Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2001-05-21
2003-09-30
Horlick, Kenneth R. (Department: 1637)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091200, C536S024330
Reexamination Certificate
active
06627402
ABSTRACT:
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 primer (Kleppe, supra). Their proposal was as follows: “The DNA duplex would be denatured to form single strands. This denaturation step would be carried out in the presence of a sufficiently large excess of the two appropriate primers. Upon cooling, one would hope to obtain two structures, each containing the full length of the template strand appropriately complexed with the primer. DNA polymerase will be added to complete the process of repair replication. Two molecules of the original duplex should result. The whole cycle could be repeated, there being added every time a fresh dose of the enzyme.” More recently, this in vitro amplification process has been further developed into the polymerase chain reaction (Mullis, et al.,
Cold Spring Harbor Symp. Quant. Biol.
51:263-273 (1986); Saiki, et ale,
Science
230:1350-1354 (1985); U.S. Patent No. 4,683,202). Although template amplification improves detection of a particular sequence because a larger amount of template is available for analysis, post amplification steps are often required to detect specific sequences within the amplified product. For example, ASO hybridization has been combined with PCR amplification for the specific detection of various disease alleles (Impraim, et al.,
Biochem. Biophys. Res. Commun.
142:710-716 (1987); Saiki, et al.,
Nature
324:163-166 (1986); Farr, et al.,
Proc. Natl. Acad. Sci. USA
85:1629-1633 (1988); Saiki, et al.,
N. Enql. J. Med.
319:537-541 (1988); Chehab, et al.,
Nature
329:293-294 (1987)). The present invention provides an alternative for the analysis of PCR amplification products to determine the presence or absence of specific sequences.
SUMMARY OF THE INVENTION
This invention provides a method for determining the presence or absence of a target nucleic acid sequence present in a sample and for discriminating between any two nucleic acid sequences even if such sequences differ only by a single nucleotide. The nucleic sequences may be single or double stranded DNA or RNA. The target sequence may be relatively pure species or a component of a nucleic acid mixture. It may be produced by an in vitro amplification such as a PCR or ligation amplification reaction or by a plurality of cycles of primer extension.
The method of the invention entails extension of a novel, two component primer on templates which may or may not include a target nucleic acid sequence. The 3′ portion of the primer is complementary to a portion of the template adjacent the target sequence. The 5′ portion of the primer is complementary to a different preselected nucleic acid sequence. Extension of the 3′ portion of the primer with a labeled deoxynucleoside triphosphates yields a labeled extension product if, but only if, the template includes the target sequence. The presence of such a labeled primer extension product is detected by hybridization of the 5′ portion to the preselected sequence. The preselected sequence is preferably immobilized on a solid support. A plurality of preselected sequences immobilized on a solid support to provide an array for concurrent screening of a plurality of labeled primer extension products is provided.
One practical embodiment of the invention relates to methods for diagnosing diseases such as sickle cell anemia or thalassemia caused by a defective allele. Kits for performing such a diagnosis are provided.
REFERENCES:
patent: 4656127 (1987-04-01), Mundy
patent: 4683195 (1987-07-01), Mullis et al.
patent: 4851331 (1989-07-01), Vary et al.
patent: 5137806 (1992-08-01), LeMaistre et al.
patent: 5981176 (1999-11-01), Wallace
patent: 6013431 (2000-01-01), Soderlund et al.
patent: 0 246 864 (1987-05-01), None
patent: 416817 (1991-03-01), None
patent: 8910414 (1989-11-01), None
patent: 9009455 (1990-08-01), None
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patent: 9113075 (1991-09-01), None
Syvanen et al. A primer-guided nucleotide incorporation assay in the genotype of apoliprotein E. Genomics vol. 8, pp. 684-69
City of Hope
Horlick Kenneth R.
Rothwell, Figg Ernst & Manbeck, PC
Wilder Cynthia
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