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

• US 7,494,791 B2
• Filed: 05/13/2005
• Issued: 02/24/2009
• 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:
  US11/128,301
• Publication Number:
  US 20060019274A1
• Time in Patent Office: 1,383 Days
• Field of Search: None
• US Class Current: 435/91.2
• 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 ore 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 nucleic acid, comprising:

(a) contacting one or more template strands of single-stranded nucleic acid with one or more oligonucleotide primers complementary to a portion of the one or more template strands;

(b) annealing at least one primer of the one or more primers to the portion of the one or more template strands to which the primer is complementary;

(c) contacting the one or more template strands with a nucleic acid polymerase and at least four different nucleoside triphosphates;

(d) extending the at least one annealed primer by the nucleic acid polymerase thereby forming one or more extension products bound to the one or more template strands;

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

(f) repeating steps (a), (b), (c), (d) and (e) to amplify the nucleic acid, wherein at least one of the one or more extension products in step (e) is used as template strands in a subsequent cycle of steps (a)-(e),

wherein, for the last cycle, step (e) is optional, and

wherein at least one of steps (b) or (d) comprises applying tension that tends to stretch the nucleic acid to the one or more template strands.

2. The method of claim 1, wherein steps (b) and (d) each comprise applying tension to the one or more template strands.

3. The method of claim 1, wherein steps (d) and (e) each comprise applying tension that tends to stretch the nucleic acid to the one or more template strands.

4. The method of claim 3, wherein applying tension in step (e) comprises applying increased amounts of the same type of tension as applied in step (d).

5. The method of claim 1, wherein steps (b) and (e) each comprise applying tension that tends to stretch the nucleic acid to the one or more template strands.

6. The method of claim 1, wherein steps (b), (d) and (e) each comprise applying tension that tends to stretch the nucleic acid to the one or more template strands.

7. The method of claim 1, wherein applying tension to the one or more template strands comprises applying mechanical tension, hydrodynamic stress, or electrical field energy to the one or more template strands.

8. The method of claim 1, wherein applying tension to the one or more template strands comprises applying a controlled amount of mechanical tension to the one or more template strands.

9. The method of claim 8, wherein applying a controlled amount of mechanical tension to the one or more template strands comprises moving a surface to which the one or more template strands are attached.

10. The method of claim 1, wherein applying tension to the one or more template strands comprises controlling hydrodynamic tension caused by fluid flow on the one or more template strands.

11. The method of claim 10, wherein the one or more template strands are anchored to a surface exposed to a fluid flow, the one or more template strands are bound to a DNA polymerase anchored to a surface exposed to a fluid flow, or the one or more template strands are retained and extended by counter-propagating fluid flows.

12. The method of claim 1, wherein applying tension to the one or more template strands comprises applying force to one or more particles connected to one or both ends of at least one strand of the one or more template strands.

13. The method of claim 1, wherein step (b) comprises applying tension to the one or more template strands within the range of about 0 to about 65 pN.

14. The method of claim 1, wherein step (b) comprises applying tension to the one or more template strands within the range of about 10 to about 65 pN.

15. The method of claim 1, wherein step (d) comprises applying tension to the one or more template strands from greater than 0 pN to about 45 pN.

16. The method of claim 1, wherein step (d) comprises applying tension to the one or more template strands and increasing the tension applied at a time corresponding to a period during which a difficult sequence on the nucleic acid is replicated.

17. The method of claim 16, wherein the nucleic acid comprises one or more segments of several residues which have a GC content greater than 50 percent and the nucleic acid is amplified at greater than 90 percent efficiency.

18. The method of claim 16, wherein the nucleic acid comprises one or more segments of several residues which have a GC content greater than 60 percent.

19. The method of claim 16, wherein the nucleic acid comprises one or more segments comprising a repeating unit of 8 or more base pairs and the nucleic acid is amplified at greater than 90 percent efficiency.

20. The method of claim 1, wherein steps (a), (b), (c), (d) and (e) are conducted at below about 65°

C.

