Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2001-04-27
2003-04-15
Horlick, Kenneth R. (Department: 1656)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091100, C536S023100
Reexamination Certificate
active
06548254
ABSTRACT:
FIELD OF THE INVENTION
The present invention is in the field of molecular beacon synthesis and use for detection of target sequences, including juxtaposed sequences produced by splicing or cloning.
BACKGROUND OF THE INVENTION
Molecular beacons (MBs) are oligonucleotides designed for the detection and quantification of target nucleic acids (e.g., target DNAs). The basic principles of molecular beacon mediated target nucleic acid detection is depicted in FIG.
1
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As depicted, 5′ and 3′ termini of the MB collectively comprise a pair of moieties which confers detectable properties of the MB. As shown, one of the termini is attached to a fluorophore and the other is attached to a quencher molecule capable of quenching a fluorescent emission of the fluorophore. For example, one example fluorophore-quencher pair can use a fluorophore such as EDANS or fluorescein, e.g., on the 5′-end and a quencher such as Dabcyl, e.g., on the 3′-end.
When the MB is present free in solution, i.e., not hybridized to a second nucleic acid, the stem of the MB is stabilized by complementary base pairing. This self-complementary pairing results in a “hairpin loop” structure for the MB in which the fluorophore and the quenching moieties are proximal to one another. In this confirmation, the fluorescent moiety is quenched by the fluorophore.
The loop of the molecular beacon is complementary to a sequence to be detected in the target nucleic acid, such that hybridization of the loop to its complementary sequence in the target forces disassociation of the stem, thereby distancing the fluorophore and quencher from each other. This results in unquenching of the fluorophore, causing an increase in fluorescence of the MB.
Further details regarding standard methods of making and using MBs are well established in the literature and MBs are available from a number of commercial reagent sources. Further details regarding methods of MB manufacture and use are found, e.g., in Leone et al. (1995) “Molecular beacon probes combined with amplification by NASBA enable homogenous real-time detection of RNA.”
Nucleic Acids Res
. 26:2150-2155; Tyagi and Kramer (1996) “Molecular beacons: probes that fluoresce upon hybridization”
Nature Biotechnology
14:303-308; Blok and Kramer (1997) “Amplifiable hybridization probes containing a molecular switch”
Mol Cell Probes
11:187-194; Hsuih et al. (1997) “Novel, ligation-dependent PCR assay for detection of hepatitis C in serum”
J Clin Microbiol
34:501-507; Kostrikis et al. (1998) “Molecular beacons: spectral genotyping of human alleles”
Science
279:1228-1229; Sokol et al. (1998) “Real time detection of DNA:RNA hybridization in living cells”
Proc. Natl. Acad. Sci. U.S.A
. 95:11538-11543; Tyagi et al. (1998) “Multicolor molecular beacons for allele discrimination”
Nature Biotechnology
16:49-53; Bonnet et al. (1999) “Thermodynamic basis of the chemical specificity of structured DNA probes”
Proc. Natl. Acad. Sci. U.S.A
. 96:6171-6176; Fang et al. (1999) “Designing a novel molecular beacon for surface-immobilized DNA hybridization studies”
J. Am. Chem. Soc
. 121:2921-2922; Marras et al. (1999) “Multiplex detection of single-nucleotide variation using molecular beacons”
Genet. Anal. Biomol. Eng
. 14:151-156; and Vet et al. (1999) “Multiplex detection of four pathogenic retroviruses using molecular beacons”
Proc. Natl. Acad. Sci. U.S.A
. 96:6394-6399. Additional details regarding MB construction and use is found in the patent literature, e.g., U.S. Pat. No. 5,925,517 (Jul. 20, 1999) to Tyagi et al. entitled “Detectably labeled dual conformation oligonucleotide probes, assays and kits;” U.S. Pat. No. 6,150,097 to Tyagi et al (Nov. 21, 2000) entitled “Nucleic acid detection probes having non-FRET fluorescence quenching and kits and assays including such probes” and U.S. Pat. No. 6,037,130 to Tyagi et al (Mar. 14, 2000), entitled “Wavelength-shifting probes and primers and their use in assays and kits.”
