Nano-PCR: methods and devices for nucleic acid amplification and detection

• US 20090246834A1
• Filed: 01/26/2009
• Published: 10/01/2009
• Est. Priority Date: 05/13/2004
• Status: Active Grant

• Current Assignee: Nanobiosym, Inc.
• Original Assignee: Nanobiosym, Inc.
• Inventors: Goel, Anita
• Granted Patent: US 8,632,973 B2
• Time in Patent Office: Days
• US Class Current: 435/91.200
• CPC Class Codes: A61K 41/00 Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
• CPC Class Codes: B01L 2200/0636 Focussing flows, e.g. to laminate flows
• CPC Class Codes: B01L 2200/0663 Stretching or orienting elongated molecules or particles
• CPC Class Codes: B01L 2300/0816 Cards, e.g. flat sample carriers usually with flow in two horizontal directions
• CPC Class Codes: B01L 2300/0861 Configuration of multiple channels and/or chambers in a single devices
• CPC Class Codes: B01L 2300/0896 Nanoscaled
• CPC Class Codes: B01L 2400/0415 electrical forces, e.g. electrokinetic
• CPC Class Codes: B01L 2400/043 magnetic forces
• CPC Class Codes: B01L 2400/0454 radiation pressure, optical tweezers
• CPC Class Codes: B01L 2400/0487 fluid pressure, pneumatics
• CPC Class Codes: B01L 3/502761 specially adapted for handling suspended solids or molecules independently from the bulk fluid flow, e.g. for trapping or sorting beads, for physically stretching molecules investigating characteristics of particles G01N15/00
• CPC Class Codes: B01L 3/502776 specially adapted for focusing or laminating flows
• CPC Class Codes: B01L 7/52 with provision for submitting samples to a predetermined sequence of different temperatures, e.g. for treating nucleic acid samples amplification or hybridisation processes per se C12Q1/68; controlling sequential reactions for synthesis B01J19/0046
• CPC Class Codes: B82Y 30/00 Nanotechnology for materials or surface science, e.g. nanocomposites
• CPC Class Codes: B82Y 5/00 Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 2523/303 Applying a physical force on a nucleic acid
• CPC Class Codes: C12Q 2523/305 Denaturation or renaturation by physical action
• CPC Class Codes: C12Q 2523/307 Denaturation or renaturation by electric current/voltage
• CPC Class Codes: C12Q 2527/101 Temperature
• CPC Class Codes: C12Q 2565/629 : being a microfluidic device
• CPC Class Codes: Y02A 50/30 : Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Abstract

Methods, devices, and compositions are described that provide for amplification of nucleic acid sequences without reliance upon temperature cycling, thus freeing the methods from conventional benchtop thermal cycling devices. Denaturation of double stranded nucleic acids, primer annealing, and precision control over primer extension by polymerase can be accomplished by applying stress to a nucleic acid. These methods can provide one or more benefits over conventional PCR methods including: precision control over the PCR process; generally improved fidelity; improved accuracy over problematic sequences such as GC-rich or tandem repeat regions; greater sequence length; increased reaction yield; reduced experimental time; greater efficiency; lower cost; greater portability; and, robustness to various environmental parameters, such as temperature, pH, and ionic strengths.

Claims

1. A method for amplifying a nucleotide sequence wherein the method comprises:

(a) contacting one or more template strands of single-stranded DNA with one or more oligonucleotide primers complementary to a portion of each template strand and;

(b) permitting hybridization of at least one primer to the complimentary portion of the template strands;

(c) contacting the template strands with a DNA polymerase and at least four different nucleoside triphosphates,

(d) permitting extension of the primers by the DNA polymerase thereby forming extension products bound to the template strands;

(e) separating the extension products from the template strands; and

thereafter repeating steps (a), (b), (c), and (d) one or more times with step (e) performed before each repetition;

wherein permitting hybridization of at least one primer to the complimentary portion of the template strands comprises, controlling the hybridization by controlling an application of stress to the template strands, or

wherein permitting extension of the primers by the DNA polymerase comprises controlling the extension of the primers by the DNA polymerase to form extension products complementary to each template strands by controlling an application of stress to the template strands, or

wherein separating the extension products from the template strands comprises separating the extension products from the template strands by applying stress to the template strands,

or a combination of these wherein stress is applied to the template strands during more than one of steps (b), (d), and (e).