Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2001-04-19
2003-08-19
Horlick, Kenneth R. (Department: 1637)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091100, C435S091200, C536S023100, C536S024300, C536S024330, C536S025300
Reexamination Certificate
active
06607888
ABSTRACT:
1. INTRODUCTION
The present invention relates to methods for analyzing nucleic acid reactions in general and transcription reactions in particular. The method utilizes individual nucleic acid molecules immobilized along their length on a planar substrate. Using optical techniques, such as epifluorescent microscopy, the reactions of individual nucleic acid molecules can be studied using the method described herein.
The present invention also relates to scalable and massively automatable methods for imaging nucleic acid reactions, either in stop-action fashion or in real time. Bayesian inference estimation methods are utilized to analyze a population of images and to produce data sets of genome-sized scale to be used in the identification of genes, promoter regions, termination regions, or virtually any other phenomena associated with transcription or reverse-transcription of nucleic acid molecules. The method can be used to fabricate maps of transcription events, to correlate these maps to restriction site maps, and to use these data to identify from where in a sequenced genome a single nucleic acid molecule originated.
2. BACKGROUND
The analysis of nucleic acid molecules at the genome level is an extremely complex endeavor which requires accurate, rapid characterization of large numbers of often very large nucleic acid molecules via high throughput DNA mapping and sequencing. The construction of physical maps, and ultimately of nucleotide sequences, for eukaryotic chromosomes currently remains laborious and difficult. This is due, in part, to the fact that current procedures for mapping and sequencing DNA were originally designed to analyze nucleic acids at the gene, rather than at the genome, level (Chumakov, et al., 1992, Nature 359:380; Maier, et al., 1992, Nat. Genet. 1:273).
2.1. DNA Sequencing
Approaches to DNA sequencing have varied widely, and have made it possible to sequence entire genomes, including portions of the human genome. The most commonly used method has been the dideoxy chain termination method of Sanger (1977, Proc. Natl. Acad. Sci. USA 74:5463). However, this method is time-consuming, labor-intensive and expensive, requiring the analysis of four sets of radioactively labeled DNA fragments resolved by gel electrophoresis to determine the DNA sequence.
To overcome some of these deficiencies, automated DNA sequencing systems were developed which used four fluorescently labeled dideoxy nucleotides to label DNA (Smith et al., 1985, Nucleic Acids Res. 13:2399-2412; Smith et al., 1986, Nature 321:674; Prober et al., 1987, Science 238:336-341, which are incorporated herein by reference). Automated slab gel electrophoresis systems enable large-scale sequence acquisition (Roach et al., 1995, Genomics 26:345-353; Venter et al., 1996, Nature 381:364-366; Profer et al., 1987, Science 238:336-341; Lake et al., 1996, Science 273:1058; Strathmann et al., 1991, Proc. Natl. Acad. Sci. USA 88:1247-1250; and the complete genomic sequence of
Saccharomyces cerevisiae
in the Stanford database). Current large-scale sequencing is largely the domain of centers where costly and complex support systems are essential for the production efforts. Efforts to deal with sequence acquisition from a large population (usually less than 1,000) is limited to relatively small numbers of loci (Davies et al., 1995, Nature 371:130-136). However, these methods are still dependent on Sanger sequencing reactions and gel electrophoresis to generate ladders and robotic sample handling procedures to deal with the attending numbers of clones and polymerase chain reacting products.
Some recently developed methods and devices for automated sequencing of bulk DNA samples that utilize fluorescently labeled nucleotides are described in U.S. Pat. No. 5,674,743; International Application Nos. PCT/GB93/00848 published Apr. 22, 1993 as WO 93/21340; PCT/US96/08633 published Jun. 4, 1996 as WO 96/39417; and PCT/US94/01156 published Jan. 31, 1994 as WO 94/18218. None of the recently developed methods is capable of sequencing individual nucleic acid molecules.
