USE OF PRODUCTS OF PCR AMPLIFICATION CARRYING ELEMENTS OF SECONDARY STRUCTURE TO IMPROVE PCR-BASED NUCLEIC ACID DETECTION

• US 20100143898A1
• Filed: 04/30/2007
• Published: 06/10/2010
• Est. Priority Date: 04/28/2006
• Status: Active Grant

• Current Assignee: Igor Kutyavin
• Original Assignee: Igor Kutyavin
• Inventors: Kutyavin, Igor
• Granted Patent: US 9,121,056 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6853 using modified primers or templates
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 2525/301 Hairpin oligonucleotides
• CPC Class Codes: C12Q 2537/143 Multiplexing, i.e. use of multiple primers or probes in a single reaction, usually for simultaneously analyse of multiple analysis
• CPC Class Codes: C12Q 2561/109 Invader technology
• CPC Class Codes: C12Q 2565/1015 labels being on the same oligonucleotide

Abstract

Particular aspects comprise amplifying target nucleic acid using PCR and an oligonucleotide primer pair wherein at least one of the primers is designed to incorporate a 5′-specialty sequence to provide for an amplification product that intramolecularly folds into a secondary structure; and detecting the amplification product by a method comprising: providing an oligonucleotide cleavage component, hybridizing said oligonucleotide cleavage component with the amplification product to form a three-strand cleavage structure wherein two strands of the three-strand cleavage structure are provided by the secondary structure of the amplification product, cleaving 3′- or 5′-strands of the three-strand cleavage structure using a duplex-specific nuclease activity resulting in a cleavage product, and detecting the cleavage product indicative of the presence of the target nucleic acid. In certain aspects both primers incorporate a 5′-specialty sequence and detecting comprises cleaving 3′- or 5′-strands of a three-strand cleavage structure using duplex-specific nuclease to provide a cleavage product.

Claims

1. A method for the detection of a target nucleic acid in a sample, comprising:

amplifying, in a reaction mixture, a target nucleic acid using PCR in the presence of a pair of oligonucleotide primers wherein at least one of the oligonucleotide primers is designed to incorporate a 5′

-specialty sequence to provide an amplification product that intramolecularly folds into a secondary structure; and

detecting the amplification product that folds into a secondary structure by a method comprising;

providing an oligonucleotide cleavage component, hybridizing said oligonucleotide cleavage component with the amplification product to form a three-strand cleavage structure wherein two strands of the three-strand cleavage structure are provided by the secondary structure of the amplification product, cleaving 3′

- or 5′

-strands of the three-strand cleavage structure using a duplex-specific nuclease activity resulting in a cleavage product, and detecting the cleavage product, wherein the presence of the cleavage product is indicative of the presence of the target nucleic acid in said sample.

3. The methods of any one of claim 1 or 2, wherein the target nucleic acid is DNA.

4. The methods of any one of claim 1 or 2, wherein the target nucleic acid is RNA and amplifying of the target nucleic acid includes a stage wherein at least one DNA copy of the RNA is synthesized using a reverse transcriptase activity.

5. The method of claim 4, wherein the reverse transcriptase activity is provided by DNA polymerase used in PCR.

6. The methods of claim 1 or 2, wherein amplifying the target nucleic acid is performed first, followed by the step of detecting the amplification product.

7. The methods of claim 1 or 2, wherein amplifying the target nucleic acid and detecting the amplification product are performed in real time.

8. The method of claim 1, wherein the oligonucleotide cleavage component provides the 3′

-strand, and the amplification product which folds into a secondary structure provides the 5′

-strand of the three-strand cleavage structure.

9. The method of claim 1, wherein the oligonucleotide cleavage component provides the 5′

-strand, and the amplification product which folds into a secondary structure provides the 3′

-strand of the three-strand cleavage structure.

10. The method of claim 8, wherein the oligonucleotide cleavage component is cleaved.

11. The method of claim 8, wherein the amplification product is cleaved.

12. The method of claim 9, wherein the oligonucleotide cleavage component is cleaved.

14. The method of any one of claims 1 and 2, comprising amplification of more than than one target nucleic acid in the same reaction mixture using, for each target sequence, a pair of oligonucleotide primers wherein at least one, or both of the oligonucleotide primers is designed to incorporate a 5′

-specialty sequence to provide an amplification product that intramolecularly folds into a secondary structure, wherein one or more of the target nucleic acids are amplified and detected.

