Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1999-03-16
2002-03-26
Horlick, Kenneth R. (Department: 1656)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091500
Reexamination Certificate
active
06361941
ABSTRACT:
Throughout this application, various publications are cited. The disclosure of these publications is hereby incorporated by reference into this application to describe more fully the state of the art to which this invention pertains.
FIELD OF THE INVENTION
This invention relates to methods of diagnosing disorders characterized by known nucleic acid mutations. The instant methods employ the use of catalytic nucleic acid molecules, and are useful in connection with diagnosing such disorders as cancer and AIDS.
BACKGROUND OF THE INVENTION
A variety of inherited and acquired diseases are associated with genetic variations such as point mutations, deletions and insertions. Some of these variations are directly associated with the presence of disease, while others correlate with disease risk and/or prognosis. There are more than 500 human genetic diseases which result from mutations in single genes (21, 22). These include cystic fibrosis, muscular dystrophy, &agr;1-antitrypsin deficiency, phenylketonuria, sickle cell anemia or trait, and various other hemoglobinopathies (21, 22). Furthermore, individuals with increased susceptibility to several common polygenic conditions, such as atherosclerotic heart disease, have been shown to have an association with the inheritance of particular DNA sequence polymorphisms.
Cancer is thought to develop due to the accumulation of genetic lesions in genes involved in cellular proliferation or differentiation. The ras proto-oncogenes, K-ras, N-ras and H-ras, and the p53 tumor suppressor gene are examples of genes which are frequently mutated in human cancers. Specific mutations in these genes leads to an increase in transforming potential. Genetic analysis would be invaluable in the clinic for assessing disease risk, diagnosis of disease, predicting a patient's prognosis or response to therapy, and monitoring a patient's progress. The introduction of such genetic tests, however, will depend on the development of simple, inexpensive, and rapid assays for genetic variations.
Methods of in vitro nucleic acid amplification have wide-spread applications in genetics and disease diagnosis. In the last decade many techniques for amplification of nucleic acid have been described. These include the polymerase chain reaction (PCR) (1-7), the ligase chain reaction (LCR) (8), the strand displacement amplification assay (SDA) (9) and transcription-mediated amplification (TMA) (10, 11) (also known as self-sustained sequence replication (SSR)). The amplification products (amplicons) produced by PCR, LCR and SDA are DNA, whereas RNA amplicons are produced by TMA. DNA or RNA templates, generated by these protocols or others, can be analyzed for the presence of sequence variation (i.e. mutation) associated with the disease to be ascertained.
As with nucleic acid amplification, catalytic nucleic acids have been studied intensively in recent years. The potential for suppression of gene function using catalytic nucleic acids as therapeutic agents is widely discussed in the literature (12-18). Catalytic RNA molecules (ribozymes) have been shown to be capable of cleaving both RNA (12) and DNA (17) molecules. Similarly, catalytic DNA molecules (DNAzymes) have also been shown to be capable of cleaving both RNA (13, 19) and DNA (18) molecules. Catalytic nucleic acid can only cleave a target nucleic acid sequence, provided that target sequence meets minimum sequence requirements. The target sequence must be complementary to the hybridizing regions of the catalytic nucleic acid and the target must contain a specific sequence at the site of cleavage. Examples of such sequence requirements at the cleavage site include the requirement for a purine:pyrmidine sequence for a class of DNAzyme cleavage (10-23 model) (19), and the requirement for the sequence uridine:H where H can equal A, C or U but not G, for the hammerhead ribozymes (23).
In addition to their therapeutic potential, catalytic nucleic acid molecules can also distinguish between targets which differ by a single point mutation (14-16). This is achieved by targeting a specific sequence which is present in wild-type but not mutant templates or vice versa. So far, this capacity for discrimination has only been exploited as a method for therapeutic manipulation of gene expression.
A review by Nollau-Wagener (24) compared several methodologies for the detection of point mutations with respect to the type of nucleic acid analyzed, the percentage of mutations detected, the time and cost of performing the assay, and problems relating to the use of toxic reagents. Each of the methodologies examined had its drawbacks. For example, denaturing gradient gel electrophoresis is time consuming, RNAase A cleavage can only detect about 70% of possible mutations, and chemical cleavage involves the use of toxic substances.
Another method, known as restriction fragment length polymorphism (RFLP), involves ascertaining whether a restriction enzyme site is present or absent at the locus of interest. In rare instances, mutations can be detected because they happen to lie within a naturally occurring restriction endonuclease recognition/cleavage site (31).
The inclusion of mismatched bases within primers used to facilitate in vitro amplification can result in the induction of artificial restriction endonuclease recognition/cleavage sites, and hence an increase in the number of loci which can be analyzed by RFLP (32). Modified primers containing mismatched bases have been used to induce artificial recognition/cleavage sites for restriction endonucleases at critical codons within the ras gene family (33-35). The general rules for designing primers which contain mismatched bases located near the 3′ termini of primers have been established (36).
Although the use of mismatched primers has expanded the utility of RFLP analysis, the technique is still limited by the fact that a minimum of four base pairs is required for recognition and cleavage by a restriction enzyme.
SUMMARY OF THE INVENTION
This invention provides a method of determining whether a subject is afflicted with a disorder characterized by the presence of a known nucleic acid mutation, which comprises the steps of (a) isolating a sample of nucleic acid molecules from the subject; (b)(i) amplifying the nucleic acid segment present in the isolated sample, which segment is known to contain the mutation in a subject afflicted with the disorder, and (ii) under suitable conditions, contacting the resulting amplified segment with a catalytic nucleic acid molecule which specifically recognizes and cleaves a target sequence present either (1) in the nucleic acid segment having the known mutation or (2) in the corresponding wild-type nucleic acid segment, but not both, with the proviso that step (ii) can be performed either subsequent to or concurrently with step (i); and (c) determining whether the catalytic nucleic acid molecule in step (b)(ii) cleaves the amplified segment, so as to determine whether the subject is afflicted with the disorder.
This invention also provides a method of determining whether a subject is afflicted with a disorder characterized by the presence of a plurality of known nucleic acid mutations, which comprises the steps of (a) isolating a sample of nucleic acid molecules from the subject; (b)(i) amplifying the nucleic acid segment present in the isolated sample, which segment is known to contain the plurality of mutations in a subject afflicted with the disorder, and (ii) under suitable conditions, contacting the resulting amplified segment with a plurality of catalytic nucleic acid molecules, each of which specifically recognizes and cleaves a target sequence present either (1) in the nucleic acid segment having the known mutation or (2) in the corresponding wild-type nucleic acid segment, but not both, with the proviso that step (ii) can be performed either subsequent to or concurrently with step (i); and (c) determining whether each of the catalytic nucleic acid molecules in step (b)(ii) cleaves the amplified segment, so as to determine whether the su
Cairns Murray J.
Fuery Caroline J.
Todd Alison V.
Horlick Kenneth R.
Johnson & Johnson Research Pty Limited
McCormack Myra
Strzelecka Teresa
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