RNase H-based assays utilizing modified RNA monomers

• US 8,911,948 B2
• Filed: 07/22/2009
• Issued: 12/16/2014
• Est. Priority Date: 04/30/2008
• Status: Active Grant

• Current Assignee: Integrated DNA Technologies (Danaher Corporation)
• Original Assignee: Integrated DNA Technologies (Danaher Corporation)
• Inventors: Walder, Joseph Alan, Behlke, Mark Aaron, Rose, Scott, Dobosy, Joseph
• Primary Examiner(s): Strzelecka, Teresa E
• Application Number:
  US12/507,142
• Publication Number:
  US 20100167353A1
• Time in Patent Office: 1,973 Days
• Field of Search: None
• US Class Current: 435/6.12
• CPC Class Codes: C12Q 1/6844 Nucleic acid amplification reactions
• CPC Class Codes: C12Q 1/6848 characterised by the means for preventing contamination or increasing the specificity or sensitivity of an amplification reaction
• CPC Class Codes: C12Q 1/6853 using modified primers or templates
• CPC Class Codes: C12Q 1/6858 Allele-specific amplification
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 2521/301 Endonuclease
• CPC Class Codes: C12Q 2525/121 incorporating both deoxyribonucleotides and ribonucleotides
• CPC Class Codes: C12Q 2525/186 incorporating a non-extendable or blocking moiety
• CPC Class Codes: C12Q 2549/101 Hot start

Abstract

The present invention pertains to novel oligonucleotide compounds for use in various biological assays, such as nucleic acid amplification, ligation and sequencing reactions. The novel oligonucleotides comprise a ribonucleic acid domain and a blocking group at or near the 3′ end of the oligonucleotide. These compounds offer an added level of specificity previously unseen. Methods for performing nucleic acid amplification, ligation and sequencing are also provided. Additionally, kits containing the oligonucleotides are also disclosed herein.

Claims

1. A method of amplifying a target DNA sequence, said method comprising the steps of:

(a) providing a reaction mixture comprising

(i) an oligonucleotide primer having a cleavage domain with a cleavage site, located within or adjacent to the cleavage domain, which is cleavable by an RNase H2 enzyme, wherein said cleavage site is positioned 5′

of a blocking group at or near the 3′

-end of the oligonucleotide primer which prevents primer extension and/or PCR,

(ii) a sample nucleic acid that may or may not have the target sequence,

(iii) a DNA polymerase,

(iv) an RNase H2 enzyme encoded by SEQ ID NO;

4, and

(v) optionally a second primer in reverse orientation to support PCR;

(b) hybridizing the blocked primer to the target DNA sequence to form a double-stranded substrate;

(c) cleaving the hybridized blocked primer with said RNase H2 enzyme to remove the blocking group from the primer; and

(d) extending the cleaved primer with the DNA polymerase.

2. The method of claim 1, wherein the blocking group is attached to the 3′

-terminal nucleotide of the primer.

3. The method of claim 1, wherein a label permitting detection of the extension reaction is attached to the oligonucleotide primer 3′

from the cleavage site.

4. The method of claim 3, wherein the label is a fluorophore.

5. The method of claim 3, wherein the label is a mass tag for detection of the amplification reaction by mass spectrometry.

6. The method of claim 1, wherein the cleavage domain comprises a continuous sequence of three or more RNA residues.

7. The method of claim 1, wherein the cleavage domain comprises one or more of the following moieties:

a DNA residue, an abasic residue, a modified nucleoside, or a modified phosphate internucleotide linkage.

8. The method of claim 1, wherein the cleavage domain comprises two adjacent RNA residues.

9. The method of claim 1, wherein the cleavage domain comprises a single RNA residue.

10. The method of claim 9, wherein a sequence flanking the cleavage site contains one or more internucleoside linkages resistant to nuclease cleavage.

11. The method of claim 10, wherein the nuclease resistant linkage is a phosphorothioate.

12. The method of claim 10, wherein the 3′

oxygen atom of the RNA residue is substituted with an amino group, thiol group, or a methylene group.

13. The method of claim 10, wherein one or both of the hydrogen atoms on the 5′

carbon atom of the nucleotide residue 3′

of the RNA residue are replaced with a bulkier substituent.

14. The method of claim 13, wherein the substituent is a methyl group.

15. The method of claim 10, wherein the nuclease resistant linkage is on the 3′

side of the cleavage site.

16. The method of claim 1, wherein the second primer comprises a cleavage domain with a cleavage site, located within or adjacent to the cleavage domain, which is cleavable by an RNase H2 enzyme, wherein said cleavage site is positioned 5′

of a blocking group at or near the 3′

-end of the second primer which prevents primer extension and/or PCR.

17. The method of claim 1, wherein the PCR assay is used to discriminate between variant alleles.

18. The method of claim 17, wherein the allelic variants are single nucleotide polymorphisms.

19. The method of claim 17, wherein a nuclease resistant linkage is incorporated on the 3′

-side of the cleavage site.

20. The method of claim 1, wherein the PCR is detected with a dye-binding assay.

21. A method of amplifying a target DNA sequence, said method comprising the steps of:

(a) providing a reaction mixture comprising

(i) an oligonucleotide primer having a cleavage domain with a cleavage site, located within or adjacent to the cleavage domain, which is cleavable by an RNase H2 enzyme, wherein said cleavage site is positioned 5′

of a blocking group at or near the 3′

-end of the oligonucleotide primer which prevents primer extension and/or PCR, and wherein said cleavage domain includes a sequence 5′

to 3′

Rx or RDx where R is an RNA residue, D is a DNA residue and x is an abasic residue,

(ii) a sample nucleic acid that may or may not have the target sequence,

(iii) a DNA polymerase,

(iv) an RNase H2 enzyme; and

(v) optionally a second primer in reverse orientation to support PCR;

(b) hybridizing the blocked primer to the target DNA sequence in said reaction mixture to form a double-stranded substrate;

(c) cleaving the hybridized blocked primer with said RNase H2 to remove the blocking group from the primer; and

(d) extending the cleaved primer with the DNA polymerase.

22. The method of claim 21 wherein said cleavage domain has the sequence RDxxD.

23. The method of claim 22 wherein x is a C3 spacer.

24. The method of claim 21 wherein the assay is used to discriminate between variant alleles.

25. The method of claim 24 wherein said allelic variants are single nucleotide polymorphisms.

26. A method of amplifying a target DNA sequence, said method comprising the steps of:

(a) providing a reaction mixture comprising

(i) an oligonucleotide primer having a cleavage domain with a cleavage site, located within or adjacent to the cleavage domain, which is cleavable by an RNase H2 enzyme, wherein said cleavage site is positioned 5′

of a blocking group at or near the 3′

-end of the oligonucleotide primer which prevents primer extension and/or PCR, and wherein said cleavage domain lacks an RNA residue and comprises one or more 2'"'"'-modified nucleosides,

(ii) a sample nucleic acid that may or may not have the target sequence,

(iii) a DNA polymerase,

(iv) an RNase H2 enzyme, and

(v) optionally a second primer in reverse orientation to support PCR;

(b) hybridizing the blocked primer to the target DNA sequence in said reaction mixture to form a double-stranded substrate;

(c) cleaving the hybridized blocked primer with said RNase H2 enzyme to remove the blocking group from the primer; and

(d) extending the cleaved primer with the DNA polymerase.

27. The method of claim 26 wherein the cleavage domain is a single 2'"'"'-fluoro nucleoside.

28. The method of claim 26 wherein the cleavage domain is two adjacent 2'"'"'-fluoro nucleoside residues.