RNASE H-BASED ASSAYS UTILIZING MODIFIED RNA MONOMERS

• US 20100167353A1
• Filed: 07/22/2009
• Published: 07/01/2010
• Est. Priority Date: 04/30/2008
• Status: Active Grant

• Current Assignee: Integrated DNA Technologies (Danaher Corporation)
• Original Assignee: Integrated DNA Technologies (Danaher Corporation)
• Inventors: Dobosy, Joseph, Behlke, Mark Aaron, Rose, Scott, Walder, Joseph Alan
• Granted Patent: US 8,911,948 B2
• Time in Patent Office: Days
• US Class Current: 435/91.200
• 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 positioned 5′

of a blocking group, said blocking group linked at or near the end of the 3′

-end of the oligonucleotide primer wherein said blocking group prevents primer extension, (ii) a sample nucleic acid that may or may not have the target sequence, (iii) a cleaving enzyme and (iv) a polymerase wherein said cleaving enzyme is a hot start cleaving enzyme which is thermostable and has reduced activity at lower temperatures;

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

c) cleaving the hybridized primer with said cleaving enzyme at a point within or adjacent to the cleavage domain to remove the blocking group from the primer; and

extending the primer with the polymerase.

2-140. -140. (canceled)