Methods and reagents for reducing non-specific amplification

• US 9,115,394 B2
• Filed: 12/11/2012
• Issued: 08/25/2015
• Est. Priority Date: 12/22/2011
• Status: Active Grant

• Current Assignee: Roche Molecular Systems Inc (Roche Holding AG)
• Original Assignee: Roche Molecular Systems Inc (Roche Holding AG)
• Inventors: Bodepudi, Veeraiah, Schoenbrunner, Nancy, Will, Stephen
• Primary Examiner(s): Horlick, Kenneth R.
• Assistant Examiner(s): THOMAS, DAVID C
• Application Number:
  US13/710,824
• Publication Number:
  US 20140057254A1
• Time in Patent Office: 987 Days
• US Class Current: 1/1
• CPC Class Codes: C07H 21/04 with deoxyribosyl as saccharide radical
• 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 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 2525/117 incorporating modified base

Abstract

The present invention provides methods and reagents for use in the amplification of nucleic acids. Amplification carried out using oligonucleotides containing modified nucleotides can result in less non-specific amplification compared to amplification carried out using unmodified oligonucleotides.

Claims

1. A method of reducing or preventing non-specific amplification of nucleic acid during a multiplex amplification reaction comprising providing multiple pairs of primer oligonucleotides capable of amplifying multiple target nucleic acid sequences;

providing multiple probe oligonucleotides, each probe oligonucleotide incorporating a N²-benzyl-dG nucleotide that blocks the extension of a primer oligonucleotide by a thermostable DNA polymerase by more than 2 nucleotides beyond the position of the N²-benzyl-dG-nucleotide if the primer oligonucleotide hybridizes to the probe oligonucleotide.

2. The method of claim 1 wherein the amplification reaction is a Taqman PCR assay and the probe oligonucleotides are 5′

nuclease probes.

3. The method of claim 1 comprising providing three or more pairs of primer oligonucleotides capable of amplifying three or more target nucleic acid sequences and providing three or more probe oligonucleotides.