Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1999-04-20
2001-01-23
Riley, Jezia (Department: 1656)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C436S501000
Reexamination Certificate
active
06177249
ABSTRACT:
BACKGROUND OF THE INVENTION
(1) Field of the Invention
This invention relates generally to nucleic acid analysis and, more particularly, to methods for detecting nucleic acid target sites with fluorescence labeled oligonucleotides and to use of the methods in DNA genotyping.
(2) Description of the Related Art
Nucleic acid analysis has become increasingly important in a number of applications including the genotyping of individuals such as in the diagnosis of hereditary diseases, the detecting of infectious agents, tissue typing for histocompatability, the identifying of individuals in forensic and paternity testing and monitoring the genetic make up of plants and animals in agricultural breeding programs (see, for example, Alford and Caskey,
Cur Opin Biotech
5:29-33, 1994 which is incorporated by reference).
One approach to nucleic acid analysis uses probes which are complementary to a nucleotide or nucleotides in the nucleic acid. These analyses are typically performed in conjunction with amplification of the DNA being tested by the polymerase chain reaction (Saiki et al.,
Science
239:487-491, 1988 which is incorporated by reference). Two variations of this approach are the Genetic bit analysis method and the oligonucleotide ligation assay.
The Genetic bit analysis method involves hybridization of an oligonucleotide to a DNA sequence immediately adjacent to a target nucleotide. The oligonucleotide then undergoes a 3′ extension with a labeled dideoxynucleoside triphosphate and the labeled oligonucleotide is subsequently detected using enzyme linked colorimetry. (Nikiforov et al,
Nucleic Acids Res
22:4167-4175, 1994 which is incorporated by reference).
The oligonucleotide ligation assay involves hybridization of a DNA sequence to two probes, one of which is labeled. One of the probes hybridizes to the nucleotides immediately contiguous to a target nucleotide and a second, allele-specific probe hybridizes to the target nucleotide and immediately contiguous nucleotides on the opposite side to the first probe. The two probes are then ligated and the resultant labeled oligonucleotide is detected using enzyme linked colorimetry (Nickerson et al.,
Proc Natl Acad Sci
87:8923-8927, 1990; U.S. Pat. Nos. 4,883,750, 4,988,617 and 5,242,794 all of which are incorporated by reference).
Both the genetic bit analysis and oligonucleotide ligation assay are time consuming and not readily adaptable to automation because they require capturing, separation, and washing of the labeled oligonucleotide followed by a multi-step detection procedure using an enzyme-linked immunosorbent assay.
In another approach, the detection of one or more nucleotides in a nucleic acid is accomplished using oligonucleotide probes labeled with two fluorescent substances in close proximity. One of the fluorophores (donor) has an emission spectrum that overlaps the excitation spectrum of the other fluorophore (acceptor) and transfer of energy takes place from the donor to the acceptor through fluorescence resonance energy transfer (T. Foster, Modern Quantum Chemistry, Istanbul Lectures, Part III, 93-137, 1965, Academic Press, New York which is incorporated by reference). The energy transfer is mediated by dipole-dipole interaction. Spectroscopically, when the donor is excited, its specific emission intensity decreases while the acceptor's specific emission intensity increases, resulting in fluorescence enhancement.
The fluorescence enhancement has been used in detection systems in which either two singly labeled oligonucleotides (Heller et al., EPO patent applications 0070685, 1983 which is incorporated by reference) or one doubly labeled oligonucleotide probe (Heller, EPO patent application 0229943, 1986 which is incorporated by reference) are first prepared and then hybridized to a target DNA or RNA sequence. The two fluorescent labels are separated by less than 22 nucleotides in the case of two singly labeled oligonucleotides or from 2 to 7 intervening base units in the case of doubly labeled oligonucleotide probes such that enhanced emission from fluorescent energy transfer can be detected from the hybridized probes.
The so-called Taqman assay uses a fluorescent energy transfer detection method which takes advantage of the decrease in emission intensity, i.e. or quenching observed in the first fluorophore. (Livak et al.,
PCR Methods and Applications
4:357-362, 1995; U.S. Pat. No. 5,528,848 which are incorporated by reference). An oligonucleotide containing the two fluorescent substances is hybridized to a target DNA sequence. The fluorescent substances are covalently linked to the oligonucleotide at a distance such that fluorescent energy transfer takes place which is then measured as a quenching of donor fluorescence. During amplification by polymerase chain reaction, the oligonucleotide is degraded thus separating the two fluorescent substances. As a result, the donor shows a loss of quenching and increase in fluorescent emission. Thus, by monitoring the loss of quenching of the donor, the target DNA sequence is detected.
One application of the TaqMan assay is in detecting single nucleotide polymorphisms, i.e. single base mutations in DNA. This method provides significant advantages over earlier assays for single nucleotide polymorphisms which were labor intensive and not readily automated. (see, for example, Botstein et al.,
Am J Human Genetics
32:314-331, 1980; Hayashi,
PCT Methods and Applications
1:34-38, 1991; Meyers et al.,
Methods in Enzymology
155:501-527, 1987; Keen et al.,
Trends in Genetics
7:5, 1991; Cotton et al.,
Proc Natl Acad Sci
85:4397-4401; Myers et al., Science 230:1242-1246, 1985; and Kwok et al.,
Genomics
23:138-144, 1994 which are incorporated by reference). Nevertheless, a significant problem with the TaqMan assay results from a relative intolerance to mismatches which is disadvantageous for allelic discrimination. (Livak et al,
PCR Methods and Applications
4:357-362, 1995 which is incorporated by reference). Thus, there remains a continuing need for an effective nucleic acid assay method that is simple to perform and readily automated.
SUMMARY OF THE INVENTION
Accordingly, therefore, the inventors herein have succeeded in discovering a new approach for detecting the presence of a target site of at least one nucleotide in a sample of nucleic acid using a fluorescent energy transfer detection method. The method involves synthesizing an oligonucleotide hybridized to a sequence of contiguous nucleotides, which include a target site of at least one nucleotide, in a nucleic acid. An essential feature of the oligonucleotide is that once formed, the oligonucleotide contains at least two fluorophores each of which is covalently bound to a separate nucleotide in the oligonucleotide. The two fluorophores are selected so that the emission spectrum of one fluorophore, referenced herein as the donor fluorophore, overlaps the excitation spectrum of the other, referenced herein as the acceptor fluorophore. The position of the two fluorophores on the oligonucleotide is such that upon release of the oligonucleotide from hybridization to the nucleic acid target site, the two fluorophores are separated by a distance that allows a fluorescence energy transfer to take place from the donor to the acceptor. The fluorescence energy transfer is then detected, either by decreased emission of the donor fluorophore or by an increase in emission of the acceptor fluorophore to indicate that the target site is present in the nucleic acid.
In one embodiment of the present invention, the oligonucleotide is formed by hybridizing a first polynucleotide which contains one of the two fluorophores to the nucleic acid. The hybridization is to a sequence of nucleotides in the nucleic acid that either includes or is immediately 3′ to the target site. The second fluorophore is covalently linked to a dideoxynucleoside triphosphate which binds to the nucleic acid immediately 5′ to the binding of the polynucleotide and is added by template directed synthesis to the 3′ end of the pol
Chen Xiangning
Kwok Pui-Yan
Howell & Haferkamp LC
Riley Jezia
Washington University
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