21. The method of claim 1, wherein the steps (a), (b), (c), (d) and (e) are repeated at least about 50 times.

22. The method of claim 21, wherein a template strand of greater than about 1000 bases is amplified.

23. The method of claim 22, wherein the method is completed in about 1 hour or less.

24. The method of claim 1, wherein the nucleic acid is amplified in a solution with a pH of 4-7 or 8-10.

25. The method of claim 1, carried out in a reaction chamber having a volume of less than about a microliter.

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

obtaining a sample of RNA,

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

separating the DNA from the RNA to form the single stranded nucleic acid.

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

obtaining a sample of DNA,

denaturing said DNA,

binding the denatured DNA to a substrate in a reaction chamber, and

flushing said reaction chamber to remove contaminants from said sample.

28. The method of claim 1 wherein, in the first instance of step (a), the one or more template strands are obtained from a single molecule of DNA.

29. The method of claim 1 wherein, in at least the first instance of step (a), the one or more template strands are contained in an unpurified sample of material.

30. A method of detecting a pathogen comprising the method of claim 1 and further comprising probing the amplified nucleic acid for the presence of a nucleotide sequence specifically associated with the pathogen.

31. The method of claim 30, wherein the pathogen is selected from the group consisting of Adeno-associated Virus, Adenovirus, Cytomegalovirus, Epstein-Barr Virus, Enterovirus, Hepatitis A Virus, Hepatitis B Virus, Hepatitis C Virus, Human Herpes Virus Type 6, Human Immunodeficiency Virus Type 1, Human Immunodeficiency Virus Type 2, Human T-Cell Lymphotropic Virus Type I, Human T-Cell Lymphotropic Virus Type II, Mycobacterium Tuberculosis, Mycoplasma, Parvovirus B-19, and Porcine Endogenous Retrovirus.

32. The method of claim 30 comprising obtaining a sample containing less than about 1000 organisms or polynucleotides per milliliter from which the template strands for the first cycle of amplification are prepared.

33. A method for amplifying a nucleic acid, comprising:

(a) contacting one or more template strands of single-stranded nucleic acid with one or more oligonucleotide primers complementary to a portion of the one or more template strands, wherein at least one primer of the one or more primers comprises one or more complexing groups capable of binding one or more molecules bound to a substrate;

(b) annealing the at least one primer with the one or more complexing groups to the portion of the one or more template strands to which the primer is complementary, thereby binding the one or more template strands to the substrate;

(c) contacting the one or more template strands with a nucleic acid polymerase and at least four different nucleoside triphosphates;

(d) extending the one or more primers by the nucleic acid polymerase thereby forming one or more extension products bound to the one or more template strands;

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

(f) repeating steps (a), (b), (c), (d) and (e) to amplify the nucleic acid, wherein, in the last cycle, step (e) is optional,

wherein at least one of the one or more extension products in step (e) is used as template strands in a subsequent cycle of steps (a)-(e), and

wherein at least one of steps (b), (d), and (e) comprises applying tension that tends to stretch the nucleic acid to the one or more template strands.

34. The method of claim 33, wherein the one or more complexing groups are activatable.

35. The method of claim 33, wherein the one or more complexing groups and the one or more molecules bound to the substrate are biotin and streptavidin, respectively.

36. The method of claim 33, wherein the one or more complexing groups comprise photoactivatable caged biotin.

37. A method for amplifying a nucleic acid, comprising:

(a) denaturing a double-stranded nucleic acid molecule by applying tension that tends to stretch the nucleic acid to one or both strands of the double-stranded nucleic acid molecule sufficient to cause separation of the double-stranded nucleic acid molecule into two single-stranded nucleic acid molecules, wherein the one or both single-stranded nucleic acid molecules to which the tension is applied becomes a template strand;

(b) contacting the one or both template strands with one or more oligonucleotide primers complementary to a portion of the template strands, a nucleic acid polymerase and at least four different nucleoside triphosphates;

