Hybridization-triggered fluorescent detection of nucleic acids

Details Hybridization-triggered fluorescent detection of nucleic acids Hybridization-triggered fluorescent detection of nucleic acids

1999-10-26

2002-10-29

Riley, Jezia (Department: 1656)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

C536S022100, C536S023100, C536S024300, C536S024330, C536S025300, C536S025320, C536S026600

Reexamination Certificate

active

06472153

ABSTRACT:

CROSS-REFERENCE TO RELATED APPLICATIONS
Not applicable.
STATEMENT OF RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH
Not applicable.
TECHNICAL FIELD
The present invention is in the field of molecular biology. More specifically, the invention is in the field of assays that utilize fluorescently-labeled probes and primers in hybridization assays for detection of nucleic acids.
BACKGROUND
The use of fluorescent molecules in the biological sciences for research and diagnostic purposes is well known. See, for example, Kirkbright “Fluorescent Indicators” in Indicators, (ed. Bishop, E.) Pergamon Press, New York, Chapter 9, pp. 685-708, 1972; and Haugland (1996)
Handbook of Fluorescent Probes and Research Chemicals
, Sixth edition, Molecular Probes, Inc., Eugene, Oreg. Fluorescent moieties have been used for non-specific labeling of single- and double-stranded nucleic acids (e.g., acridine, ethidium bromide) and for labeling of nucleic acid probes that are used in sequence-specific detection of nucleic acid targets. In general, when fluorescent nucleic acid binding molecules and/or fluorescently-labeled probes are used for nucleic acid detection, unbound fluorescent material must be removed from the system, prior to analysis, to maximize detection of a signal. If unbound material is not removed, background fluorescence leads to a reduction in the signal:noise ratio.
Compositions which are fluorescent when bound to double-stranded DNA, but which do not fluoresce (or fluoresce at a different wavelength) when unbound, have been described. See, for example, Haugland, supra, pp. 144-156 and 161-174, especially pp. 161-165. Although such compositions may exhibit fairly general sequence preferences (e.g., for AT-rich vs. GC-rich target sequences), they are not capable of either sequence-specific detection of a target or of mismatch discrimination between targets having related but non-identical sequences. In addition, such compositions cannot be used for multiplex detection of target sequences (i.e., simultaneous detection of more than one target sequence).
Several new analytical techniques depend on sequence-specific detection and mismatch discrimination using fluorescence as a readout. For instance, homogeneous detection methods for monitoring the accumulation of specific PCR products have recently been developed. One of these assays utilizes an oligonucleotide probe which contains a fluorescent molecule at its 5′ end and a fluorescence quencher at its 3′ end. Because of the presence of the quencher, the oligonucleotide probe does not exhibit fluorescence, or exhibits relatively low fluorescence, in the single-stranded state. The assay exploits the 5′→3′ nuclease activity of Taq DNA polymerase to hydrolyze such a probe after it has formed a sequence-specific duplex with a target nucleic acid. Hydrolysis releases the fluorescent molecule from the 5′ end of the probe, removing it from proximity with the quencher, thereby allowing increased fluorescence to occur. Lee et al. (1993)
Nucleic Acids Res
. 16:3761-3766. In another recently-developed technique, microvolume multi-sample fluorimeters with rapid temperature control have been developed for use with 5′-nuclease assays using double-labeled fluorescent probes. Wittwer et al. (1997)
Biotechniques
22:176-181. U.S. Pat. No. 5,871,908 describes a homogeneous assay in which fluorescent signal varies with a temperature gradient and the variation is detected in real time. However, all of these assays involve post-hybridization detection steps, often involving the use of enzymes, which are costly, time-consuming and can be difficult to regulate, in terms of their activity.
There is thus a need for sensitive and straightforward methods and compositions for sequence-specific detection of nucleic acid targets; in particular fluorescent detection. Besides the advantages of using fluorescent molecules as an alternative to radioisotopes, improvements in speed, economy and convenience would attend the development of a method in which the hybridization event itself provided a direct readout, without requiring subsequent detection steps, such as enzymatic treatment of hybridized material.
Tyagi et al. (1996)
Nature Biotechnol
14:303-308 described probes containing a fluorophore and a quencher molecule which, in the unhybridized state, form a hairpin which brings the fluorophore and the quencher into proximity so that fluorescence is quenched. Upon hybridization, the hairpin structure is disrupted and fluorescence is observed. Such probes require the attachment of both a fluorophore and a quencher, and also must contain regions of self-complementarity, which may interfere with their ability to hybridize to their target.
Minor groove binding agents that non-covalently bind within the minor groove of double stranded DNA have been described. Zimmer et al. (1986)
Prog. Biophys. Molec. Biol
. 47:31-112; Levina et al. (1996)
Antisense & NucL. Acid Drug Develop
. 6:75-85. Hybridization assays using an oligonucleotide coupled to a minor groove binder (MGB) have been described in U.S. Pat. No. 5,801,155, and in International Patent Application No. PCT/US99/07487. These publications describe the ability of minor groove binders, when conjugated to an oligonucleotide, to increase the ability of the oligonucleotide to distinguish between a perfectly-matched target sequence and a target sequence with a single-nucleotide mismatch. This heightened discriminatory ability of MGB-oligonucleotide conjugates is reflected in a greater difference in melting temperature (T
m
) between matched and mismatched duplexes formed with an MGB-oligonucleotide conjugate, on the one hand, and matched and mismatched duplexes formed with an unmodified oligonucleotide, on the other. The aforementioned U.S. Pat. No. 5,801,155, and International Patent Application No. PCT/US99/07487 additionally disclose that a duplex comprising a MGB-oligonucleotide conjugate has a higher melting temperature than a duplex of identical sequence comprising an unmodified oligonucleotide. This property of duplexes comprising a MGB-oligonucleotide conjugate allows more facile detection of related mismatched sequences with a MGB-oligonucleotide probe, and enables the use of shorter oligonucleotide probes in PCR amplification reactions, if the probe is conjugated to a MGB. These publications also describe the use of an oligonucleotide coupled to a minor groove binder, a fluorophore and a fluorescent quencher, in hydrolyzable probe assays.
Intercalating agents are, generally speaking, flat aromatic molecules that bind non-covalently to double-stranded DNA or RNA by positioning themselves between adjacent base pairs of the duplex. Gago (1998)
Method
14:277-292. U.S. Pat. No. 4,835,283 and PCT publication WO 98/50541, for example, disclose oligonucleotides that are covalently bound to an intercalating group. Oligonucleotides conjugated to either minor groove binders or intercalating groups can be used in hybridization assays.
Hoechst 33258 and 33342 are examples of fluorescent dyes that bind in the minor groove of DNA duplexes. A conjugate consisting of an oligonucleotide coupled to a Hoechst-like minor groove binder has been observed to show increased fluorescence upon hybridization to a single-stranded target. O'Donnell et al. (1995)
Biorg. Med. Chem
. 3:743-750; and Wiederholt et al. (1996)
J. Amer. Chem. Soc
. 118:7055-7062. This conjugate consisted solely of an oligonucleotide bound to a MGB.
EP 231 495 discloses a polynucleotide compound comprising at least two entities, which upon hybridization is capable of generating a change in property of the hybrid.
SUMMARY OF THE INVENTION
The present invention provides methods and compositions for improved hybridization detection and mismatch discrimination by fluorescence. In the practice of the invention, an increase in fluorescent signal, a change in fluorescence excitation and/or emission, and/or some other change in fluorescence properties occurs after hybridization of an oligonucleotide, appropriat

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