15. The methods of any one of claims 1, 2 and 14, wherein amplifying and detecting of the target nucleic acids are performed to measure the amount of the target nucleic acids in the sample.

16. The methods of any one of claims 1, 2 and 14, wherein amplifying and detecting of the target nucleic acids are performed to determine polymorphic variations of the target nucleic acids in the sample.

17. The methods of any one of claims 11, 12 and 13, wherein the duplex-specific cleavage is provided by 5′

-nuclease activity.

18. The methods of any one of claims 1 and 2, wherein the three-strand cleavage structure is an optimal cleavage structure for 5′

-nuclease activity.

19. The method of claim 17, wherein the 5′

-nuclease activity is provided by a DNA polymerase used in amplifying said target nucleic acid.

20. The method of claim 19, wherein the DNA polymerase activity compirses Taq polymerase.

21. The method of claim 17, wherein the 5′

-nuclease activity is provided by an enzyme that does not express a DNA polymerase activity.

22. The method of claim 21, wherein the enzyme activity that does not express a DNA polymerase activity comprises FEN.

23. The method of claim 21, wherein the enzyme activity that does not express a DNA polymerase activity comprises cleavase.

24. The method of claim 10, wherein the duplex-specific cleavage is provided by a 3′

-nuclease activity.

25. The method of claim 24, wherein the 3′

-nuclease activity is provided by an Endonuclease IV or equivalent activity.

26. The method of claim 8, wherein the three-strand cleavage structure is an optimal cleavage structure for Endonuclease IV.

27. The methods of any one of claims 1 and 8, wherein the oligonucleotide cleavage component and the oligonucleotide primer that does not incorporate the 5′

-specialty sequence are the same molecule.

28. The method of claim 8, wherein the oligonucleotide cleavage component is created during extension of an oligonucleotide primer hybridized to the amplification product that intramolecularly folds into a secondary structure, and wherein cleavage of the three-strand cleavage structure is provided by a 5′

-nuclease activity.

29. The methods of any one of claims 10 and 12, wherein the cleavage is performed in a cycling mode.

30. The methods of any one of claims 1 and 2, wherein oligonucleotide primers that are designed to incorporate a 5′

-specialty sequence are fully extendable in PCR.

31. The methods of any one of claims 1 and 2, wherein one of the oligonucleotide primers that are designed to incorporate a 5′

-specialty sequence is partially extendable in PCR provided that said 5′

-specialty sequence is coupled to the 5′

-end of the primer through a non-extendable linker.

32. The methods of any one of claims 1 and 2, wherein at least one of the oligonucleotide components is immobilized on a solid support during amplification and/or detection stages.

33. The method of claim 17, wherein the cleavage product comprises or is a flap oligonucleotide.

34. The method of claim 33, wherein the flap oligonucleotide serves as an oligonucleotide cleavage component in a different cleavage reaction in the reaction mixture providing a 3′

-strand to different three-strand cleavage structure that is cleaved by a 5′

-nuclease resulting in a different cleavage product, and detecting the different cleavage product, wherein the presence of the different cleavage product is indicative of the presence of the flap oligonucleotide and the target nucleic acid in the sample.

35. The method of claim 34, wherein the different cleavage product contains a label and the label is used in detecting of the different cleavage products.

36. The method of claim 35, wherein the label comprises a fluorescent label.

2. A method for the detection of a target nucleic acid in a sample, comprising:

amplifying, in a reaction mixture, a target nucleic acid using PCR in presence of a pair of oligonucleotide primers wherein both oligonucleotide primers are designed to incorporate a 5′

-specialty sequence to provide amplification products that intramolecular fold into three-strand cleavage structures; and

detecting at least one of the amplification products by a method comprising;

cleaving 3′

- or 5′

-strands of the three-strand cleavage structure using a duplex-specific nuclease activity resulting in a cleavage product, and detecting the cleavage product, wherein the presence of the cleavage product is indicative of the presence of the target nucleic acid in the sample.

13. The method of claim 2, wherein the 5′

-strand of the three-strand cleavage structure is cleaved.