(c) reducing the tension applied to the one or both template strands in step a) to permit the one or more primers to anneal to the corresponding portions of the template strands to which the one or more primers are complementary;

(d) controlling the tension applied to the one or both template strands so as to permit extension of the one or more primers by the nucleic acid polymerase, thereby forming one or more double-stranded nucleic acid molecules in which one or more extension products are bound to the one or more template strands; and

(e) repeating steps (a), (b), (c), and (d) to amplify the nucleic acid,

wherein at least one of the one or more double-stranded nucleic acid molecules formed in step (d) in a cycle of steps (a)-(d) is used as the double-stranded nucleic acid molecule in step (a) in a subsequent cycle of steps (a)-(d), so that at least one extension product formed in step (d) is used as a template strand in a subsequent cycle of steps (a)-(d).

38. The method of claim 37, wherein at least one of applying, reducing, and controlling the tension comprises binding the nucleic acid molecules to which the tension is applied to a substrate or one or more particles or both and controlling movement of said substrate or the one or more particles or both.

39. The method of claim 37, wherein at least one of applying, reducing, and controlling the tension comprises binding the nucleic acid molecules to which the tension is applied to one or more particles and controlling movement of said one or more particles by optical or magnetic manipulation.

40. The method of claim 37, wherein at least one of applying, reducing, and controlling the tension comprises modulating fluid flow over the nucleic acid molecules to which the tension is applied.

41. The method of claim 37, wherein at least one of said nucleic acid molecules is bound by a polymerase that is bound to the substrate, and wherein fluid is flowed over the substrate.

42. The method of claim 37, wherein at least one of said nucleic acid molecules is bound to a substrate.

43. The method of claim 37, wherein at least one of said nucleic acid molecules is stretched in a fluid flow velocity gradient.

44. The method of claim 37, wherein at least one of said nucleic acid molecules is stretched by a counter propagating fluid flow.

45. The method of claim 37, wherein step (d) comprises increasing the tension at a time corresponding to a period during which a difficult sequence on the one or more template strands is replicated.

46. A method comprising:

(a) providing a sample of double-stranded DNA (dsDNA) containing a target sequence, oligonucleotide primers complimentary to the 3′

ends of the target sequence and its complement;

at least four different nucleoside triphosphates and a DNA polymerase;

(b) denaturing the dsDNA into single-stranded DNA (ssDNA) molecules by applying tension that tends to stretch the nucleic acid to one or both strands of the dsDNA sufficient to cause the dsDNA to melt, wherein the one or both resulting ssDNA molecules becomes a template strand;

(c) reducing the tension applied to the one or both template strands sufficient to permit hybridization of at least one of the primers to its complementary sequence in the template strands;

(d) modulating the tension applied to the one or both template strands to a level allowing the DNA polymerase to extend the at least one primer to form an extension product, thereby forming one or more molecules of dsDNA; and

(e) repeating steps (b) through (d) to amplify the dsDNA provided in step (a), wherein at least one of the one or more molecules of dsDNA formed in step (d) in a cycle of steps (b) through (d) is denatured in step (b) in a subsequent cycle of steps (b) through (d), so that at least one extension product formed in step (d) is used as a template strand in a subsequent cycle of steps (a)-(d).

47. The method of claim 46, wherein the entire process is carried out at ambient temperature.

48. The method of claim 46, wherein the process is carried out at a temperature above about 20°

C. and below about 60°

C.

49. The method of claim 46, wherein the process is carried out at room temperature and the tension in step b) is above about 65 pN, in step c) the tension is reduced to between about 0 pN to about 60 pN, and in step d) the tension is modulated between about 0 pN to about 45 pN.

50. The method of claim 46, wherein the tension is caused and controlled by electrically, magnetically, or optically manipulating one or more particles attached to the DNA, by moving a surface to which the DNA is attached, by controlling a fluid flow to which the DNA is exposed, or a combination of these methods.

