Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1998-10-28
2001-09-11
Whisenant, Ethan (Department: 1655)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091200, C536S023100, C536S024300
Reexamination Certificate
active
06287766
ABSTRACT:
FIELD OF THE INVENTION
BACKGROUND OF THE INVENTION
The determination of the DNA base sequence of the human genome will have a major impact on biomedical science in the next century. The completion of the first complete human DNA will enhance a range of applications from genetic mapping of disease-associated genes to diagnostic tests for disease susceptibility and drug response. The determination of base composition at specific, variable DNA sites known as single nucleotide polymorphisms (SNPs) is especially important. The current generation of sequence determination methods are too slow and costly to meet large-scale SNP analysis requirements. Thus, there is a need for faster, more efficient methods for analyzing genetic sequences for SNPs.
SNPs have a number of uses in mapping, disease gene identification, and diagnostic assays. All of these applications involve the determination of basEs composition at the SNP site. Conventional sequencing can provide this information, but is impractical for screening a large number of sites in a large number of individuals. Several alternative methods have been developed to increase throughput.
Two techniques have been developed to determine base composition at a single site, minisequencing (See, e.g., “Minisequencing: A Specific Tool For DNA Analysis And Diagnostics On Oligonucleotide Arrays,” by Tomi Pastinen et al., Genome Research 7, 606 (1997)), and oligo-ligation (See, e.g., “Single-Well Genotyping Of Diallelic Sequence Variations By A Two-Color ELISA-Based Oligonucleotide Ligation Assay,” by Vincent O. Tobe et al., Nuclear Acids Res. 24, 3728 (1996)). In minisequencing, a primer is designed to interrogate a specific site on a sample template, and polymerase is used to extend the primer with a labeled dideoxynucleotide. In oligo-ligation, a similar primer is designed, and ligase is used to covalently attach a downstream oligo that is variable at the site of interest. In each case, the preference of an enzyme for correctly base-paired substrates is used to discriminate the base identity that is revealed by the covalent attachment of a label to the primer. In most applications these assays are configured with the primer immobilized on a solid substrate, including microplates, magnetic beads and recently, oligonucleotides microarrayed on microscope slides. Detection strategies include direct labeling with fluorescence detection or indirect labeling using biotin and a labeled streptavidin with fluorescent, chemiluminescent, or absorbance detection.
Oligonucleotide microarrays or “DNA chips” have generated much attention for their potential for massively parallel analysis. The prospect of sequencing tens of thousands of bases of a small sample in just a few minutes is exciting. At present, this technology has limited availability in that arrays to sequence only a handful of genes are currently available, with substantial hardware and consumable costs. In addition, the general approach of sequencing by hybridization is not particularly robust, with the requirement of significant sequence-dependent optimization of hybridization conditions. Nonetheless, the parallelism of an “array” technology is very powerful. and multiplexed sequence determination is an important element of the new flow cytometry method.
Accordingly, it is an object of the present invention to provide a method for determining the base composition at specific sites in a strand of DNA using microspheres and flow cytometry, wherein the specificity of enzymes for discriminating base composition is combined with the parallel analysis of a fluorescent microsphere array.
Additional objects, advantages, and novel features of the invention will be set forth in part in the description which follows, and in part will become apparent to those skilled in the art upon examinations of the following or may be learned by practice of the invention. The objects and advantages of the invention may be realized and attained by means of the instrumentalities and combinations particularly pointed out in the appended claims.
SUMMARY OF THE INVENTION
To achieve the forgoing and other objects, and in accordance with the purposes of the present invention as embodied and broadly described herein, the method for determining the base composition at specific sites on a DNA strand hereof includes the steps of: preparing an oligonucleotide primer bearing an immobilization or capture tag, fluorescently labeled dideoxynucleotides; extending the oligonucleotide primer using DNA polymerase with the fluorescent dideoxynucleotide; specifically binding the tagged primers to microspheres; and measuring microsphere fluorescence by flow cytometry.
Preferably, the oligonucleotide primers are designed to anneal to the DNA sample under investigation immediately adjacent to the site of interest so as to interrogate the next nucleotide base on the DNA sample.
It is also preferred that the primers have on their 5′ terminus one of: (a) an amino or other functional group suitable for covalent coupling to a microsphere; (b) a biotin group suitable for binding to avidin or streptavidin immobilized on a microsphere; or (c) an oligonucleotide tag that is complementary to an oligonucleotide capture probe immobilized on a microsphere surface.
In another aspect of the present invention, in accordance with its objects and purposes, the method for determining the base composition at specific sites on a DNA strand hereof includes the steps of: preparing an oligonucleotide primer bearing an immobilization or capture tag, fluorescently labeled dideoxynucleotides; preparing a fluorescent reporter oligonucleotide; enzymatically ligating the oligonucleotide primer to the fluorescent reporter oligonucleotide; specifically binding the tagged primers to microspheres; and measuring microsphere fluorescence by flow cytometry.
Benefits and advantages of the present invention include a sensitive, homogenous, and flexible method for determining DNA base composition at specific sites.
REFERENCES:
patent: 4988617 (1991-01-01), Landegren et al.
patent: WO9606950 (1996-03-01), None
patent: WO9967414 (1999-12-01), None
Shumaker et al., Human Mutation 7 : 346-354 (1996).*
Ugozzoli et al., GATA 9(4) : 107-112 (1992).*
Vlieger et al., Analytical Biochemistry 205 : 1-7 (1992).*
T. Pastinen et al., “Minisequencing: A Specific Tool for DNA Analysis and Diagnostics on Oligonucleotide Arrays,” Genome Research 7,606 (1997).
V. O. Tobe et al., “Single-Well Genotyping of Diallelic Sequence Variation by a Two-Color ELISA-Based Oligonucleotide Ligation Assay,” Nuclear Acids Res. 24,3728 (1996).
Cai Hong
Nolan John P.
White P. Scott
Freund Samuel M.
The Regents of the University of California
Whisenant Ethan
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