DNA-based analyses are used routinely in a wide spectrum of settings, including clinical hematology, molecular genetics, microbiology and immunology. Many current techniques rely on PCR amplification of a polynucleotide of interest (hereinafter “target molecule”) in conjunction with several types of post-amplification detection techniques. Other non-PCR based amplification techniques are well known in the art including, but not limited to, oligo ligation assay (OLA), ligase chain reaction (LCR), transcription-mediated amplification (TMA), and strand displacement amplification (SDA). Additionally, these techniques are amenable to mixing. That is, the product of one amplification reaction can be used as the target of another amplification reaction, which allows great sensitivity with an additional step that tends to increase sensitivity.
One preferred amplification format is known as a real-time homogeneous assay. A real-time assay is one that produces data indicative of the presence or quantity of a target molecule during the amplification process, as opposed to the end of the amplification process. A homogeneous assay is one in which the amplification and detection reagents are mixed together and simultaneously contacted with a sample, which may contain a target nucleic acid molecule. Thus, the ability to detect and quantify DNA targets in real-time homogeneous systems as amplification proceeds is centered in single-tube assays in which the processes required for target molecule amplification and detection take place in a single “close-tube” reaction format. For example, current techniques that use PCR amplification and have these features are generally known as Real-Time PCR techniques. Similarly, non-PCR-based technologies are also within the skill of the ordinary artisan and are amenable to homogeneous detection methods.
In most amplification and detection techniques a probe is used to detect an amplification product. Several probe systems known in the art utilize a fluorophore and quencher. For example, molecular beacon probes are single-stranded oligonucleic acid probes that can form a hairpin structure in which a fluorophore and a quencher are usually placed on the opposite ends of the oligonucleotide. At either end of the probe short complementary sequences allow for the formation of an intramolecular stem, which enables the fluorophore and the quencher to come into close proximity. The loop portion of the molecular beacon is complementary to a target nucleic acid of interest. Binding of this probe to its target nucleic acid of interest forms a hybrid that forces the stem apart. This causes a conformation change that moves the fluorophore and the quencher away from each other and leads to a more intense fluorescent signal. Molecular beacon probes are, however, highly sensitive to small sequence variation in the probe target (Tyagi S. and Kramer F. R., Nature Biotechnology, Vol. 14, pages 303-308 (1996); Tyagi et al., Nature Biotechnology, Vol. 16, pages 49-53(1998); Piatek et al., Nature Biotechnology, Vol. 16 pages 359-363 (1998); Marras S. et al., Genetic Analysis: Biomolecular Engineering, Vol. 14, pages 151-156 (1999); Tapp I. et al, BioTechniques, Vol 28, pages 732-738 (2000)).
Unlike molecular beacon probes, some single-stranded linear probes possessing also a quencher and a fluorophore attached at opposite ends of an oligonucleotide do not form a hairpin structure. Instead, this kind of linear oligonucleotide probes in solution behaves like a random coil, its two ends occasionally come close to one another, resulting in a measurable change in energy transfer. However, when the probe binds to its target, the probe-target hybrid forces the two ends of the probe apart, disrupting the interaction between the two terminal moieties, and thus restoring the fluorescent signal from the fluorophore. In addition, single-stranded linear probes can be designed as “TaqMan probes”, that bind to target strands during PCR and thus can be enzymatically cleaved by the 5′→3′ exonuclease activity of the Taq DNA polymerase during the primer extension phase of the PCR cycle resulting in an increase in fluorescence in each cycle proportional to the amount of specific product generated. It has been reported that long single-stranded linear probes suffer from high “background” signals, while shorter ones are sensitive to single-base mismatches (Lee L. G. et al., Nucleic Acids Research, Vol. 21, pages 3761-3766 (1993); Täpp I. et al. (above); U.S. Pat. Nos. 6,258,569; 6,030,787).
Double-stranded linear probes are also known in the art. Double-stranded linear probes have two complementary oligonucleotides. The probes described in the prior art have been of equal length, in which at least one of the oligonucleotides acts as a probe for a target sequence in a single-stranded conformation. The 5′ end of one of the oligonucleotides is labeled with a fluorophore and the 3′ end of the other oligonucleotide is labeled with a quencher, e.g., an acceptor fluorophore, or vice versa. When these two oligonucleotides are annealed to each other, the two labels are close to one another, thereby quenching fluorescence. Target nucleic acids, however, compete for binding to the probe, resulting in a less than proportional increase of probe fluorescence with increasing target nucleic acid concentration (Morrison L. et al., Anal. Biochem., Vol. 183, pages 231-244 (1989); U.S. Pat. No. 5,928,862).
Double-stranded linear probes modified by shortening one of the two complementary oligonucleotides by few bases to make a partially double-stranded linear probe, are also known in the art. In such double-stranded linear probes in the prior art, the longer oligonucleotide has been end-labeled with a fluorophore and the slightly shorter oligonucleotide has been end-labeled with a quencher. In the double-stranded form, the probe is less fluorescent due to the close proximity of the fluorophore and the quencher. In the presence of a target, however, the shorter quencher oligonucleotide is displaced by the target. As a result, the longer oligonucleotide (in the form of probe-target hybrid) becomes substantially more fluorescent.
The double-stranded probes known in the prior art having oligonucleotides of unequal lengths display complete discrimination between a perfectly matched target and single nucleotide mismatch targets. Also, these probes do not have optimal reaction kinetics especially when low quantities of target nucleic acid are present. (Li et al., Nucleic Acids Research, Vol. 30, No. 2, e5 (2002))
The detection of viral RNAs presents certain challenges, which are not presented by the desire to detect DNAs of interest. The probes of the prior art are suitable for the detection of viral RNAs, but could be improved. First, some viral RNA targets are prone to rapid mutation in the bodies of their hosts. To ensure that mutated viral RNA sequences are detected along with so-called “wild-type” sequences, nucleic acid probes used to detect viral RNAs should be tolerant of mismatches, yet still specific enough to avoid interaction with non-target nucleic acids. (i.e., false-positive results). Many of the probes of the prior art are sensitive to single-nucleotide changes, and therefore, are not optimal for the detection of viral nucleic acids.
Additionally, viral RNAs often must be reverse transcribed into DNA before amplification of a nucleic acid sequence of interest. Unfortunately, it has been discovered by the present inventors that some prior art nucleic acid probes can interfere with the reverse transcription (i.e., enzymatic copying of RNA sequences into DNA sequences).
It is also desirable that nucleic acid probes be capable of sensitively detecting both small and large quantities of nucleic acids of interest. Some nucleic acids probes of the prior art are not well suited to detecting small quantity of nucleic acids of interest. Other nucleic acids probes of the prior art are not well suited to the sensitive detection of large quantities of nucleic acids.
In view of the above, there is a need for a probe in which: a) the sequences can be readily manipulated, b) the oligonucleotides are easy to design without the limitation of being capable of forming stem or loop, c) there is high tolerance to mismatches, and/or d) the oligonucleotides are suitable for real-time RT-PCR reactions.
USPTO Backgrounds
USPTO Backgrounds