51. 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 double-stranded nucleic acid,

wherein at least one of the double-stranded nucleic acid molecules produced in step (c) in a cycle of steps (a)-(c) is denatured in step (a) in a subsequent cycle of steps (a)-(c), so that at least one extension product formed in step (c) is used as a template strand in a subsequent cycle of steps (a)-(d), and

wherein at least one of steps (b) and (c) comprises applying tension that tends to stretch the nucleic acid to the nucleic acid.

52. The method of claim 51, wherein applying tension comprises applying mechanical tension, hydrodynamic stress, or electromagnetic force, or any combination thereof.

53. The method of claim 51, wherein steps (a) though (d) are performed at temperature below 80°

C.

54. The method of claim 51, wherein steps (a) though (d) are performed at temperature below 30°

C.

55. The method of claim 51, wherein the method is performed at an ambient temperature.

56. The method of claim 51, wherein tension that tends to stretch the nucleic acid applied in step (a) is equal to or greater than 65 pN.

57. The method of claim 51, wherein tension applied in step (b) is equal to or less than 50 pN.

58. The method of claim 51, wherein tension applied in step (b) is from 30 pN to 50 pN, inclusive.

59. The method of claim 51, wherein tension applied in step (c) is equal to or less than 45 pN.

60. The method of claim 51, wherein tension applied in step (b) is from 35 pN to 45 pN, inclusive.

61. The method of claim 51, wherein the single-stranded template strands produced in the first instance of step (a) are produced by denaturing a single molecule of double-stranded nucleic acid.

62. The method of claim 51, wherein step (a) comprises applying tension to the nucleic acid.

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

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

(b) annealing primers to the template strands;

(c) extending the primers by a template-strand-driven polymerase to produce extension products while applying mechanical tension to the primer or the template strands, thereby forming double-stranded nucleic acid molecules; and

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

wherein at least one of the double-stranded nucleic acid molecules produced in step (c) in a cycle, of steps (a)-(c) is denatured in step (a) in a subsequent cycle of steps (a)-(c), so that at least one extension product formed in step (c) is used as a template strand in a subsequent cycle of steps (a)-(d).

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

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

(b) annealing primers to the template strands;

(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 double-stranded nucleic acid,

wherein at least one of the double-stranded nucleic acid molecules produced in step (c) in a cycle of steps (a)-(c) is denatured in step (a) in a subsequent cycle of steps (a)-(c), so that at least one extension product formed in step (c) is used as a template strand in a subsequent cycle of steps (a)-(d), and

wherein at least one of steps (a)-(c) comprises applying hydrodynamic tension to the nucleic acid.

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

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

(b) annealing primers to the template strands;

(c) extending the primers by a template-strand-driven polymerase to produce extension products while applying electromagnetic tension to the primer or the template strands, thereby forming double-stranded nucleic acid molecules; and

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

wherein at least one of the double-stranded nucleic acid molecules produced in step (c) in a cycle of steps (a)-(c) is denatured in step (a) in a subsequent cycle of steps (a)-(c), so that at least one extension product formed in step (c) is used as a template strand in a subsequent cycle of steps (a)-(d).

66. The method of claim 65, wherein step (a) or step (b) comprises applying electromagnetic tension to the nucleic acid.

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

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

(b) annealing primers to the template strands;

(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 double-stranded nucleic acid,

wherein step (a) is isothermal and comprises applying mechanical or hydrodynamic tension to the nucleic acid;

and wherein at least one of the double-stranded nucleic acid molecules produced in step (c) in a cycle of steps (a)-(c) is denatured in step (a) in a subsequent cycle of steps (a)-(c), so that at least one extension product formed in step (c) is used as a template strand in a subsequent cycle of steps (a)-(d).

68. The method of claim 67, wherein steps (b) through (c) are isothermal.

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

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

(b) annealing primers to the template strands;

(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 double-stranded nucleic acid,

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

wherein step (c) comprises applying adjustable tension that tends to stretch the nucleic acid to the nucleic acid, said adjustable tension modulating proofreading exonuclease activity of the nucleic acid polymerase.

