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

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.

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.

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).

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.

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.