Setting of multiple priming oligonucleotides for solid gel amplification in hydrogels

• US 8,986,959 B2
• Filed: 08/29/2011
• Issued: 03/24/2015
• Est. Priority Date: 08/30/2010
• Status: Active Grant

• Current Assignee: The Governors of the University of Alberta (University of Alberta)
• Original Assignee: The Governors of the University of Alberta (University of Alberta)
• Inventors: Atrazhev, Alexey
• Primary Examiner(s): MARSCHEL, ARDIN H
• Application Number:
  US13/819,420
• Publication Number:
  US 20130157278A1
• Time in Patent Office: 1,303 Days
• Field of Search: 435/6.1, 435/6.12, 435/91.2
• US Class Current: 435/91.2
• CPC Class Codes: C12Q 1/6844 Nucleic acid amplification reactions
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 2565/501 being an array of oligonucleotides
• CPC Class Codes: C12Q 2565/518 characterised by the immobilisation of the nucleic acid sample or target
• CPC Class Codes: C12Q 2565/629 being a microfluidic device

Abstract

The present invention provides for a novel system and method for amplification and detection of nucleic acids within a microfluidic device wherein multiple nucleotides capable of priming PCR are present within the system and substantially sequestered within separate hydrogel posts therein.

Claims

1. A method for detecting a nucleic acid molecule within a hydrogel post array comprising

a) Depositing on a substantially planar surface plural aqueous solutions each containing at least one nucleic acid capable of initiating an amplification of a selected template nucleic acid and a viscosity increasing solute, said plural aqueous solutions not being in fluid communication with each other and forming a multiplicity of deposits;

b) Allowing said aqueous solutions to evaporate in said deposits;

c) Establishing an array of hydrogel posts, each hydrogel post containing a cell-free, enzymatic, nucleic-acid amplification system, said hydrogel posts comprising the array being arranged so as any given post impinges upon only one deposit;

d) distributing on at least one of said hydrogel posts nucleic acid molecules, at least one of which comprises a template nucleic acid for said amplification system;

e) incubating said hydrogel posts under conditions promoting the synthesis of an amplified nucleic acid product by said amplification system from said at least one template;

wherein said amplification system comprises at least two non-immobilized nucleic acids capable of promoting synthesis of amplified nucleic acid product from said template.

2. The method of claim 1 wherein the viscosity increasing solute is selected from the group consisting of monosaccharide, disaccharide, polysaccharide, linear polyacrylamide, polyvinyl pyrrolidone, and polyethyleneglycol.

3. The method of claim 2 wherein the solute is a disaccharide selected from the group consisting of sucrose, maltose or trehalose.