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

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

(b) annealing primers to the template strands;

(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 double-stranded nucleic acid,

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

wherein primers used in step (b) are activatable primers comprising complexing groups for immobilizing extension products obtained in step (c) wherein tension that tends to stretch the nucleic acid is applied in at least one cycle.

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

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

(b) annealing primers to the template strands;

(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 double-stranded nucleic acid,

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

wherein at least one of steps (a)-(c) comprises applying tension that tends to stretch the nucleic acid to the nucleic acid; and

wherein primers used in step (b) are activatable primers comprising complexing groups for immobilizing extension products obtained in step (c).

72. The method of claim 71, wherein the tension applied during at least one of steps (a)-(c) is mechanical tension.

73. A method of performing nucleic acid amplification, 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 double-stranded nucleic acid,

wherein at least one of the double-stranded nucleic acid molecules produced in step (c) in a cycle of steps (a)-(c) is denatured in step (a) in a subsequent cycle of steps (a)-(c), so that at least one extension product formed in step (c) is used as a template strand in a subsequent cycle of steps (a)-(d),

wherein at least one of steps (b) and (c) comprises applying tension that tends to stretch the nucleic acid to the nucleic acid, and

wherein the number of copies of the template strand increases exponentially, as a power of n, after steps (a) through (c) are repeated n times, wherein n is an integer greater than 1.

74. A method for controlling the stringency of annealing of a primer to a nucleic acid template strand, comprising:

in a plurality of cycles of nucleic acid amplification,

annealing a primer to a nucleic acid template strand, wherein tension that tends to stretch the nucleic acid is adjustably applied to said nucleic acid template strand or to said primer to control the stringency of annealing of said primer to said nucleic acid template strand; and

wherein an extension product formed by extending the primer by a nucleic acid polymerase is used as a nucleic acid template strand in a subsequent cycle of nucleic acid amplification.

75. A method for lowering the error rates of extending a primer by a nucleic acid polymerase, comprising:

in a plurality of cycles of nucleic acid amplification,

extending a primer annealed to a nucleic acid template strand by a nucleic acid polymerase to produce an extension product comprising double-stranded nucleic acid molecules, wherein tension that tends to stretch the nucleic acid is adjustably applied to said nucleic acid template strand or to said primer to lower the error rates of extending said primer by nucleic acid polymerase, wherein the extension product is used as a nucleic acid template strand in a subsequent cycle of nucleic acid amplification.

76. A method for controlling the stringency of annealing of a primer to a GC-rich nucleic acid template strand, comprising:

in a plurality of cycles of nucleic acid amplification,

annealing a primer to a GC-rich nucleic acid template strand, wherein tension that tends to stretch the nucleic acid is adjustably applied to said GC-rich nucleic acid template strand or to said primer to control the stringency of annealing of said primer to said GC-rich nucleic acid template strand; and

wherein an extension product formed by a nucleic acid polymerase is used as a nucleic acid template strand in a subsequent cycle of nucleic acid amplification.

77. A method for lowering the error rates of extending a primer on a GC-rich template strand by a nucleic acid polymerase, comprising:

in a plurality of cycles of nucleic acid amplification,

extending a primer annealed to a GC-rich nucleic acid template strand by a nucleic acid polymerase to produce an extension product comprising double-stranded nucleic acid molecules, wherein tension that tends to stretch the nucleic acid is adjustably applied to said GC-rich nucleic acid template strand or to said primer to lower the error rates of extending said primer on said GC-rich nucleic acid template strand,

wherein the extension product is used as a nucleic acid template strand in a subsequent cycle of nucleic acid amplification.

