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

• US 8,632,973 B2
• Filed: 01/26/2009
• Issued: 01/21/2014
• Est. Priority Date: 05/13/2004
• Status: Active Grant

• Current Assignee: Nanobiosym, Inc.
• Original Assignee: Nanobiosym, Inc.
• Inventors: Goel, Anita
• Primary Examiner(s): Kim, Young J
• Application Number:
  US12/321,825
• Publication Number:
  US 20090246834A1
• Time in Patent Office: 1,821 Days
• Field of Search: None
• US Class Current: 435/6.11
• 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 of sequencing a nucleic acid molecule, comprising:

(a) providing a sample, including;

a nucleic acid target sequence,

a nucleic acid polymerase;

a primer complementary to the nucleic acid target sequence; and

one or more nucleotides;

(b) annealing the primer to its complementary nucleic acid target sequence;

(c) extending the primer to form an extension product;

(d) optionally repeating steps (b) through (d) to form further extension products; and

(e) determining the sequence of the nucleic acid target sequence,

wherein at least step (c) comprises applying tension that stretches the nucleic acid target sequence and/or the primer extension product.

2. The method of claim 1, wherein the nucleic acid target sequence is an RNA, a double-stranded DNA or a single-stranded DNA.

3. The method of claim 1, wherein determining the sequence of the nucleic acid target sequence is performed by detecting length of the extension product.

4. The method of claim 1, wherein the one or more nucleotides are optionally labeled by at least one detectable chemical moiety.

5. The method of claim 1, wherein the one or more nucleotides are labeled by at least one base-specific fluorescent moiety and wherein determining the sequence of the nucleic acid target sequence is performed by detecting the at least one base-specific fluorescent moiety.

6. The method of claim 1, wherein the nucleic acid target sequence is amplified prior to performing steps (a) through (e).

7. The method of claim 1, further comprising, prior to the first instance of step (a):

obtaining an RNA,

contacting the RNA with a reverse transcriptase in the presence of a composition sufficient to permit synthesis of a complementary DNA strand, and

separating the complementary DNA strand from the RNA to form a single-stranded nucleic acid.

8. A method of performing nucleic acid amplification, comprising:

(a) denaturing a double-stranded nucleic acid to produce a single-stranded template strand;

(b) annealing at least one primer to the template strand;

(c) extending the at least one primer by a template-strand-driven polymerase to produce extension products, thereby forming double-stranded nucleic acid molecules; and

(d) repeating steps (a)-(c) to amplify the double-stranded nucleic acid,

wherein at least one extension product formed in step (c) is used as the template strand in a subsequent cycle of steps (a)-(c),

wherein at least step comprises applying tension that stretches the nucleic acids to the template strand and/or the at least one primer, and

wherein during the extension step the tension is adjusted according to the position of the polymerase along the template strand.

9. The method of claim 8, wherein the position of the polymerase along the template strand is monitored.

10. The method of claim 9, wherein the position of the polymerase along the template strand is computed.

11. The method of claim 9, wherein the template strand includes a difficult sequence on the nucleic acid and the steps of monitoring the position of the polymerase along the template strand corresponds to determining a location of the difficult sequence.

12. The method of claim 8, wherein the tension is applied to the template strand during step (c) and further wherein a position of the polymerase varies along the template strand during step (c).

13. A method of performing nucleic acid amplification by a polymerase chain reaction, comprising:

(a) denaturing a double-stranded nucleic acid to produce single-stranded template strands;

(b) annealing primers to the template strands; and

(c) extending the primers by a template-strand-driven polymerase to produce extension products, thereby forming double-stranded nucleic acid molecules; and

(d) repeating steps (a)-(c) to amplify the nucleic acid,

wherein at least one extension product formed in step (c) is used as a template strand in at least one subsequent cycle of steps (a)-(c), and

wherein at least step (c) comprises applying tension that stretches the nucleic acid to the nucleic acid.

14. The method of claim 13, further including applying tension to the template strands during the step of annealing the primer to the template strand.

15. The method of claim 13, further including applying tension to the template strands during the step of extending the primers by a template-strand-driven polymerase.

16. A method for lowering the error rates of extending a plurality of primers on one or more template strands by one or more nucleic acid polymerases, comprising:

extending the plurality of primers annealed to the one or more template strands by one or more nucleic acid polymerases to produce an extension product comprising double-stranded nucleic acid molecules,

wherein tension that stretches the nucleic acids is applied to said one or more template strands or to said plurality of primers to lower the error rates of extending said plurality of primers on said one or more template strands.

17. A method for amplifying cDNA, comprising:

(a) isolating RNA from a sample;

(b) performing reverse transcription thereby obtaining a cDNA template;

(c) amplifying the cDNA template by a method, including;

(i) optionally denaturing a double stranded nucleic acid template to produce a single stranded template strand;

(ii) annealing at least one primer to the nucleic acid template strand;

(iii) extending the one or more primer(s) by a nucleic acid polymerase to produce extension products, thereby forming double stranded nucleic acid molecules; and

(iv) repeating steps (i)-(iii) to amplify the cDNA, wherein tension that stretches the nucleic acid is applied during at least step (iii).

18. A method for controlling hybridization of a primer and a nucleic acid template, comprising:

annealing one or more primers to one or more nucleic acid templates, wherein the annealing step further comprises applying tension that stretches said nucleic acid templates or said primers,

thereby controlling hybridization of the primers and the nucleic acid templates;

anchoring one end of each of said nucleic acid templates to a surface; and

flowing a fluid past the surface with a fluid flow rate sufficient to apply said tension by hydrodynamic force.

19. A method for controlling hybridization of a primer and a nucleic acid template, comprising:

annealing one or more primers to one or more nucleic acid templates, wherein the annealing step further comprises applying tension that stretches said nucleic acid templates or said primers,

thereby controlling hybridization of the primers and the nucleic acid templates;

establishing a stagnation point in a fluid flow;

suspending each of said nucleic acid templates and said primers in the stagnation point, and

wherein the tension is applied by hydrodynamic stress generated by the fluid flow.