Nucleic Acid Detection Method Involving the Direct Generation of a Measurable Signal

• US 20070292868A1
• Filed: 06/16/2005
• Published: 12/20/2007
• Est. Priority Date: 06/16/2005
• Status: Active Grant

• Current Assignee: Biotools Biotechnological & Medical Laboratories S.A.
• Original Assignee: Biotools Biotechnological & Medical Laboratories S.A.
• Inventors: Limones Lopez, Gemma, Madejon Seiz, Antonio, Rodriguez Gil, Sonia, Calvo Macarro, Francisco, Franco De Sarabia Rosado, Pedro
• Granted Patent: US 7,919,244 B2
• Time in Patent Office: Days
• US Class Current: 435/6.1
• CPC Class Codes: C12Q 1/6823 Release of bound markers
• CPC Class Codes: C12Q 2521/319 Exonuclease
• CPC Class Codes: C12Q 2525/185 incorporating bases where the precise position of the bases in the nucleic acid string is important
• CPC Class Codes: C12Q 2565/1015 labels being on the same oligonucleotide

Abstract

A nucleic acid detection method involving the direct generation of a measurable signal, by the action of an enzyme with 3′-5′ nuclease activity, and the applications for it, the signal generated can be detectable and quantifiable and can be carried out in real-time, in this method, the nucleic acid is placed in contact with at least one oligonucleotide that does not hybridize perfectly with it, so that the enzyme will split it at the unpaired bases generating the signal, the oligonucleotide can be labeled.

Claims

1. Method to detect nucleic acids, DNA or RNA, comprising directly generating a measurable signal wherein the signal is produced by the activity of an enzyme with 3′

-5′

nuclease activity when this makes contact with a nucleic acid substrate, with at least one oligonucleotide capable of hybridizing with it, leaving one or more bases unpaired at the 3′

end of the chain, or adjacent bases, which are split off by the activity of the enzyme 3′

-5′

nuclease, generating the measurable signal.

2. Detection method according to claim 1 wherein the oligonucleotide can be labeled in at least one base in any position along the chain.

3. Method according to claim 2 wherein the oligonucleotide is labeled at the 3′

end or adjacent bases.

4. Method according to claim 2 wherein both ends, 3′

and 5′

, of the oligonucleotide are labeled.

5. Method according to claim 2 wherein the label/s are done with fluorides.

6. Method according to claim 4 wherein the 5′

end is labeled with a fluoride quencher and the 3′

with a fluoride reporter.

7. Method according to claim 1 wherein the enzymes, oligonucleotides and nucleic acid substrate are in an aqueous solution.

8. Method according to claim 1 wherein the oligonucleotide is bound to a solid support.

9. Method according to claim 1 wherein the nucleic acid substrate is bound to a solid support.

10. Method according to claim 1 wherein the nucleic acid substrate is a DNA chain that is generated by previous gene amplification steps, in vitro transcription (cDNA), or isothermal amplification.

11. Method according to claim 1 wherein the nucleic acid substrate is derived from animal or plant biological samples, cell cultures, food products, samples of water, soil or air.

12. Method according to claim 1 wherein it is performed coupled to a nucleic acid amplification system, such as primer extension, PCR or isothermal amplification systems.

13. Method according to claim 12 wherein the PCR system is a PCR system in real-time.

14. Method according to claim 1 wherein it is used to detect and discriminate between nucleic acid sequences that differ in one base, with the oligonucleotide presenting the mutation of interest at its 3′

end, generating the detectable signal when the nucleic acid substrate presents the mutation.

15. Method according to claim 1 wherein it is used to detect the presence of mutations in nucleic acid encoding codons, and the oligonucleotide presents the codon to be analyzed at the 3′

end, generating a detectable signal when the nucleic acid presents mutations in this codon.

16. Method according to claim 1 wherein, in the presence of a mixture of nucleotides and an enzyme with polymerase activity, the oligonucleotide or oligonucleotides used to generate the signal hybridize with the nucleic acid substrate, so that nucleic acid extension occurs simultaneously to generation of the signal.

17. Method according to claim 1 wherein, in the presence of a mixture of nucleotides, an enzyme with polymerase activity, an enzyme with chain displacement activity and/or 5′

-3′

nuclease activity, and at least two primers, the oligonucleotide binds to the nucleic acid of interest and functions as a probe generating the signal by the 3′

-5′

nuclease activity, without acting as a primer of the extension reaction.

18. Method according to claim 16 wherein the enzyme with polymerase activity is a polymerase with proofreading 3′

-5′

nuclease activity.

19. Method according to claim 18 wherein the polymerase with proofreading 3′

-5′

nuclease activity is thermostable.

20. Method according to claim 15 wherein the oligonucleotide presents modifications in the binding of its bases by non-phosphodiester bonds or by the inclusion of spacers, to protect these positions from the 3′

-5′

nuclease activity.

21. Method according to claim 20 wherein the oligonucleotide is modified at its 3′

end to protect the hydrolysis of certain positions.

22. Method according to claim 17 wherein the oligonucleotide is modified to inhibit the 5′

-3′

nuclease activity of the polymerase with chain displacement activity.

23. Method according to claim 17 wherein the oligonucleotide is modified at its 3′

end to block the extension reaction.

24. Method according to claim 1 wherein this is carried out in a hybridization system in the absence of a polymerization reaction in at least one hybridization/catalysis reaction.

25. Method according to claim 24 wherein the hybridization/catalysis reaction is monitored in systems in real-time.

26. A kit to determine a nucleic acid sequence in a sample, said kit comprising an oligonucleotide that contains the sequence complementary to the nucleic acid sequence to be determined with one or more non-complementary bases at the 3′

end of the chain, a polymerase enzyme 3′

-5′

nuclease proofreading activity.

27. A kit according to claim 26 further comprising at least one oligonucleotide primer of the nucleic acid.

28. A kit according to claim 26 wherein the oligonucleotide is blocked at the 3′

end.

29. A kit according to claim 26 wherein the oligonucleotide is labeled at the 3′

end with a fluoride reporter and at the 5′

end with a fluoride quencher.

30. A kit according to claim 26 further comprising deoxynucleotides.