Techniques for sequencing large genomes of DNA have relied upon the construction of Yeast Artificial Chromosomes (“YAC”) contiguous sequences. Preliminary physical maps of a large fraction of the human genome have been generated via YACs (Cohen et al., 1993, Nature 366:698-701). However, extensive high resolution maps of YACs have not been widely generated, due to the high frequency of rearrangement/chimerism among YACs, the low complexity of fingerprints generated by hybridization approaches, and the extensive labor required to overcome these problems. Ordered maps of YACs have been optically made by using a spermine condensation method (to avoid shearing the DNA) and fixing the clones in molten agarose onto derivatized glass surfaces (Cai et al., 1995, Natl. Acad. Sci. USA 92:5164-5168). There have been several proposals for the rapid attainment of sequence data from clones that minimize or obviate the need for shotgun sequencing approaches or subcloning of large insert clones (Smith et al., 1994, Nature Genet. 7:40-47; Kupfer et al., 1995, Genomics 27:90-100; Chen et al., 1993, Genomics 17:651-656 and Roach et al., 1995, Genomics 26:345-353). Several of these approaches advocate the generation of “sequence sampled maps” (Smith et al., 1994, Nature Genet. 7:40-47 and Venter et al., 1996, Nature 381:364-366) which require fingerprinting of clones, or large numbers of subclones, to achieve good target coverage while simultaneously generating a fine-scale map.
A recent development has been the proposal of DNA sequencing of aligned and oriented Bacterial Artificial Chromosomes (“BAC”) contiguous sequences (Venter et al., 1996, Nature 381:364-366); (see also Smith et al., 1994, Nature Genetics 7:40-47; Kupfer et al., 1995, Genomics 27:90-100; and Chen et al., 1993, Genomics 17:651-656). BACs offer the advantage of considerably greater stability than YACs, are more easily physically managed due to their smaller size (
~
500 kb to 2 Mb versus
~
100 to 200 kb, respectively), and are more compatible with automated DNA purification procedures (Kim et al., 1996, Proc. Natl. Acad. Sci. USA 93:6297-6301; Kim et al., 1994, Genomics 24:527-534; and Schmitt et al., 1996, Genomics 33:9-20). Further approaches for the optical analysis of BAC clones were also developed (Cai et al., 1998, Proc. Natl. Acad. Sci. USA 95:3390-3395).
Limitations of these approaches described above include low throughput, DNA fragmentation (preventing subsequent or simultaneous multimethod analyses), and difficulties in automation. Despite the potential utilities of these and other approaches, it is increasingly clear that current molecular approaches were developed primarily for characterization of single genes, not entire genomes, and are, therefore, not optimally suited to the analysis of polygenic diseases and complex traits, especially on a population-wide basis (Risch et al., 1996, Science 273:1516-1517).
2.2. Visualization and Surface Mounting of Single DNA Molecules
Single molecule approaches represent a subset of current physical and genetic mapping approaches constitute the two major approaches to genomic analysis, and are critical to mapping and cloning of disease genes and to direct sequencing efforts. Such methods of visualization of single DNA molecules include fluorescence microscopy in solution (Yanagida et al., 1986, in
Applications of fluorescence in the biomedical sciences
Taylor et al. (eds), Alan Liss, New York, pp 321-345; Yanagida et al., 1983, Cold Spring Harbor Symp. Quantit. Biol. 47:177; Matsumoto et al., 1981, J. Mol. Biol. 132:501-516; Schwartz et al., 1989, Nature 338:520-522; and Houseal et al., 1989, Biophys. J. 56:507-516); FISH (Manuelidis et al., 1982, J. Cell. Biol. 95:619; Lawrence et al., 1988, Cell 52:51; Lichter et al., 1990, Science 247:64; Heng et al., 1992, Proc. Natl. Acad. Sci. USA 89:9509; van den Engh et al., 1992, Science 257:1410); visualization by scanning tunneling microscopy or atomic force microscopy techniques (Keller et al., 1989, Proc. Nat
Schwartz David C.
Wu Tian
DeWitt Ross & Stevens S.C.
Horlick Kenneth R.
Kim Young
Leone, Esq. Joseph T.
Wisconsin Alumni Research Foundation
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