Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2000-12-07
2003-04-29
Benzion, Gary (Department: 1634)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091200, C435S808000, C536S025320
Reexamination Certificate
active
06555317
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
Nucleic acid hybridization has been employed for investigating the identity and establishing the presence of nucleic acids. Hybridization is based on complementary base pairing. When complementary single stranded nucleic acids are incubated together, the complementary base sequences pair to form double stranded hybrid molecules. The ability of single stranded deoxyribonucleic acid (ssDNA) or ribonucleic acid (RNA) to form a hydrogen bonded structure with a complementary nucleic acid sequence has been employed as an analytical tool in molecular biology research. The availability of radioactive nucleoside triphosphates of high specific activity and the
32
P labeling of DNA with T4 polynucleotide kinase has made it possible to identify, isolate, and characterize various nucleic acid sequences of biological interest.
Nucleic acid hybridization has great potential in diagnosing disease states associated with unique nucleic acid sequences. These unique nucleic acid sequences may result from genetic or environmental change in DNA by insertions, deletions, point mutations, or by acquiring foreign DNA or RNA by means of infection by bacteria, molds, fungi, and viruses. Nucleic acid hybridization has, until now, been employed primarily in academic and industrial molecular biology laboratories. The application of nucleic acid hybridization as a diagnostic tool in clinical medicine is limited because of the frequently very low concentrations of disease related DNA or RNA present in a patient's body fluid and the unavailability of a sufficiently sensitive method of nucleic acid hybridization analysis.
One method for detecting specific nucleic acid sequences generally involves immobilization of the target nucleic acid on a solid support such as nitrocellulose paper, cellulose paper, diazotized paper, or a nylon membrane. After the target nucleic acid is fixed on the support, the support is contacted with a suitably labeled probe nucleic acid for about two to forty-eight hours. After the above time period, the solid support is washed several times at a controlled temperature to remove unhybridized probe. The support is then dried and the hybridized material is detected by autoradiography or by spectrometric methods.
When very low concentrations must be detected, the above method is slow and labor intensive, and nonisotopic labels that are less readily detected than radiolabels are frequently not suitable.
A method for the enzymatic amplification of specific segments of DNA known as the polymerase chain reaction (PCR) method has been described. This in vitro amplification procedure is based on repeated cycles of denaturation, oligonucleotide primer annealing, and primer extension by thermophilic polymerase, resulting in the exponential increase in copies of the region flanked by the primers. The PCR primers, which anneal to opposite strands of the DNA, are positioned so that the polymerase catalyzed extension product of one primer can serve as a template strand for the other, leading to the accumulation of a discrete fragment whose length is defined by the distance between the hybridization sites on the DNA sequence complementary to the 5′ ends of the oligonucleotide primers.
Other methods for amplifying nucleic acids are single primer amplification, ligase chain reaction (LCR), nucleic acid sequence based amplification (NASBA) and the Q-beta-replicase method. Regardless of the amplification used, the amplified product must be detected.
Genetic recombination involves the exchange of DNA strands between two related DNA duplexes. The branch point between two duplex DNAs that have exchanged a pair of strands is thought to be an important intermediate in homologous recombination. This branch point is otherwise referred to as the Holliday junction. Movement of the Holliday junction by branch migration can increase or decrease the amount of genetic information exchanged between homologues. In vitro strand exchange is protein mediated, unlike the spontaneous migration that occurs in vitro.
There is a great demand for simple universal high-throughput methods for detection of differences in related nucleic acid sequences regardless of the exact nature of the difference. This demand is becoming more and more urgent due to the ongoing rapid discovery of new disease related mutations brought about by the progress of the Human Genome Project. A detection method for mutations that is not dependent on the exact location of the mutation is valuable in the case of diseases that are known to result from various mutations within a given sequence. Moreover, such a method will be useful for verification of sequence homology as related to various applications in molecular biology, molecular medicine and population genetics.
Some of the current methods are either targeted for sets of known mutations, such as, for example, the Reverse Dot Blot method, or involve gel-based techniques, such as, for example, single stranded conformational polymorphism (SSCP), denaturing gradient gel electrophoresis (DGGE) or direct sequencing as well as a number of methods for the detection of heteroduplexes. Accordingly, such methods are laborious and time consuming.
Various methods for mutation detection have been developed in the recent years based on amplification technology. The detection of sequence alterations is based on one of the following principles: allele-specific hybridization, chemical modification of mismatched bases with subsequent strand cleavage, nuclease cleavage at mismatches, recognition of mismatches by specific DNA binding proteins, changes in electrophoretic mobility of mismatched duplexes in gradients of denaturing agents, conformation-induced changes in electrophoretic mobility of single-stranded DNA sometimes combined with conformation-specific nuclease cleavage. Some of these methods are too laborious and time-consuming and many depend on the nature of base alteration.
It is desirable to have a sensitive, simple, inexpensive method for detecting differences in nucleic acids such as mutations, preferably, in a homogeneous format. The method should minimize the number and complexity of steps and reagents. Such a method would be suitable for a large scale population screening.
2. Description of the Related Art
Formation of a single base mismatch that impedes spontaneous DNA branch migration is described by Panyutin, et al., (1993)
J. Mol. Biol.,
230:413-424.
The kinetics of spontaneous DNA branch migration is discussed by Panyutin, et al., (1994)
Proc. Natl. Acad. Sci. USA,
91: 2021-2025.
European Patent Application No. 0 450 370 A1 (Wetmur, et al.,) discloses branch migration of nucleotides.
A displacement polynucleotide assay method and polynucleotide complex reagent therefor is discussed in U.S. Pat. No. 4,766,062 (Diamond, et al.,).
A strand displacement assay and complex useful therefor is discussed in PCT application WO 94/06937 (Eadie, et al.,).
PCT application WO/86/06412 (Fritsch, et al.,) discusses process and nucleic acid construct for producing reagent complexes useful in determining target nucleotide sequences.
A process for amplifying, detecting and/or cloning nucleic acid sequences is disclosed in U.S. Pat. Nos. 4,683,195, 4,683,202, 4,800,159, 4,965,188 and 5,008,182. Sequence polymerization by polymerase chain reaction is described by Saiki, et al., (1986)
Science,
230: 1350-1354. Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase is described by Saiki, et al,
Science
(1988) 239:487.
SUMMARY OF THE INVENTION
One method in accordance with the present invention is directed to the detection of the presence of a difference between two related nucleic acid sequences. In the method a complex is formed comprising both of the nucleic acid sequences in double stranded form. Both members of at least one pair of non-complementary strands within the complex have labels. The association of the labels as part of the complex is determined as an indication of the presence of the difference between the two
Kurn Nurith
Lishanski Alla
Ullman Edwin F.
Benzion Gary
Dade Behring Marburg GmbH
Gattari Patrick G.
Tung Joyce
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