Methods and Compositions for High Sensitivity Fluorescent Mutation Detection with Mismatch Cutting Dna Endonucleases

• US 20080113354A1
• Filed: 11/14/2005
• Published: 05/15/2008
• Est. Priority Date: 11/12/2004
• Status: Abandoned Application

• Current Assignee: Transgenomic, Inc. (Precipio, Inc.)
• Original Assignee: Transgenomic, Inc. (Precipio, Inc.)
• Inventors: Gerard, Gary F., Shi, Yanggu
• Application Number:
  US11/718,761
• Publication Number:
  US 20080113354A1
• Time in Patent Office: Days
• US Class Current: 435/6
• 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 2525/155 incorporating/generating a new priming site
• CPC Class Codes: C12Q 2525/161 incorporating target specific and non-target specific sites
• CPC Class Codes: C12Q 2537/113 Heteroduplex formation
• CPC Class Codes: C12Q 2549/119 using nested primers
• CPC Class Codes: C12Q 2563/107 fluorescence

Abstract

Methods and kits are provided with DNA substrates having a fluorescent label positioned at a nucleotide internal from its 5′ end for use with CEL nuclease to determine whether a DNA sequence contains mutations or polymorphic changes.

Claims

1. A PCR primer for generating fluorescent PCR products in mismatch cutting DNA endonuclease mutation detection, said primer comprising a nucleotide sequence with a 5′

end and 3′

end and a fluorescent label attached to a nucleotide of the nucleotide sequence which is internal with respect to a 5′

end of the nucleotide sequence.

2. The PCR primer of claim 1 wherein the nucleotide sequence is a universal sequence.

3. A single reaction amplification/fluorescent labeling polymerase chain reaction (PCR) comprising a plurality of cycles at an annealing temperature with primers of claim 1 followed by a plurality of cycles of amplification.

5. An amplification/fluorescent labeling nested polymerase chain reaction (PCR) comprising amplifying a target gene by standard PCR and using the resulting PCR product as a template in a separate PCR reaction with primers of claim 1.

7. A method for detecting mutations in a DNA sequence with a mismatch cutting DNA endonuclease comprising amplifying and fluorescence labeling a DNA sample in accordance with the amplification/fluorescent labeling nested polymerase chain reaction (PCR) of claim 3, digesting the DNA with a mismatch cutting DNA endonuclease;

and detecting fluorescently any digested DNA fragments indicative of a mutation in the DNA sequence.

8. The method of claim 7 wherein fluorescence detection is performed by capillary electrophoresis, gel electrophoresis or high pressure liquid chromatography.

9. The method of claim 7 wherein the DNA sample is treated to reduce the salt concentration without concentrating the DNA in the sample prior to detecting fluorescently any digested DNA fragments by capillary electrophoresis.

10. The method of claim 7 wherein the mismatch cutting DNA endonuclease is from the CEL nuclease family of DNA endonucleases.

11. The method of claim 7 wherein the mismatch cutting DNA endonuclease is CEL I nuclease.

12. The method of claim 7 wherein the mismatch cutting DNA endonuclease is CEL II nuclease.

13. A kit for single reaction or nested amplification/fluorescent labeling polymerase chain reaction (PCR) comprising a primer of claim 1.

14. The kit of claim 13 further comprising rules for designing target sequence specific PCR primers for embedding internal fluorescent dye in PCR products by universal fluorescence priming, PCR DNA polymerase or PCR reaction components.

15. A kit for detecting mutations in a DNA sequence with a mismatch cutting DNA endonuclease, said kit comprising a primer of claim 1 and a mismatch cutting DNA endonuclease.

16. The kit of claim 15 wherein the mismatch cutting DNA endonuclease is CEL nuclease.

17. A method for detecting mutations in a DNA sequence with a mismatch cutting DNA endonuclease comprising amplifying and fluorescence labeling a DNA sample in accordance with the amplification/fluorescent labeling nested polymerase chain reaction (PCR) of claim 5, digesting the DNA with a mismatch cutting DNA endonuclease;

and detecting fluorescently any digested DNA fragments indicative of a mutation in the DNA sequence.

18. The method of claim 17 wherein fluorescence detection is performed by capillary electrophoresis, gel electrophoresis or high pressure liquid chromatography.

19. The method of claim 17 wherein the DNA sample is treated to reduce the salt concentration without concentrating the DNA in the sample prior to detecting fluorescently any digested DNA fragments by capillary electrophoresis.

20. The method of claim 17 wherein the mismatch cutting DNA endonuclease is from the CEL nuclease family of DNA endonucleases.

21. The method of claim 17 wherein the mismatch cutting DNA endonuclease is CEL I nuclease.

22. The method of claim 17 wherein the mismatch cutting DNA endonuclease is CEL II nuclease.

23. The single reaction amplification/fluorescent labeling polymerase chain reaction (PCR) of claim 3 wherein the nucleotide sequence of the primer is a universal sequence.

24. The single reaction amplification/fluorescent labeling polymerase chain reaction (PCR) of claim 5 wherein the nucleotide sequence of the primer is a universal sequence.

25. A kit for single reaction or nested amplification/fluorescent labeling polymerase chain reaction (PCR) comprising a primer of claim 2.

26. The kit of claim 25 further comprising rules for designing target sequence specific PCR primers for embedding internal fluorescent dye in PCR products by universal fluorescence priming, PCR DNA polymerase or PCR reaction components.

27. A kit for detecting mutations in a DNA sequence with a mismatch cutting DNA endonuclease, said kit comprising a primer of claim 2 and a mismatch cutting DNA endonuclease.

28. The kit of claim 27 wherein the mismatch cutting DNA endonuclease is CEL nuclease.

4. (canceled)

6. (canceled)