis, and treatment of human genetic diseases.
In addition to the human genome, the mouse genome is being sequenced. Genbank provides about 1.2% of the 3-billion-base mouse genome and a rough draft of the mouse genome is expected to be available by 2003 and a finished genome by 2005. The Drosophila Genome Project has also been completed recently. Thus far, genomes of more than 30 organisms have been sequenced.
Traditional nucleic acid sequencing methods include the chemical cleavage method (or the Maxam-Gilbert method) and the chain termination method (or the Sanger method) (Sambrook et al.,
Molecular Cloning: A Laboratory Manual,
2
nd
Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989). The basic strategy for the chemical cleavage method is to specifically cleave the end-labeled DNA at only one type of nucleotide, which produces a set of labeled fragments. These labeled fragments are then separated according to their size by electrophoresis. The DNA sequence can be directly read off an autoradiogram. The chain termination method utilizes a DNA polymerase to make complementary copies of the single-stranded DNA being sequenced in the presence of a suitable primer and four deoxynucleoside triphosphates (dNTPs), of which at least one is labeled. In addition, a small amount of the 2′, 3′-dideoxynucleoside triphosphate of one of the bases is added to the sequencing reaction, which generates a series of truncated chains. Each truncated chain is terminated by the dideoxy analog at positions occupied by the corresponding base, because of the absence of a 3′-OH group. Electrophoresis separates these truncated chains according to their sizes, thus indicating the positions at which dideoxy incorporation occurs and in turn the corresponding normal nucleotide. Although the efficiency of the traditional sequencing methods has been improved by automation, the use of gel electrophoresis in both methods presents a limitation on the rate of sequencing.
While the traditional chemical cleavage and chain termination sequencing methods are capable of identifying the sequence of all nucleotides in a target nucleic acid, it is quite sufficient in many cases to know the sequence identity of a single nucleotide (or a few nucleotides) at a predetermined site, i.e., the detection of known sequence variations. During the past decade, the development of array-based hybridization technology has received great attention. This high throughput method, in which hundreds to thousands of polynucleotide probes immobilized on a solid surface are hybridized to target nucleic acids to gain sequence and function information, has brought economical incentives to many applications. See, e.g., McKenzie, S., et al.,
European Journal of Human Genetics
416-429 (1998), Green et al.,
Curr. Opin. in Chem. Biol.
2:404-410 (1998), Gerhold et al.,
TIBS,
24:168-173 (1999), Young,
Cell
102:9-15 (2000), and U.S. Pat. Nos. 5,700,637, 6,054,270, 5,837,832, 5,744,305, and 5,445,943.
DNA array-based sequencing technology generally falls into two categories. The first category is sequencing by polynucleotide hybridization. Sets of polynucleotide probes, that differ by having A, T, C, or G substituted at or near the central position, are immobilized on a solid support by in situ synthesis or by deposition of pre-synthesized polynucleotide probes. Labeled target nucleic acids containing the sequences of interest will hybridize best to perfectly matched polynucleotide probes, whereas sequence variations will alter the hybridization pattern, thereby allowing the determination of mutations and polymorphic sites (Wang, D., et al.,
Science
280:1077-1082 (1998), Lipshutz, R., et al.,
Nature Genetics Supplement
21:20-24 (1999), and Drmanac et al.,
Nature Biotechnology
16:5-58 (1998)).
Alternatively, the de novo sequencing of target nucleic acids by polynucleotide hybridization may also be accomplished. For example, an array of all possible 8-mer polynucleotide probes may be hybridized with fluorescently labeled target nucleic acids, generating large amounts of overlapping hybridization data. The reassembling of this data by computer algorithm can determine the sequence of target nucleic acids. See, e.g., Drmanac, S. et al.,
Nature Biotechnology
116:54-58 (1998), Drmanac, S. et al.
Genomics
4:114-28 (1989), and U.S. Pat. Nos. 5,202,231, 5,492,806, 5,525,464, 5,667,972, 5,695,940, 5,972,619, 6,018,041, and 6,025,136.
The second category is sequencing by primer extension reactions (also known as minisequencing). Typically, a DNA polymerase is used specifically to extend an interrogation primer, which anneals to the nucleic acids immediately 3′ of the single base substitution of interest, with a single labeled nucleoside triphosphate complementary to the single base substitution (Syvänen,
Human Mutation
13:1-10 (1999), Syvänen et al.,
Genomics
8:684-692 (1990), Sokolov,
Nucleic Acids Res.
18:3671 (1990), and Kuppuswami et al.,
Proc. Natl. Acad. Sci. USA
88:1143-1147 (1991)).
While methods of hybridization and primer extension-based nucleic acid sequencing have gained widespread acceptance in commercial areas, there are many limitations to the existing methods. The current methods for determining polynucleotide variations in a target nucleic acid employ discrete amplification steps and sequencing steps (Landegren et al.,
Genome Res.
8:769-776 (1998)). Thus, additional amount of time and labor is required to separate amplification products from the amplification primers and dNTPs before the sequencing reaction. Further, it is estimated that at least about 3,000,000 single nucleotide polymorphisms (SNPs) exist in an individual's genome. As SNPs are dispersed throughout the genome, it is necessary to amplify a large number of discrete regions in the genome so that each SNP can be analyzed. Accordingly, the genetic analysis of a single individual's SNPs can require more than 3,000,000 amplification reactions be carried out and the product of each amplification reaction be analyzed. In addition, genetic analysis of a disease may require extensive genotyping of hundreds of thousands of individuals. Therefore, the number of separate amplification and sequencing reactions can be in the millions. The cost in terms of time, labor, equipment, laboratory space and reagents for carrying out discrete amplification and sequencing reactions on a large-scale is prohibitively high. Finally, the designing, optimizing and manufacturing of probe-immobilized arrays can be costly as well. For example, photolithographic synthesis of an array with N-mer polynucleotides typically requires 4×N different chrome photolithographic masks (i.e., 100 different chrome masks for a 25-mer synthesis) (Singh-Gasson et al.,
Nature Biotechnol.
17:974-
Berninger Mark
Brennan Thomas M.
Chatelain François
Halluin Albert P.
Howrey Simon Arnold & White , LLP
Metrigen, Inc.
Siew Jeffrey
LandOfFree
Method and apparatus for performing large numbers 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 performing large numbers of..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Method and apparatus for performing large numbers of... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-3117567