Organic compounds -- part of the class 532-570 series – Organic compounds – Diazo or diazonium
Reexamination Certificate
2000-10-26
2003-03-11
Shah, Mukund J. (Department: 1624)
Organic compounds -- part of the class 532-570 series
Organic compounds
Diazo or diazonium
C536S025320, C536S026600, C544S243000, C544S244000
Reexamination Certificate
active
06531581
ABSTRACT:
BACKGROUND OF THE INVENTION
Many procedures in medical research and recombinant DNA technology rely on the labeling of nucleotides that are then incorporated into oligo or polynucleotide probes. Commonly used labels have included radioactive isotopes, biotin, iminobiotin, haptens, fluorescent dyes and electron-dense reagents.
Problems with radioisotope labeling include the risk to the people handling the radioisotope-labeled material, and the need for elaborate and expensive safety precautions to be taken and maintained during the preparation, utilization and disposal of the radioactive material. Radioisotopes and radio-labeled nucleotides or polynucleotides are very expensive to purchase and use, due in part because of the safety precautions required and the problems in safely disposing of radioactive hazardous waste. In addition, the probe's structural integrity and sensitivity can be rapidly degraded during storage due to radioactive decay and radiochemical decomposition.
Oligo and polynucleotides can also be labeled with biotin and iminobiotin, haptens and fluorescent dyes for the direct detection of nucleotides. For example Ward et al. in U.S. Pat. Nos. 4,711,955; 5,449,767, 5,328,824; 5,476,928; herein incorporated by reference, describe the labeling of nucleotides with a hapten, biotin, or iminobiotin. The hapten is detected by a labeled antibody and the biotin or iminobiotin is detected by a labeled avidin or strepavidin.
Ruth, U.S. Pat. No. 4,948,882, herein incorporated by reference, describes the derivatization of nucleotides with fluorescent dyes, biotin, and antigens. Again, as in Ward et al. the biotin is detected by avidin, antigens are detected by labeled antibodies, and the fluorescent dyes are directly detected by spectral techniques.
The labeled nucleotides of Ruth are incorporated into oligo or polynucleotides by conventional phosphoramidite chemistry. The synthesized oligo or polynucleotides are then used as probes to detect DNA sequences. It is important to note that these labeled nucleotides are directly detectable when the probe is used in contrast to the labeled pyrimidines of the invention.
Labeling of nucleic acid probes with fluorophores facilitates microscopic analysis of chromosomes and their genetic structure by fluorescent in situ hybridization (FISH). A method of FISH probe preparation and signal detection is described by Ward et al. In the area of DNA diagnostics, automated platforms based on labeled synthetic oligonucleotides immobilized on silicon chips work by fluorescence detection and are capable of the parallel analysis of many samples and mutations. The methods used in preparing labeled, chemically activated nucleotide precursors for oligonucleotide synthesis is discussed and demonstrated by Ruth. Nucleic acid amplification methods such as PCR have become very important in genetic analysis and the detection of trace amounts of nucleic acid from pathogenic bacteria and viruses. Analysis of many PCR reactions by standard electrophoretic methods becomes tedious, time consuming and does not readily allow for rapid and automated data acquisition. PCR has been adapted to use fluorescent molecules by incorporation of fluorescent labeled primers or nucleotides into the product which is then directly detected or by the use of fluorescent probes that are then detected. Removal of unincorporated, labeled substrates is usually necessary and can be accomplished by filtration, electrophoretic gel purification or chromatographic methods. However, the large amount of sample handling required by these analytical techniques make these purification methods labor intensive, not quantitative and they invariably leads to serious contamination problems. Affinity capture of PCR products by strepavidin coated beads or micro titer wells requires incorporation of biotin labels in addition to the fluorophores and still involves transfer steps that can lead to contamination. Instrumentation utilizing both gel electrophoresis and laser excitation optics represents an improvement in data acquisition but cannot handle large numbers of samples, retains the comparatively prolonged separation times characteristic of gels and still requires sample transfer.
The use of fluorescent energy transfer, oligonucleotide primers containing hairpin secondary structure are described in U.S. Pat. No. 5,866,336, and in pending U.S. patent application Ser. Nos.: 08/837,034 filed Apr. 11, 1997 and 08/891,516 filed Jul. 11, 1997, each of which is entitled
Nucleic Acid Amplification of Oligonucleotides with Molecular Energy Transfer Labels and Methods Based Thereon
. The contents of all three of these documents are herein incorporated by reference. These documents solve the background problems associated with unincorporated, labeled substrates, alleviates sample transfer problems and enables the use of a homogeneous PCR assay for the analysis of many samples without cross contamination by amplicon. The unincorporated primer contains a target-specific, single stranded region with a 3′-hydroxyl terminus from which polymerase catalyzed elongation occurs. Under native conditions the unincorporated primer contains a tract of self complimentary nucleotides in the 5′ region that are hydrogen bonded into a hairpin conformation. The 5′-hydroxy terminus is modified with a fluorophore. The 5′-deoxynucleotide is adenosine (dA). The last base of the double stranded hairpin stem region is a deoxyuridine (dU) which is base paired to the 5′-dA. The aromatic azo dye 4-dimethylaminoazobenzene-4′-sulfonyl chloride (dabsyl) is linked via a spacer arm to the C-5 carbon of the dU base. Hairpins can be extremely stable structures for their size, having high Tm's and strongly negative free energies. A hairpin is an intramolecular formation and is much more kinetically and entropically favored than the formation of a hybridized duplex. Under these structural conditions the fluorophore and the DABSYL are tightly held in close proximity to each other. At these short molecular distances the fluorophore and DABSYL can have orbital contact and overlap, being able to form relatively weak chemical interactions such as it complexes, hydrogen bonding or salt complex formation. This orbital interaction promote very efficient transfer of excitation energy from the fluor to the dabsyl. The DABSYL acceptor is not fluorescent and dissipates much of the donated or transferred energy as heat. While resonance energy transfer plays a role in the fluorescein or other fluorophore quenching other mechanisms of energy transfer can operate over these short distances as well and can account for the very efficient quenching of the fluor by the quencher.
After target hybridization and polymerase extension, the primer becomes a template for the next round of DNA replication. Polymerase displaces the 5′-end of the hairpin and copies the remainder. This process opens the hairpin conformation and the primer enters into the standard double helical B DNA conformation. The fluorophore and DABSYL are then separated by more than 60 Angstroms. The incorporated primer is now capable of producing strong fluorescent emissions when exposed to the appropriate excitation wavelengths. Unincorporated primer remains as a hairpin and produces very little flourescent emission for the reasons previously stated.
The hairpin, energy transfer primers are synthesized by standard automated phosphoramidite chemistry. See Caruthers, U.S. Pat. Nos.: 4,415,734 and 4,458,066 herein incorporated by reference. Linkage of the DABSYL moiety requires post synthetic modification. During oligonucleotide synthesis a commercially available dU phosphoramidite containing an aliphatic amino group linked to the base is incorporated. After deprotection and desalting the crude oligonucleotide is purified by reverse phase HPLC followed by solvent removal. The terminal amino group of the linker is reacted with a molar excess of commercially available dabsyl-succinimidyl ester or DABSYL sulfonyl chloride under alkaline conditions for
Boal Jila
Nardone Glenn
Nazarenko Irena
Foley & Lardner
McKenzie Thomas
Serologicals, Inc.
Shah Mukund J.
LandOfFree
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