Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Patent
1996-07-15
1999-03-09
Zitomer, Stephanie W.
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
435 912, 435196, 536 243, 935 77, 935 78, C12Q 168, C12P 1934, C12N 916
Patent
active
058798862
DESCRIPTION:
BRIEF SUMMARY
This application is a 371 (National Stage) of PCT/FR94/01068 filed Sep. 9, 1994 which claims priority to the French application, 93/10821, filed Sep. 10, 1993.
The object of the present invention is a method for detecting molecules containing nucleotide mismatches and the location of these mismatches, and its application to the detection of base substitutions or deletions in nucleotide sequences.
BACKGROUND OF THE INVENTION
The majority of diseases linked to with genome modifications, either of the host organisms or of infectious organisms, are very often due to a simple change of one or more nucleotides. Considerable effort has therefore been devoted to develop methods for detecting not only already known mutations but also unknown mutations.
However, the methods available for searching for unknown point mutations in DNA regions of significant size have many disadvantages which render their use difficult.
For example, electrophoresis gels with a denaturation gradient require computer-assisted optimizations of the target region and electrophoresis conditions adapted to each DNA fragment (Myers et al. (1987), Methods Enzymol, 155, 501-527).
The technique of single strand conformation polymorphism (SSCP) (Orita et al. (1989)--Genomics, 5, 874), despite its experimental simplicity, has shown a sensitivity for the detection of mutations in DNA fragments of around 150 base pairs.
However, neither of these methods gives precise information concerning the location of the mutation in the DNA fragment.
Although direct sequencing methods are becoming increasingly rapid, they remain costly and lengthy in searching for unknown mutations and are not reliable in the case of heterozygous point mutations.
Another method involving chemical cleavage at the site of the mismatches (CCM) such as described by Cotton et al. (Proc. Natl. Acad. Sci., USA (1988) 85, 4397-4401) is in principle well suited to the detection of mutations, independently of the length and the composition of the sequence of the region of interest. It has been used with success in a large number of studies (Cotton (1993) Mutation Research 285, 125-144).
This method has enabled the detection of mutations in fragments amplified by enzymatic routes and of a length of one kilobase.
No present method is likely to be used routinely for various reasons: lack of reliability (a significant number of false negatives) and also because the complexity of the methods which thus do not lend themselves to automated operation.
For example in the conventional CCM method, the heteroduplexes, in other words the double-stranded DNA, resulting from a matching between two heterologous DNA molecules, are formed between the patient's DNA amplified by the enzymatic route, and a DNA fragment representing the wild type sequence labeled at one end by use of a radioactive isotope. The heteroduplex DNA molecules are formed with the DNA corresponding to the sequence of the wild type labeled at the ends of each of its strands, coding or non-coding, and are then chemically treated in parallel reactions in order to reveal the mismatches.
In theory, all the mutations ought to be able to be detected by the use of two probes, after specific cleavage of the non-matched cytosines and thymines induced by hydroxylamine and osmium tetroxide respectively, since a non-matched cytosine or thymine should be present at the site of the mutation, either on the coding strand or on the non-coding strand of the probe labeled with a radioactive isotope.
In practice, cleavage at the site of some mismatches can occur incorrectly, because of the nature of the non-matched bases and the environment of these bases.
The probes used in the conventional CCM method thus do not allow for four types of heteroduplex existing simultaneously. For example, Cotton et al. (1993, cited above) describe only one or two types of heteroduplex.
A further disadvantage of this technique is the necessity for repeating the radioactive labeling of the primers for chain polymerization (PCR) and the size markers.
In addition, this techn
REFERENCES:
patent: 5217863 (1993-06-01), Cotton et al.
patent: 5376526 (1994-12-01), Brown et al.
patent: 5556750 (1996-09-01), Modrich et al.
Wood et al., "HLA-DR/Dw Matching By PCR Fingerprinting: The Origin of PCR Fingerprints and Further Applications", European J. of Immunogenetics, vol. 18, pp. 147-153 (1991).
Friedl et al., "Single-step Screening Method for the Most Common Mutation in Familial Adenomatous Polyposis," Human Molecular Genetics, vol. 2, No. 9, pp. 1481-1482 (1993).
Cai et al., "A Rapid and Simple Electrophoretic Method for the Detection of Mutations . . . ", Human Genetics, vol. 87, No. 6, pp. 728-730 (1991).
Lu et al., "Detection of Single DNA Base Mutations with Mismatch Repair Enzymes," Genomics, vol. 14, No. 2, pp. 249-255 (1992).
R. Cotton, "Current Methods of Mutation Detection," Mutation Research, vol. 285, pp. 125-144 (1993).
Myers et al., Methods Enzymol, vol. 155, pp. 501-527 (1987).
Orita et al., Genomics, vol. 5, p. 874 (1989).
Cotton et al., Proc. Nat'l Acad. Scie., USA, vol. 85, pp. 4397-4401 (1988).
Biasotto Michel
Meo Tommaso
Tosi Mario
Verpy Elisabeth
Institut National de la Sante et de la Recherche Medicale
Institut Pasteur
Zitomer Stephanie W.
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