Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1999-04-30
2001-11-27
Zitomer, Stephanie (Department: 1656)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091200, C536S023100, C536S024300, C536S025320, C204S450000
Reexamination Certificate
active
06322980
ABSTRACT:
FIELD OF THE INVENTION
The field of this invention is the detection of single nucleotide polymorphisms.
BACKGROUND OF THE INVENTION
As the human genome is elucidated, there is, and will continue to be comparisons of the sequences of different individuals. It is believed that there will be about one polymorphism per 1,000 bases, so that one may anticipate that there will be a extensive number of differences between individuals. By single nucleotide polymorphism (snp's) is intended that there will be a prevalent nucleotide at the site, with one or more of the remaining bases being present in a substantially smaller percent of the population.
For the most part, the snp's will be in non-conding regions, primarily between genes, but will also be present in exons and introns. In addition, the great proporton of the snp's will not affect the phenotype of the individual, but will clearly effect the genotype. The snp's have a number of properties of interest. Since the snp's will be inherited, individual snp's and/or snp patterns may be related to genetic defects, such as deletions, insertions and mutations, involving one or more bases in genes. Rather than isolating and sequencing the target gene, it will be sufficient to identify the snp's involved.
In addition, the snp's may be used in forensic medicine to identify individuals. While other genetic markers are available, the large number of snp's and their extensive distribution in the chromosomes, make the snp's an attractive target. Also, by determining a plurality of snp's associated with a specific phenotype, one may use the snp pattern as an indication of the phenotype, rather than requiring a determination of the genes associated wit the phenotype.
The need to identify a large number of bases distributed over potentially centimorgans of DNA offers a major challenge. Any method should be accurate, reasonably economical in limiting the amount of reagents required and providing for a single assay, which allows for differentiation of the different snp's.
BRIEF DESCRIPTION OF THE RELATED ART
Holland (
Proc. Natl. Acad. Sci. USA
(1991):88:7276) discloses that the exonuclease activity of the thermostable enzyme Thermus aquaticus DNA polymerase in PCR amplification to generate specific detectable signal concomitantly with amplification.
The TAQMAN assay is discussed by Lee in
Nucleic Acid Research
(1993) 21:16 3761).
White (Trends Biotechnology (1996) 14(12); 478-483) discusses the problems of multiplexing in the TAQMAN assay.
Marino,
Electrophoresis
(1996) 17:1499 desribes low-strigency-sequence specific PCR (LSSP-PCR). A PCR amplified sequence is subjected to single primer amplification under conditions of low stringency to produce a range of different length amplicons. Different patterns are obtained when there are differences in sequence. The patterns are unique to an individual and of possible value for identity testing.
Single strand conformation a polymorphism (SSCP) yields similar results. In this method the PCR amplified DNA is denatured and sequence dependent conformations of the single strands are detected by their differing rates of migration during gel electrophoresis. As with LSSP-PCR above, different patterns are obtained that signal differences in sequence. However, neither LSSP-PCR nor SSCP gives specific sequence information and both depend on the questionable assumption that any base that is changed in a sequence will give rise to a conformational change that can be detected.
Pastinen,
Clin. Chem,
(1996) 42:1391 amplifies the target DNA and immobilizes the amplicons. Multiple primers are then allowed to hybridize to sites 3′ and contiguous to an SNP site of interest. Each primer has a different size that serves as a code. The hybridized primers are extended by one base using a fluorescently labeled dideoxynucleoside triphosphate. The size of each of the fluorescent products that is produced, determined by gel electrophoresis, indicates the sequence and, thus, the location of the SNP. The identity of the base at the SNP site is defined by the triphosphate that is used. A similar approach is taken by Haff,
Nucleic Acids Res.
(1997) 25:3749 except that the sizing is carried out by mass spectroscopy and thus avoids the need for a label. However, both method have the serious limitation that screening for a large number of sites will require large, very pure primers that can have troublesome secondary structures and be very expensive to synthesize.
Hacia,
Nat. Genet.
(1996) 14:441 uses a high density array of oligonucleotides. Labeled DNA samples were allowed to bind to 96,600 20-base oligonucleotides and the binding patterns produced from different individuals were compared. The method is attractive in that SNP's can be directly identified but the cost of the arrays is high.
Fan (Oct. 6-8, 1997, IBC, Annapolis Md.) has reported results of a large scale screening of human sequence-tagged sites. The accuracy of single nucleotide polymorphism screening was determined by conventional ABI resequencing.
Allele specific oligonucleotide hybridization along with mass spectroscopy has been discussed by Ross in
Anal. Chem.
(1997) 69:4197.
Brenner and Lener,
PNAS
(1992) 89:5381, suggested that compounds prepared by combinatorial synthesis can each be labeled with a characteristic DNA sequence. If a given compound proves of interest, the corresponding DNA label is amplified by PCR and sequence thereby identifying the compound.
W. Clark Still, in U.S. Pat. No. 5,565,324 and in Accounts of Chem. Res., (1996) 29:155, uses a releasable mixture of halocarbons on beads to code for a specific compound on the bead that is produced during synthesis of a combinatorial library. Beads bearing a compound of interest are treated to release the coding molecules and the mix is analyzed by gas chromatography with flame ionization detection.
SUMMARY OF THE INVENTION
Snp's are detected by employing a combination of a primer and fluorescent snp detector sequence in the presence of primer extension reagents, where the polymerase includes 5′-3′ exonuclease activity. The fluorescent snp detector sequence has at least one nucleotide which is substituted with an electrophoretic tag. One combines the target DNA, which will usually have been processed, with the primer extension reagents and at least one oligonucleotide pair for each snp of interest under conditions for primer extension. After sufficient time for primer extension to occur with degradation of detector sequences bound to target DNA, the electrophoretic tags, which are fluorescent or are made fluorescent, are separated and detected. By having a different electrophoretic tag for each snp, with each electrophoretic tag having a different electrophoretic mobility, one can readily determine the snp's which are present in the target DNA.
Electrophoretic tags are also provided for use in the snp detection method.
REFERENCES:
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patent: 5552028 (1996-09-01), Madabhushi et al.
patent: 5565324 (1996-10-01), Still et al.
patent: 5567292 (1996-10-01), Madabhushi et al.
patent: 5580732 (1996-12-01), Grossman et al.
patent: 5624800 (1997-04-01), Grossman et al.
patent: 5703222 (1997-12-01), Grossman et al.
patent: 5807682 (1998-09-01), Grossman et al.
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patent: 5916426 (1999-06-01), Madabhushi et al.
patent: 5989871 (1999-11-01), Grossman et al.
Brenner and Lerner (1992)PNAS89:5381-83.
Fan (Oct. 6-8, 1997) IBC, Annapolis MD.
Hacia (1996)Nat. Genet.14:441-47.
Haff (1997)Nucleic Acids Res.25:3749-50.
Holland (1991)Proc. Natl. Acad. Sci. USA88:7276-80.
Lee (1993)Nucleic Acid Research21:16 3761-66.
Marino (1996)Electrophoresis17:1499-04.
Pastinen (1996)Clin. Chem.42:1391-97.
Ross (1997)Anal. Chem.69:4197-4202.
Still et al. (1996)Accounts of Chem. Res.29:155-63.
White (Dec. 1996)Tibtech14(12): 478-83.
ACLARA BioSciences, Inc.
Tung Joyce
Zitomer Stephanie
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