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

• Current Assignee: Fox Chase Cancer Center
• Original Assignee: Fox Chase Cancer Center
• Inventors: Yeung, Anthony T.
• Primary Examiner(s): Jones, W. Gary
• Assistant Examiner(s): Atzel, Amy
• Application Number:
  US08/658,322
• Time in Patent Office: 979 Days
• Field of Search: 435/6, 435/91.2, 435/183, 435/195, 435/196, 530/350
• US Class Current: 435/6.18
• CPC Class Codes: C12N 9/22 Ribonucleases RNAses, DNAses catalytic nucleic acids C12N15/113
• CPC Class Codes: C12Q 1/683 involving restriction enzymes, e.g. restriction fragment length polymorphism [RFLP]
• CPC Class Codes: C12Q 2521/514 Mismatch repair protein
• CPC Class Codes: C12Q 2537/113 Heteroduplex formation

Abstract

An endonuclease and its method of use for the detection of mutations in targeted polynucleotide sequences are provided, which facilitate the localization and identification of mutations, mismatches and genetic polymorphisms.

Claims

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.

2. The method as claimed in claim 1 wherein said endonuclease is from celery.

3. The method as claimed in claim 1 wherein said polynucleotide is DNA.

4. The method as claimed in claim 2 wherein the sequences subjected to said endonuclease activity are further subjected to the activity of a protein, said protein being selected from the group consisting of DNA ligase, DNA polymerase, DNA helicase, 3'"'"'-5'"'"' DNA Exonuclease, DNA binding proteins that bind to DNA termini or a combination of said proteins, thereby reducing non-specific DNA cleavage.

5. The method as claimed in claim 2 wherein the sequences subjected to said endonuclease activity are further subjected to DNA polymerase activity, so as to reduce non-specific DNA cleavage.

6. The method as claimed in claim 2 wherein target DNA is analyzed in the presence of a multiplicity of pooled samples.

7. The method as claimed in claim 2 wherein said polynucleotide is cDNA.

8. The method as claimed in claim 1, wherein said polynucleotides are analyzed on a DNA sequencing gel thereby identifying the location of the mutation in a target DNA strand relative to DNA sequencing molecular weight markers.

9. The method as claimed in claim 1 wherein said determination is employed as an assay for detection of cancer.

10. The method as claimed in claim 1 wherein said determination is employed as an assay for detection of birth defects.

12. The method as claimed in claim 2 wherein the sequences subjected to said endonuclease activity are further subjected to the activity of a protein, said protein being selected from the group consisting of DNA ligase, DNA polymerase, DNA helicase, 3'"'"'-5'"'"' DNA Exonuclease, DNA binding proteins that bind to DNA termini or a combination of said proteins, thereby stimulating turnover of said endonuclease.

13. The method as claimed in claim 2 wherein said sequences subjected to said endonuclease activity are further subjected to DNA polymerase activity, thereby stimulating turnover of said endonuclease.

11. A method for determining a mutation in a target sequence of 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, exposed to endonuclease and analyzed for the presence of said mutation, the improvement comprising the use of a mismatch endonuclease enzyme from celery, 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;

c) recognition of a mutation in a target polynucleotide said recognition being substantially unaffected by flanking polynucleotide sequences; and

d) recognition of polynucleotide loops and insertions between said hybridized polynucleotides.

14. A mismatch endonuclease enzyme for determining a mutation in a target sequence of single stranded mammalian polynucleotide with reference to a non-mutated sequence in a polynucleotide that is hybridizable with the polynucleotide including said target sequence, said enzyme being isolated from a plant source and effective to:

a) detect 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) recognize polynucleotide loops and insertions in said hybridized polynucleotides;

c) catalyze formation of a substantially single-stranded nick at the DNA site containing a mismatch;

d) recognize a mutation in a target polynucleotide sequence, said recognition being substantially unaffected by flanking DNA sequences.

15. An enzyme as claimed in claim 14, wherein said enzyme is CEL I.

16. An enzyme as claimed in claim 14, said enzyme being in substantially pure form.

17. An enzyme as claimed in claim 15, said enzyme being in substantially pure form.