Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1998-09-03
2002-10-15
Marschel, Ardin H. (Department: 1631)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C536S022100, C536S025300, C436S501000
Reexamination Certificate
active
06465175
ABSTRACT:
TECHNICAL FIELD
This invention relates generally to nucleic acid chemistry and hybridization assays. More particularly, the invention relates to a method for detecting a target nucleic acid in a sample using an oligonucleotide probe bearing a detectable label.
BACKGROUND
Nucleic acids have joined antigens and antibodies as key molecular targets for human diagnostic assays. In particular, due to the recent development of a variety of technical capabilities, microorganisms can now be detected and quantified at unprecedented low levels in clinical specimens.
A variety of concepts have provided the basis for a number of different probe-target hybridization detection systems.
Glazer et al. (1992)
Nature
359:959 describe fluorescent intercalation complexes of polycationic ligands with double-stranded DNA (dsDNA) for high-sensitivity DNA detection. The fluorescent homodimers intercalate into dsDNA to form a complex that is stable to electrophoresis, thus allowing detection and quantitation of DNA after separation with minimum background interference.
Tyagi et al. (1996)
Nature Biotech.
14:303-308 describe a probe that is optically silent in solution but fluoresce upon hybridization with a complementary target. The probe is a single-stranded oligonucleotide that possesses a stem-and-loop structure. The loop is a sequence complementary to the target. One arm of the stem has a fluorescent moiety attached and the other arm has a nonfluorescent quenching moiety attached. In the nonhybridized state, the stem keeps the two moieties in sufficiently close proximity so that the fluorescent moiety is quenched by the nonfluorescent moiety. Upon hybridization, the consonant conformational change in the probe forces the arm sequences apart and allows the fluorescent moiety to be detected.
Mergny et al. (1994)
Nucleic Acids Res.
22:920-928 describes the use of fluorescence energy transfer to probe for primary or secondary structural features of single-stranded DNA. Two probes that hybridize to adjacent sequence of the target DNA respectively carry donor and acceptor dyes. Hybridization of the two probes to the target leads to fluorescence excitation energy transfer between the donor and acceptor dyes.
Davis et al. (1996)
Nucleic Acids Res.
24:702-706 describe the use of DNA constructs containing one or two fluorescein molecules in flow cytometry. The fluorescein molecules were attached to the 3′-end of a DNA probe through an 18-atom spacer arm that resulted in a 10-fold increase in fluorescence intensity compared to the DNA probe to which fluorescein was directly attached to the 3′-end of the probe.
In addition, many forms of target amplification have been introduced for enhancing detection sensitivity since the polymerase chain reaction was first developed. These methods can be used for producing greater quantities of the target nucleic acid and assays using these methods generally use conventional detection schemes.
In contrast, direct analysis of target nucleic acids with great sensitivity has been accomplished using signal amplification. This method has been applied to the quantitation of many organisms and mRNAs. As few as 50 picomoles of the human immunodeficiency virus genome has been quantified in human plasma samples. The key molecule in the amplification method is a branched DNA (bDNA) that permits the specific incorporation of many labeled probes.
Two types of bDNA molecules have been used. The first type is a large network comprised of DNA oligomers assembled in solution from oligonucleotides containing three equally distributed N
4
-[N-(6-aminocaproyl-2-aminoethyl)]-5-methyl-2′-deoxycytidine residues coupled to one another through homobifunctional amine-reactive cross-linking agents. Such “amplification multimer” (AM) networks permitted the specific hybridization of many alkaline phosphatase-labeled DNA probes per hybridization probe-target nucleic acid complex. These AMs gave significant signal amplification. As the multimer was increased in size, a limit to the signal amplification was achieved, possibly due to the location of complementary oligonucleotide sequences buried in the spherical cross-linked structure which may be inaccessible to the large alkaline phosphatase-labeled probes.
The second type of bDNA are “comb-type” molecules that comprise a linear primary oligonucleotide with a plurality of secondary side sequences having sites complementary to labeled DNA probes. As with the AMs, alkaline phosphatase-labeled DNA probes provide an amplified signal that does not match the theoretical signal amplification, possibly due to steric considerations.
The signal from fluorescently labeled DNA-probes can also be amplified using multiply labeled oligomers. However, due possibly to quenching effects, the incorporation of additional labels into a labeled probe does not produce a linear increase in fluorescent signal. For example, a two-fold increase in the number of labels per probe only yields a 1.2-fold increase in signal output. It would be possible to obtain an amplified fluorescent signal using a bDNA molecule as a scaffold for labeled DNA probes. However, again proximity of fluorophors may result in quenching-limited signal output. A highly sensitive fluorescent bDNA signal amplification-based assay would result if the quenching phenomenon were minimized.
SUMMARY OF THE INVENTION
The present invention provides methods and probes for detecting nucleic acid analytes in a sample. In general, the methods represent nucleic acid hybridization assays, such as fluorescent in situ hybridization assays, polymerase chain reaction assays, ligase chain reaction assays, competitive hybridization assays, strand displacement assays, and the like. In particular, the methods represent solution phase sandwich hybridization assays which involve binding the analyte to a solid support, labeling the analyte, and detecting the presence of label on the support. Preferred methods involve the use of amplification multimers which enable the binding of significantly more label in the analyte-probe complex, enhancing assay sensitivity and specificity. The probes comprise an oligonucleotide sequence to which has been conjugated, directly or indirectly through a linker, a dye that when in solution emits detectable radiation. Upon hybridization of the direct oligonucleotide-dye conjugate to a complementary oligonucleotide sequence the detectable radiation is substantially quenched. The detectable signal from an oligonucleotide-linker-dye conjugate is not appreciably quenched upon hybridization to a complementary oligonucleotide.
In a first aspect of the invention, a method is provide for detecting an oligonucleotide of interest in a sample. The method comprises (a) providing an oligonucleotide probe comprising (i) a nucleic acid sequence complementary to a nucleic acid sequence in the oligonucleotide of interest, and (ii) a label that, when the probe is in single-stranded, nonhybridized form, provides a detectable fluorescent signal, but which, when the probe hybridizes to a complementary nucleic acid strand, does not fluoresce, (b) combining the probe with the sample suspected of containing the oligonucleotide of interest under hybridizing conditions, while monitoring emitted fluorescence from the probe, and (c) correlating any decrease in fluorescence which occurs throughout step (b) with the presence or quantity of the oligonucleotide of interest.
In a related aspect of the invention, an improved solution phase sandwich hybridization assay for detecting an nucleic acid analyte in a sample, comprising: (a) binding the analyte indirectly to a solid support; (b) labeling the analyte; and (c) detecting the presence of label on the support, in which the improvement comprises incorporating a label probe system comprising (i) a label extender molecule having a first segment L-1 capable of hybridizing to a nucleic acid sequence in the analyte and a second segment L-2, (ii) an amplification multimer containing a nucleic acid sequence M-1 capable of hybridizing to nucleic acid sequen
Fiss Ellen
Horn Thomas
Law Say-Jong
Schroeder Hartmut R.
Sells Todd
Bayer Corporation
Hartrum J. Elin
Marschel Ardin H.
Reed Dianne E.
Reed & Associates
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