Methods for amplification of nucleic acids utilizing clamp oligonucleotides

• US 10,202,629 B2
• Filed: 03/14/2014
• Issued: 02/12/2019
• Est. Priority Date: 03/15/2013
• Status: Active Grant

• Current Assignee: Aegea Biotechnologies, Inc.
• Original Assignee: Aegea Biotechnologies, Inc.
• Inventors: Arnold, Lyle J.
• Primary Examiner(s): Thomas, David C
• Application Number:
  US14/773,365
• Publication Number:
  US 20160130623A1
• Time in Patent Office: 1,796 Days
• CPC Class Codes: C12P 19/34 Polynucleotides, e.g. nucleic acids, oligoribonucleotides
• CPC Class Codes: C12Q 1/6844 Nucleic acid amplification reactions
• CPC Class Codes: C12Q 2521/501 Ligase
• CPC Class Codes: C12Q 2525/307 Circular oligonucleotides
• CPC Class Codes: C12Q 2549/126 using oligonucleotides as clamps

Abstract

The present invention provides methods of amplifying a target nucleic acid utilizing a clamp oligonucleotide comprising a first target-binding region on the 3′-terminus and a second target-binding region on the 5′-terminus and tether region in between. The tether region may comprise a variety of user-defined sequences or elements that allow for further manipulation of the target nucleic acid. Such as, for example, capture followed by amplification, identification and/or sequencing. The target-binding regions bind to the target nucleic acid, the 3′-terminus functions as a primer to initiate extension across the target nucleic acid sequence and ligation of the gap results in formation of a circularized nucleic acid. This circular template can be used in a variety of processes, including amplification and sequencing.

Claims

1. A non-rolling amplification method for amplifying a target nucleic acid, said method comprising the steps of:

annealing first and second target binding regions to a target nucleic acid in a first mixture comprising a clamp oligonucleotide and a target nucleic acid wherein said clamp oligonucleotide comprises said first target binding region on the 3′

-terminus and said second target binding region on the 5′

-terminus and user-defined tether region between said first and said second target binding regions, wherein said tether region comprises one or more capture elements and extending said clamp oligonucleotide across said target nucleic acid to produce a first duplex containing a first nucleic acid and said target nucleic acid;

ligating the ends of said first nucleic acid to produce a circularized nucleic acid; and

optionally removing said circularized nucleic acid from said target nucleic acid;

capturing said circular nucleic acid on a solid support wherein said solid support contains a capture means to bind said capture element of said tether region, in a second mixture comprising a primer able to bind said at least one primer binding site of said tether region and expanding said primer to produce a second nucleic acid duplex containing a second nucleic acid and said first nucleic acid; and

disassociating said second nucleic acid from said first nucleic acid thereby amplifying said target nucleic acid.

2. The method according to claim 1, wherein said tether region further comprises one or more primer sites, one or more anchor sites, one or more barcodes, one or more adaptor sites, one or more detection sites, one or more sequencing primer binding sites, or combinations thereof.