Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Patent
1994-02-04
1999-11-09
Zitomer, Stephanie S.
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
435 912, 536 2433, 935 77, 935 78, C12Q 168, C12P 1934, C07H 2104, C07H 2102
Patent
active
059811765
DESCRIPTION:
BRIEF SUMMARY
FIELD OF INVENTION
The present invention relates to a method for detecting and for discriminating between nucleic acid sequences if present in a sample. More specifically, the invention relates to a method for determining if a particular DNA or RNA sequence is present in a sample. The invention also relates to discriminating between sequences which differ from each other by as little as a single nucleotide. The DNA or RNA may be single or double stranded, and may be a single species or a component of a mixture of nucleic acids. The method of the invention utilizes a novel primer design. The sequence of the primer is composed of two portions, the 3' portion is a primer specific for the target nucleic acid sequence and the 5' portion is complementary to a preselected nucleic acid sequence. Extension of the 3' portion of the primer with labeled deoxynucleosides triphosphate yields a labeled extension product if, but only if, the template includes the target sequence. The labeled extension product is detected by hybridization of the 5' portion to the preselected sequence.
BACKGROUND OF THE INVENTION
The genome of an organism is unique. Not only do the genomes of different species differ, but the genomes of different individuals within a species differ (with the exception of identical twins or clones). These differences provide individual and species specific characteristics which can be used for identification by nucleic acid biochemical techniques such as hybridization and polymerase mediated reactions, both dependent for their specificity on precise base pairing.
The goal of nucleic acid based diagnostics is the detection of specific nucleic acid sequences. This goal often requires the detection of a specific sequence in the presence of other sequences. In certain cases it is necessary to discriminate between closely related sequences, even sequences which differ by only a single nucleotide. Prior art methods for doing so are described in various publications. For example, the use of allele-specific oligonucleotide (ASO) hybridization probes for the detection of specific nucleic acid sequences has been described (Wu et al., DNA 8:135-142 (1989); Thein, et al., Br. J. Haematol. 70:225-231 (1988); Connor, et al., Proc. Natl. Acad. Sci. USA 80:278-282 (1983); Studencki, et al., Am. J. Hum. Genet. 37:42-51 (1985); Pirastu, et al., N.Engl.J.Med. 309:284-287 (1983); Orkin, et al., J.Clin.Invest. 71:775-779 (1983); Thein and Wallace, The use of synthetic oligonucleotides as specific hybridization probes in the diagnosis of genetic disorders. In Human genetic diseases: A practical approach. K. E. Davies, ed. (Oxford; IRL Press), pp. 33-50 (1986)). This approach allows the discrimination between nucleic acids which differ by as little as a single nucleotide (e.g., alleles). Individual hybridization reactions are required for each allele to be detected. Erlich, et al., Eur.J.Immunogenet. 18:33-55 (1991) and Zhang, et al., Nucleic Acids Res. 19:3929-3933 (1991) have recently described the use of immobilized ASO probes. In this method, a set of ASO probes is immobilized on a membrane and hybridized with labeled polymerase chain reaction (PCR) products. Under appropriate conditions hybridization is allele specific. Each hybridization can analyze only a single amplification reaction. The present invention allows for the detection of specific sequences in a sample. Because the template specific step and the detection step are each controlled by specific but independent base pairing requirements, the overall process allows detection of multiple templates and multiple samples simultaneously.
The concept of in vitro DNA amplification was first proposed by Khorana and coworkers in 1971 (Kleppe, et al., J.Mol.Biol. 56:341-361 (1971)). Realizing that total chemical synthesis of a gene would result in a finite amount of product, a procedure for in vitro replication was proposed. Their procedure was based on extensive studies of the repair replication reaction, the in vitro replication of a DNA template using a complementary p
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City of Hope
Zitomer Stephanie S.
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