78. A method of improving fidelity of nucleic acid amplification, 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 double-stranded nucleic acid,

wherein at least one of the double-stranded nucleic acid molecules produced in step (c) in a cycle of steps (a)-(c) is denatured in step (a) in a subsequent cycle of steps (a)-(c), so that at least one extension product formed in step (c) is used as a template strand in a subsequent cycle of steps (a)-(d), and

wherein at least one of steps (a) through (c) comprises adjustably applying tension that tends to stretch the nucleic acid to the nucleic acid to improve fidelity of nucleic acid amplification.

79. A method for amplifying a nucleic acid, comprising:

(a) contracting one or more template strands of single-stranded nucleic acid with one or more oglinucleotide primers complementary to a portion of the one or more template strands;

(b) annealing at least one primer of the one or more primers to the portion of the one or more template strands to which the primer is complementary;

(c) contacting the one or more template strands with a nucleic acid polymerase and at least four different nucleoside triphosphates;

(d) extending the at least one annealed primer by the nucleic acid polymerase while applying mechanical tension, hydrodynamic tension, or electromagnetic tension to the one or more primer or the one or more template strands, thereby forming one or more extension products bound to the one or more template strands, wherein mechanical tension, hydrodynamic tension, or electromagnetic tension tends to stretch the nucleic acid;

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

(f) repeating steps (a)-(e) to amplify the nucleic acid,

wherein at least one of the one or more extension products in step (e) is used as template strands in a subsequent cycle of steps (a)-(e), wherein, for the last cycle, step (e) is optional.

80. A method for amplifying nucleic acid, comprising:

(a) contacting one or more template strands of single-stranded nucleic acid with one or more oligonucleotide primers complementary to a portion of the one or more template strands;

(b) annealing at least one primer of the one or more primers to the portion of the one or more template strands to which the primer is complementary;

(c) contacting the one or more template strands with a nucleic acid polymerase and at least four different nucleoside triphosphates;

(d) extending the at least one annealed primer by the nucleic acid polymerase thereby forming one or more extension products bound to the one or more template strands;

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

(f) repeating steps (a)-(e) to amplify the nucleic acid,

wherein at least one of the one or more extension products in step (e) is used as template strands in a subsequent cycle of steps (a)-(e),

wherein, for the last cycle, step (e) is optional, and

wherein at least one of steps (a)-(e) comprises applying hydrodynamic tension to the one or more template strands.

81. A method for amplifying a nucleic acid, comprising:

(a) contacting one or more template strands of single-stranded nucleic acid with one or more oligonucleotide primers complementary to a portion of the one or more template strands;

(b) annealing at least one primer of the one or more primers to the portion of the one or more template strands to which the primer is complementary;

(c) contacting the one or more template strands with a nucleic acid polymerase and at least four different nucleoside triphosphates;

(d) extending the at least one annealed primer by the nucleic acid polymerase, thereby forming one or more extension products bound to the one or more template strands;

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

(f) repeating steps (a)-(e) to amplify the nucleic acid,

wherein at least one of the one or more extension products in step (e) is used as template strands in a subsequent cycle of steps (a)-(e),

wherein, for the last cycle, step (e) is optional, and

wherein tension that tends to stretch the nucleic acid is applied and varied during each cycle of steps (a)-(e).

82. A method for amplifying a nucleic acid, comprising:

(a) contacting one or more template strands of single-stranded nucleic acid with one or more oligonucleotide primers complementary to a portion of the one or more template strands;

(b) annealing at least one primer of the one or more primers to the portion of the one or more template strands to which the primer is complementary;

(c) contacting the one or more template strands with a nucleic acid polymerase and at least four different nucleoside triphosphates;

(d) extending the at least one annealed primer by the nucleic acid polymerase, thereby forming one or more extension products bound to the one or more template strands;

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

(f) repeating steps (a)-(e) to amplify the nucleic acid,

wherein at least one of the one or more extension products in step (e) is used as template strands in a subsequent cycle of steps (a)-(e),

wherein, for the last cycle, step (e) is optional,

wherein tension that tends to stretch the nucleic acid is applied and varied during each cycle of steps (a)-(e), and

wherein each step (a) through (e) is isothermal.