Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1999-11-02
2001-07-31
Brusca, John S. (Department: 1631)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S007100, C435S283100, C435S285200, C435S286500, C530S351000, C536S023100
Reexamination Certificate
active
06268147
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates generally to the fields of molecular biology and nucleic acid analysis. More specifically, the present invention relates to a novel method of nucleic acid analysis using tandem hybridization approaches.
2. Description of the Related Art
The emerging field of DNA technology is bringing powerful analytical capabilities in research and clinical laboratories. A prime example is the identification of mutations causing genetic diseases, such as the detection of &Dgr;F508 mutation responsible of cystic fibrosis disease (Riordan et al,
Science
245:1066-1073, 1989). Many technical approaches have been devised to search for the presence of one or a few mutations in a single assay. However, it is now evident that many genetic diseases can be caused by a wide variety of mutations in the gene altered, as with cystic fibrosis, where more than 200 different mutations in the CFTR gene have been detected in CF patients (Tsui,
Trends Genet
. 8:392-398, 1992). Several analytical approaches have recently been proposed to simultaneously investigate multiple mutations in a single assay. For example, fluorescently labeled allele-specific oligonucleotides have been used in the analysis of several mutations by PCR (Heller, In
The Polymerase Chain Reaction
, Mullis et al, Eds., Birkhäusen, Boston, pp. 134-141, 1994) or the ligase chain reaction (Jou et al,
Human Mutat
. 5:86-93, 1995; Eggerdin,
Human Mutat
. 5:153-165, 1995). Oligonucleotide hybridization shows promise as a rapid and sensitive method for simultaneous analysis of large numbers of mutations (Conner et al,
Proc. Natl. Acad. Sci., USA
. 80:278-282, 1983; Southern, International patent application PCT GB 89/00460, 1988; Beattie et al,
Clin. Chem
. 39:719-722, 1992; Southern et al, Genomics 13:1008-1017, 1992; Maskos & Southern,
Nucl. Acids Res
. 20:1675-1678, 1992; Mirzabekov,
Trends Biotechnol
. 12:27-32, 1994; Case-Green et al, In
Innovation and Perspectives in Solid Phase Synthesis, Proc
. 3
rd International Symposium on Solid Phase Synthesis
, Epton, Ed., Mayflower Worldwide Ltd., Birmingham, U.K., pp. 77-82, 1994; Pease et al,
Proc. Natl. Acad. Sci., U.S.A
. 91:5022-5026, 1994; Nikiforov et al,
Nucl. Acids Res
. 22:4167-4175, 1994; Beattie et al,
Molec. Biotechnol
. 4:213-225, 1995; Beattie et al,
Clin. Chem
. 41:700-706, 1995; Parinov et al,
Nucl. Acids Res
. 24:2998-3004, 1996; Yershov et al,
Proc. Natl. Acad. Sci., U.S.A
. 93:4913-4918, 1996; Hacia et al,
Nature Genetics
14:441-447, 1996; Southern,
Trends Genet
. 12:110-115, 1996). Various strategies have been described which enable simultaneous analysis of numerous oligonucleotide hybridization reactions. The most common strategy employed to separately analyze the hybridization of numerous oligonucleotide probes to a nucleic acid analyte is to immobilize each oligonucleotide at a specific, addressable site on a surface, then to label the analyte nucleic acid, hybridize it to the oligonucleotide array, and measure the relative quantity of label bound at each position across the array, using a CCD imaging system, a scanning confocal microscope, phosphorimager, exposure of X-ray film, etc. The inverse situation to the use of oligonucleotide arrays to analyze a nucleic acid sample is to immobilize numerous nucleic acid samples in a two-dimensional array, then to analyze the binding of a DNA probe to each of the arrayed analytes. Included in the latter approach are membrane hybridizations using size-separated nucleic acid fragments (as in Southern blots and Northern blots) and slot blots and dot blots in which each analyte is placed onto the membrane at a specific location. In addition, high density arrays of genomic clones, cDNAs, gene-specific amplicons or other PCR products, immobilized onto membranes or onto glass or silicon surfaces, are frequently used in hybridizations with oligonucleotide probes or longer nucleic acid fragments, for genome mapping, genotyping and gene expression profiling. Another approach to multiplex DNA hybridization is to immobilize each DNA probe to microbeads color-coded with a specific “signature” of fluorophores, then to hybridize the analyte nucleic acid labeled with a molecular tag with the bead mixture and analyze the mixture by flow cytometry, using the fluorescent signature to resolve each probe and the molecular tag to quantitate the binding of analyte to each probe (FlowMetrix method of Luminex, Inc.).
