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Mismatch endonuclease and its use in identifying mutations in targeted polynucleotide strands

  • US 5,869,245 A
  • Filed: 06/05/1996
  • Issued: 02/09/1999
  • Est. Priority Date: 06/05/1996
  • Status: Expired due to Term
First Claim
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1. A method for determining a mutation in a target sequence of a single stranded polynucleotide with reference to a non-mutated sequence of a polynucleotide that is hybridizable with the polynucleotide including said target sequence, wherein said polynucleotides are amplified, labeled with a detectable marker, hybridized to one another, subjected to the activity of an endonuclease and analyzed for the presence of said mutation, the improvement comprising the use of a mismatch endonuclease enzyme of plant origin, the activity of said enzyme comprising:

  • a) detection of all mismatches whether known or unknown between said hybridized polynucleotides, said detection occurring over a pH range of 5-9, said enzyme exhibiting substantial activity over the entire pH range;

    b) catalytic formation of a substantially single-stranded nick at a target sequence containing a mismatch; and

    c) recognition of a mutation in a target polynucleotide sequence, said recognition being substantially unaffected by flanking polynucleotide sequences.

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