Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1995-06-07
2001-04-24
Horlick, Kenneth R. (Department: 1656)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S007100, C436S094000, C436S501000, C536S023100
Reexamination Certificate
active
06221581
ABSTRACT:
BACKGROUND OF THE INVENTION
Nucleic acid hybridization assays are used as a tool for the detection and identification of a target genetic material such as DNA or RNA. Such detection and identification can be for a specific DNA or RNA sequence or specific gene or a point mutation or deletion of a DNA or RNA sequence or gene. A number of techniques exist to carry out such assays. (see
Methods In Enzymology,
Vol. 68, R. Wu (Ed) pp. 379-469, 1979; and Dunn, A. R., and Sambrook, J.,
Methods In Enzymology,
Vol. 65, Part 1, pp. 468-478, 1980). One of the most widely used procedures is called the Southern blot filter hybridization method (Southern, E., J. Mol. Biol. 98, 503, 1975). This procedure is usually used to identify a particular DNA fragment separated from a mixture of DNA fragments by electrophoresis. The procedure is generally carried out by isolating a sample of DNA from some microorganism. The isolated DNA is subjected to a restriction endonuclease digestion and electrophoresed on a gel (agarose, acrylamide, etc.). When the gel containing the separated DNA fragments is put in contact (blotted with a nitrocellulose filter sheet or diazotized paper, etc.), the fragments are transferred and become bound to the nitrocellulose sheet. The gel-transfer nitrocellulose sheet containing the DNA fragments is then heated to denature the DNA. At this point the sheet is treated with a solution containing a denatured labeled DNA probe and hybridization is allowed to take place. The unhybridized labeled DNA probe is then washed away. The label of the DNA probe is then detected.
It is known to carry out a homogeneous hybridization assay based upon non-radiative energy transfer. This hybridization assay system utilizes a chemiluminescent catalyst and an absorber/emitter moiety. The system involves the use of two polynucleotide reagent strands in such a way that the hybridization assays carried out are in a homogeneous fashion. This means that the target polynucleotide sequence can be detected and identified in solution without the need to carry out any immobolization procedures. The method comprises contacting the target genetic material, under hybridization conditions, with first and second single stranded polynucleotide reagent segments which are complementary to substantially mutually exclusive portions of the target single stranded polynucleotide. The first reagent segment has a chemiluminescent catalyst and the second reagent segment has an absorber/emitter moiety positioned such that, upon hybridization with the target single stranded polynucleotide, the chemilumnescent catalyst and absorber/emitter moiety are close enough in proximity to permit non-radiative energy transfer. The single stranded polynucleotide sample is then contacted with chemiluminescent reagents effective for causing light emission in the presence of the chemiluminescent catalyst. The quantity of light emitted by the absorber/emitter moiety is then measured by an appropriate instrument which thereby indicates the presence of the sample single stranded polynucleotide. This method is disclosed in European Patent Application Number 0 070 685, published Jan. 26, 1983.
SUMMARY OF THE INVENTION
This invention provides methods for the detection of a target genetic material having a desired base sequence or gene. Also disclosed are methods for the detection of mutations, such as a point mutation or the detection of a gene or base. Also provided are components for use in such methods.
The methods are based upon techniques which utilize two labeled single stranded polynucleotide segments which are complementary to the same or the opposite strands of the target genetic material. The methods of the invention result in the formation of a double hybrid and/or a multihybrid, defined hereinbelow.
The method of detection of the double hybrid and the multihybrid is dependent upon the choice of label.
Each single stranded polynucleotide segment can be either part of the same polynucleotide segment i.e., one probe which comprises two polynucleotide segments, of interest or can be two separate polynucleotide segments, i.e., two probes with each probe comprising a polynucleotide segment of interest. The label of each probe can be a particle, a moiety which is capable of generating a signal, either directly, e.g., a radioactive label, or indirectly, e.g., an enzyme-linked system or a system wherein each label alone can not create a signal, but when such labels are brought into contact a signal can be generated.
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Kidd et al.,Nature304, 230-234 (Jul. 1983).*
Huang, “Monoclonal Antibody Specific for Double-Stranded Conformation of DNA:A Non-Radioactive Method For Detection of DNA/DNA Hybrids”, Rapid Detection & Identification of Infectious Agents Symposium, Oct. 9, 1983.*
Ranki et al.,Gene21, 77-85 (1983).*
Ballard et al.,Mol. Imunol.19(6),793-799 (1982).*
Ranki M. et al.,Gene21:77-85 (1983).
Urdea, M.S., “Application of a Rapid Non-Radioactive Nucleic Acid Analysis System to the Detection of Sexually Transmitted Disease Organisms and Their Associated Microbial Resistances,” Abstracts of the Third San Diego Conference, Practical Aspects of Molecular Probes (1988).
Ballard D.W. and Voss, Jr., E.W.,Mol. Imunol.19(6):793-799 (1982).
Kidd V.J. et al.,Nature304:230-239 (Jul. 1983).
Nobrega, F.G., et al., “A Rapid Method for Detecting Specific RNA Transcripts by Hybridization to DNA Probes in Solution,”Analytical Biochemistry131:141-145 (1983).
Flavel, R.A., et al., “DNA-DNA Hybridization on Nitrocellulose Filters,”Eur. J. Biochem47:535-543 (1974).
Spiegelman, G.B., et al., “Kinetics of Ribonucleic Acid-Deoxyribonucleic Acid Membrane Filter Hybridization,”Biochemistry12(6):1234-1242(1973).
David E. Kennell, “Principles and Practices of Nucleic Acid Hybridization,”Progress in Nucleic Acid Research and Molecular Biology,vol. II (Davidson, J.N. and Cohn,W.E., Eds.), Academic Press, New York and London (1971).
Engelhardt Dean L.
Rabbani Elazar
Enzo Diagnostics, Inc.
Fedus, Esq. Ronald C.
Horlick Kenneth R.
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