MBs are gaining wide spread acceptance as robust reagents for detecting and quantitating nucleic acids, including in real time (e.g., MBs can be used to detect targets as they are formed). A variety of commercial suppliers produce standard and custom molecular beacons, including Chruachem (chruachem.com), Oswel Research Products Ltd. (UK; oswel.com), Research Genetics (a division of Invitrogen, Huntsville Ala. (resgen.com)), the Midland Certified Reagent Company (Midland, Tex. mcrc.com) and Gorilla Genomics, LLC (Alameda, Calif.). A variety of kits which utilize molecular beacons are also commercially available, such as the Sentinel™ Molecular Beacon Allelic Discrimination Kits from Stratagene (La Jolla, Calif.) and various kits from Eurogentec SA (Belgium, eurogentec.com) and Isogen Bioscience BV (The Netherlands, isogen.com).
Despite such widespread acceptance and commercial development of MBs and related technologies, there remain a number of areas for improvement in the design, manufacture, synthesis, and purification of MBs. For example, in the area of single nucleotide polymorphism (SNP) detection, one typically designs, tests and synthesizes MBs separately for each SNP. This is, of course, inefficient and expensive at several levels. For example, the design and testing process is labor intensive. Additionally, it is difficult to scale the amount of MB actually needed to the synthesis scheme used to make the MB. That is, it can be difficult to scale a synthetic reaction down to produce only as much material as is actually needed—which, with the evolution of modern laboratory systems that run and detect reactions in nanoliter volumes, can be extremely small indeed.
Further in this regard, there are a number of specific difficulties with current synthetic schemes for making MBs. First, MB oligonucleotides require labels on both the 5′ and 3′ ends of the oligonucleotide. Adding 5′ and 3′ labels to oligonucleotides increases their cost dramatically, since specialized CPG (controlled pore glass) supports for solid-phase synthesis are typically used for the 3′ attachment, and specialized phosphoramidites are required for the 5′ attachment. Second, MBs are generally long oligonucleotides (typically greater than 30 nucleotides in length). The longer an oligonucleotide, the lower the percentage of final oligonucleotide product which is full-length, due to the compounding likelihood of synthesis failure at each base. Oligonucleotide purity, therefore, decreases as a function of oligonucleotide length, reducing the effectiveness of the MB and increasing the requirement for purification following synthesis. Indeed, typically, MB oligonucleotides are purified to operate according to specifications. Purification of oligonucleotides that differ by one or a few bases in length is best achieved by polyacrylamide gel electrophoresis (PAGE)-based methods, which are relatively labor intensive and, therefore, expensive. Finally, once designed and synthesized, there is a significant probability that a given MB will be ineffective, due to interfering secondary structure in its own loop region, or interfering secondary structure in the sequence of the target DNA to which the MB hybridizes, which interferes with the hybridization of the MB and target sequence.
The present invention uses modular synthesis strategies to overcome scalability, purification and synthesis issues noted above and to substantially decrease the amount of time needed to design and test MBs. Libraries, kits, devices, ligation mixtures and methods to achieve these goals are provided.
A fuller understanding of the invention will be provided by review of the following.
SUMMARY OF THE INVENTION
The present invention uses ligation-based assembly to make MBs. That is, MB components such as the stem, loop, label and label quenching moieties are made separately and then assembled by chemical or enzymatic ligation. Basic approaches include template-based, multiple template based and non-template based ligation assembly reactions. The MBs and components used to make the MBs can
Beckman Kenneth B.
Mancebo Ricardo
Gorilla Genomics, Inc.
Horlick Kenneth R.
Quine Jonathan Alan
Quine Intellectual Property Law Group P.C.
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