Simultaneous hybridization of a DNA sample to numerous oligonucleotide probes attached to a solid support material (“DNA chip,” or “genosensor”) has been proposed as a powerful research tool in various kinds of DNA sequence analysis, including sequencing by hybridization, scanning for known or unknown mutations in a gene of known nucleotide sequence, genotyping of organisms, and genome mapping (Southern, International patent application PCT GB 89/00460, 1988; Beattie et al,
Clin. Chem
. 39:719-722, 1992; Southern et al,
Genomics
13:1008-1017, 1992; Maskos & Southern,
Nucl. Acids Res
. 20:1675-1678, 1992; Mirzabekov,
Trends Biotechnol
. 12:27-32, 1994; Case-Green et al, In
Innovation and Perspectives in Solid Phase Synthesis, Proc
. 3
rd International Symposium on Solid Phase Synthesis
, Epton, Ed., Mayflower Worldwide Ltd., Birmingham, U.K., pp. 77-82, 1994; Pease et al,
Proc. Natl. Acad. Sci., U.S.A
. 91:5022-5026, 1994; Nikiforov et al,
Nucl. Acids Res
. 22:4167-4175, 1994; Beattie et al,
Molec. Biotechnol
. 4:213-225, 1995; Beattie et al,
Clin. Chem
. 41:700-706, 1995; Parinov et al,
Nucl. Acids Res
. 24:2998-3004, 1996; Yershov et al,
Proc. Natl. Acad. Sci., U.S.A
. 93:4913-4918, 1996; Hacia et al,
Nature Genetics
14:441-447, 1996; Southern,
Trends Genet
. 12:110-115, 1996; Bains & Smith,
J. Theor. Biol
. 135:303-307, 1988; Drmanac et al,
Genomics
4:114-128, 1989; Khrapko et al,
FEBS Lett
. 256:118-122, 1989; Khrapko et al,
DNA Sequence
1:375-388, 1991; Bains,
Genomics
11:294-301, 1991; Fodor et al,
Science
251:767-773, 1991; Drmanac & Crkvenjakov,
Int. J. Genome Res
. 1:59-79, 1992; Drmanac et al,
Science
260:1649-1652, 1993; Bains,
DNA Sequence
4:143-150, 1993; Meier-Ewert et al,
Nature
361:375-376, 1993; Broude et al,
Proc. Natl. Acad. Sci., USA
91:3072-3076, 1994; Hoheisel,
Trends Genet
. 10:79-83, 1994; Drmanac & Drmanac,
BioTechniques
17:328-336, 1994; Lamture et al,
Nucl. Acids Res
. 22:2121-25, 1994; Caetano-Anolles,
Nature Biotechnol
. 14:1668-1674, 1996; Lockhart et al,
Nature Biotechnol
. 14:1675-1680, 1996; Milner et al,
Nature Biotechnol
. 15:537-541, 1997). Despite widespread interest generated about the various multiplex hybridization technologies, several technical challenges remain to be solved before these techniques can reach their full potential and be successfully implemented in a robust fashion. One problem, anticipated from the beginning, is the spontaneous formation of secondary structure in the single stranded target nucleic acid, making certain stretches of target sequence poorly accessible to hybridization (Case-Green et al, In
Innovation and Perspectives in Solid Phase Synthesis, Proc
. 3
rd International Symposium on Solid Phase Synthesis
, Epton, Ed., Mayflower Worldwide Ltd., Birmingham, U.K., pp. 77-82, 1994; Beattie et al,
Clin. Chem
. 41:700-706, 1995; Milner et al,
Nature Biotechnol
. 15:537-541, 1997). This problem may be especially difficult when short oligonucleotide probes are used, wherein the hybridization temperature is too low to disrupt some regions of intrastrand secondary structure. Many applications of membrane-, chip- or bead-based hybridization technologies, especially those requiring base mismatch discrimination, may require short probes. One strategy for minimizing the secondary or higher order structure in the DNA target is to fragment the target sequence to very small size. However, such cleavage is difficult to control and does not solve the problem in the case of strong hairpin loops occurring within a short target sequence. The strategy of conv
Beattie Kenneth Loren
Rodriguez Rogelio Maldonado
Adler Benjamin Aaron
Brusca John S.
Siu Stephen
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