Nucleic acid mutation assays

Details Nucleic acid mutation assays Nucleic acid mutation assays

1996-10-28

1999-07-06

Horlick, Kenneth R.

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

435 912, 424 941, 2041828, 536 243, C12Q 168, C12P 1934, A61K 3843, C25B 100

Patent

active

059196238

ABSTRACT:
A method for detecting and locating mutations in DNA involves forming heteroduplex molecules by hybridizing single strands derived from a sample of target DNA under test with single strands derived from a sample of non-mutant reference nucleic acid so that any mutation causing an alteration in one or more nucleotide bases in the target DNA produces a base pair mismatch in the corresponding heteroduplex molecule. The nucleic acid mixture is then reacted with a mismatch-binding protein such as the mismatch repair enzyme Mut"S" which recognizes and binds to any such resultant mismatch site. Subsequent treatment with an exonuclease having unidirectional activity degrades duplex molecules free of mismatches but mismatch-containing heteroduplex molecules are protected by the mismatch-binding protein bound to the mismatch sited therein and this limits the extent of the exonuclease degradation. The degradation products are then analyzed, e.g. by get electrophoresis, to determine the size of residual single-stranded nucleic acid fragments and hence to establish the location of the mutation. This method has useful applications in medical diagnosis and biotechnology.

REFERENCES:
patent: 5459039 (1995-10-01), Modrich et al.
patent: 5556750 (1996-09-01), Modrich et al.
Su et al., Escherichia coliMutS-encoded protein binds to mismatched DNA base pairs, Proc. Natl. Acad. Sci. USA, vol. 83, pp. 5057-5061, 1986.
Lishanski et al., Mutation detection by mismatch binding protein, MutS, in amplified DNA: Application to the cystic fibrosis gene, vol. 91, pp. 2674-2678, 1994.
Ng et al. Mutational and In Vitro protein-binding studies on centromere DNA from Saccharomyces cerevisiae, vol. 7(12), pp. 4522-4534, 1987.
Proc. Natlacad. Sci., vol. 83, no. 14, Jul. 1986 Natlacad Sci. Washington, DC, UC;, pp. 5057-5061, S.-S. Su and P. Modrich "Escherichia colimutS-encoded protein binds to mismatched DNA base pairs", cited in appln., see p. 5060,I.col,line 8-p. 5061,rt.col.,line2; figures 3,5.
Proc. Natlacad Sci.,vol.91,no.7, Mar. 29, 1994 Natlacad Sci., Washington,DC,US.; pp. 2674-2678,A. Lishanski et al. "mutation detection by mismatch binding protein, Muts, in amplified DNA: Application to the cystic fibrosis gene". the whole document.
Nucleic Acids Res. (1994), 22(13),2710-11, Soden:Narhad;ISSN: 0305-1048, 1994, Ellis ,La. et al "MutS binding protects heteoduplex DNA from exonuclease digestion in vitro: a simple method for detecting mutations", the whole document.
J. Biolchem., vol 263, no 14, May 1, 1988 Am. Soc.Biochem.Molbiol, Inc., Baltimore,US;pp. 6829-6835;S.-S.SU et al "Mispair specificity of methyl-directed DNA mismatch correction in vitro". see p.6833,I col.line 20-p. 6834,rt col,line 22; figures 3,4.

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