Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Patent
1989-05-31
1993-03-02
Moskowitz, Margaret
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
435 40, 435173, 435289, 435290, 435291, 435300, 435310, 435803, 435973, 436501, 436809, 935 19, 935 78, 935 86, C12Q 168
Patent
active
051908565
DESCRIPTION:
BRIEF SUMMARY
The invention relates to a method for the detection of single base mutations in multiple loci in human genomic DNA.
One aspect of the invention relates to a method of performing screening programs for genomic DNA for the detection of single base mutations.
Another aspect of the invention relates to a device for carrying out such screening programs.
The ability to detect single base mutations in human genomic DNA is of fundamental importance in the diagnosis of genetic diseases and in the detection of both induced and spontaneous mutations in malignant cell lines. Southern blotting technique to detect the absence or presence of restriction enzyme recognition sequences is limited by the fact that most single based substitutions do not alter a restriction endonuclease recognition sequence.
A recently developed method significantly increases the number of single base mutations that can be detected in a particular DNA fragment. The method is based on the melting behaviour of the DNA fragments and the use of denaturing gradient gelelectrophoresis as shown by Fischer, S. G. and Lerman, L. S. (1983) Proc. Natl. Acad. Sci. USA 80: 1579-83; Myers, R. M., Fischer, S. G., Maniatis, T. and Lerman, L. S. (1985) Nucl. Acids Res. 13: 3111-3129; Lerman, L. S., Silverstein, K. and Grinfeldt, E. in Molecular Biol. of Homo Sapiens, Cold Spring Harbor Lab. (1986) pp. 285-297. DNA fragments differing by single base substitutions can be separated from each other by electrophoresis in polyacrylamide gels containing an ascending gradient of the DNA denaturants urea and formamide. Two identical DNA fragments differing by only one single base pair, will initially move through the polyacrylamide gel at a constant rate. As they migrate into a critical concentration of denaturant, specific domains within the fragments melt to produce partially denatured DNA. Melting of a domain is accompanied by an abrupt decrease in mobility. The position in the denaturant gradient gel at which the decrease in mobility is observed corresponds to the melting temperature of that domain. Since a single base substitution within the melting domain results in a melting temperature difference, partial denaturation of the two DNA fragments will occur at different positions in the gel. DNA molecules can therefore be separated on the basis of very small differences in the melting temperature.
However, the previously described techniques to analyse genomic DNA fragments by the use of denaturing gradient gelelectrophoresis (DGGE) are based on two steps prior to electrophoresis: 1) denaturing of DNA fragments and 2) re-annealing of strands to a radioactive labelled probe (Myers, R. M. and Maniatis, T. (1986) in Molecular Biol. of Homo sapiens. Cold Spring Harbor Lab. pp. 275-284; Noll, W. W. and Collins, M. (1987) Proc. Natl. Acad. Sci. USA 84: 3339-3343). These techniques therefore limit the number of different loci to be analysed to one in each run and only minor sequence variations in a small DNA region can be detected. The probes that can be used are limited to genomic probes if not only exons are to be analysed. The object of the present invention is to develop a method for efficient transfer of partially melted genomic DNA fragments from the polyacrylamide gel, to be able to analyse for mutations in several different loci on the same DNA sample using a number of different probes on the same blot. The problems with the previously described transfer techniques have been that only a few percent of the fragments were transferred from these DG gels. The method of the invention should therefore greatly facilitate the identification of DNA polymorphisms not detected by restriction enzymes in a large number of different loci. This technique should therefore also be useful in screening for mutations within any DNA fragment where a probe exists.
The invention will now be illustrated with reference to the accompanying figures in which:
FIG. 1 shows an apparatus which may be used in the invention,
FIG. 2 is a schematic diagram of the plasmid NJ1/4.1,
FIG. 3a is an autor
REFERENCES:
patent: 4292161 (1981-09-01), Hoefer et al.
Bethesda Research Labs Catalog (1985) p. 80.
Reiser et al., (1978) Biochem. and Biophys. Res. Comm., vol. 85, No. 3, pp. 1104-1112.
Myers et al., (1985) Nuc. Acids Res., vol. 13, No. 9, pp. 3131-3145.
Fischer, S. G. and Lerman, L. S., Proc. Natl. Acad. Sci., USA, vol. 80 pp. 1579-1583, Mar. 1983.
Borresen, A. L. (1986), Annals of Clinic. Res., vol. 18, pp. 258-263.
R. M. Myers, et al., (Molecular Biology of Homo Sapiens, Cold Spring Harbor Lab., 1986, pp. 275-284).
L. S. Lerman, et al., (Molecular Biology of Homo Sapiens, Cold Spring Harbor Lab., 1986, pp. 285-297).
Marschel Ardin H.
Moskowitz Margaret
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