Nano-PCR: methods and devices for nucleic acid amplification and detection
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Publication Number
US-7494791-B2
Publication Date
2009-02-24
Expiration Date
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.
Core Innovation
The invention provides nucleic acid amplification methods that use tension to drive nucleic acid denaturation and to promote primer annealing and polymerase extension instead of relying on thermal cycling. Template strands of single-stranded nucleic acid are contacted with oligonucleotide primers, primers are annealed, nucleic acid polymerase contacts the template strands with at least four different nucleoside triphosphates, and primers are extended to form one or more extension products bound to the one or more template strands. One or more extension products is separated from the template strands and is used as template strands in a subsequent cycle to amplify the nucleic acid, with step (e) optional in the last cycle.
In alternative embodiments, double-stranded nucleic acid molecules are denatured by applying tension that tends to stretch the nucleic acid sufficient to cause separation into two single-stranded nucleic acid molecules, where the single-stranded nucleic acid molecules become template strands. Tension is then reduced and controlled to permit primer hybridization and extension by a template-strand-driven polymerase, forming double-stranded nucleic acid molecules with extension products bound to template strands. These double-stranded nucleic acid molecules and/or extension products are then reused as templates in subsequent cycles, so that at least one extension product formed in a prior cycle becomes a template strand in a subsequent cycle.
The invention further incorporates adjustably applied tension that tends to stretch the nucleic acid to control primer annealing stringency and to lower error rates and improve fidelity, including for GC-rich nucleic acid templates. Complexing groups associated with primers are used to immobilize extension products obtained from step (c), including activatable complexing groups such as biotin and streptavidin and photoactivatable caged biotin. In some embodiments, denaturation is isothermal by applying mechanical or hydrodynamic tension, and in other embodiments each step of the amplification cycle is isothermal.
Claims Coverage
The provided material includes eight independent claims. Across these claims, the inventive coverage centers on cyclic nucleic acid amplification where tension that tends to stretch nucleic acid replaces thermal denaturation and is used to control annealing stringency, extension fidelity, and/or isothermal operation, with extension products reused as templates in subsequent cycles.
Cyclic amplification with tension-assisted denaturation, annealing, and extension using reused extension products
Repeating cycles of contacting template strands with oligonucleotide primers, annealing primers, contacting with a nucleic acid polymerase and at least four different nucleoside triphosphates, extending the annealed primer to form extension products bound to template strands, and separating extension products, wherein at least one extension product in step (e) is used as template strands in a subsequent cycle, and wherein for the last cycle step (e) is optional and at least one of steps (b) or (d) comprises applying tension that tends to stretch the nucleic acid to the template strands.
Primer annealing via activatable substrate-binding complexing groups under tension with cyclic template reuse
Repeating steps of annealing primers with one or more complexing groups capable of binding molecules bound to a substrate to template strands, contacting template strands with a nucleic acid polymerase and at least four different nucleoside triphosphates, extending the primers to form extension products bound to template strands, separating extension products, and reusing at least one extension product as template strands in a subsequent cycle, where in the last cycle separation step (e) is optional and at least one of steps (b), (d), or (e) comprises applying tension that tends to stretch the nucleic acid to the template strands.
Tension-driven denaturation and tension-controlled primer annealing and extension with cycle-to-cycle dsDNA template reuse
Denaturing a double-stranded nucleic acid by applying tension sufficient to separate into two single-stranded nucleic acid molecules that become template strands, contacting template strands with oligonucleotide primers, a nucleic acid polymerase and at least four different nucleoside triphosphates, reducing the tension to permit primer annealing, controlling the tension to permit extension by the nucleic acid polymerase thereby forming one or more double-stranded nucleic acid molecules with extension products bound to template strands, and repeating to amplify wherein at least one double-stranded nucleic acid molecule formed in step (d) is used as the double-stranded nucleic acid molecule in step (a) in a subsequent cycle.
Tension-controlled dsDNA melting and isothermal-capable cyclic amplification with template reuse
Providing a sample of double-stranded DNA containing a target sequence, oligonucleotide primers complementary to the 3′ ends of the target sequence and its complement, at least four different nucleoside triphosphates and a DNA polymerase; denaturing the dsDNA into ssDNA molecules by applying tension sufficient to cause the dsDNA to melt where resulting ssDNA becomes a template strand; reducing tension to permit hybridization of at least one primer; modulating tension to a level allowing the DNA polymerase to extend the primer to form an extension product thereby forming one or more molecules of dsDNA; and repeating steps (b) through (d) to amplify wherein at least one dsDNA molecule formed in a cycle is denatured in step (b) in a subsequent cycle so at least one extension product is used as a template strand in a subsequent cycle.
PCR-like cyclic amplification with tension applied during annealing and/or extension and template reuse
Denaturing a double-stranded nucleic acid to produce single-stranded template strands, annealing primers to the template strands, extending the primers by a template-strand-driven polymerase to produce extension products thereby forming double-stranded nucleic acid molecules; repeating steps (a) through (c) to amplify the double-stranded nucleic acid wherein at least one double-stranded nucleic acid molecule produced in step (c) in a cycle is denatured in step (a) in a subsequent cycle so that at least one extension product formed in step (c) is used as a template strand in a subsequent cycle, and wherein at least one of steps (b) and (c) comprises applying tension that tends to stretch the nucleic acid to the nucleic acid.
Mechanical-tension-assisted polymerase extension in cyclic amplification with ds template reuse
Denaturing a double-stranded nucleic acid to produce single-stranded template strands, annealing primers to the template strands, extending the primers by a template-strand-driven polymerase while applying mechanical tension to the primer or the template strands thereby forming double-stranded nucleic acid molecules; repeating steps (a) through (c) to amplify wherein at least one of the double-stranded nucleic acid molecules produced in step (c) in a cycle is denatured in step (a) in a subsequent cycle so that at least one extension product formed in step (c) is used as a template strand in a subsequent cycle.
Hydrodynamic-tension-assisted amplification by cyclic denaturation, annealing, and extension with template reuse
Repeating steps including denaturing a double-stranded nucleic acid to produce single-stranded template strands, annealing primers to the template strands, extending the primers by a template-strand-driven polymerase to produce extension products thereby forming double-stranded nucleic acid molecules, and repeating steps (a) through (c) to amplify wherein at least one double-stranded nucleic acid molecule produced in step (c) in a cycle is denatured in step (a) in a subsequent cycle so that at least one extension product formed in step (c) is used as a template strand in a subsequent cycle, and wherein at least one of steps (a) through (c) comprises applying hydrodynamic tension to the nucleic acid.
Electromagnetic-tension-assisted amplification with cyclic denaturation and extension product template reuse
Denaturing a double-stranded nucleic acid to produce single-stranded template strands, annealing primers to the template strands, extending the primers by a template-strand-driven polymerase while applying electromagnetic tension to the primer or the template strands thereby forming double-stranded nucleic acid molecules; repeating steps (a) through (c) to amplify wherein at least one double-stranded nucleic acid molecule produced in step (c) in a cycle is denatured in step (a) in a subsequent cycle so that at least one extension product formed in step (c) is used as a template strand in a subsequent cycle.
Isothermal denaturation by mechanical or hydrodynamic tension with cyclic template reuse
In a method of amplifying nucleic acid by denaturing a double-stranded nucleic acid to produce single-stranded template strands, annealing primers to the template strands, extending the primers by a template-strand-driven polymerase to produce extension products thereby forming double-stranded nucleic acid molecules, and repeating steps (a) through (c) to amplify, 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 is denatured in step (a) in a subsequent cycle so that at least one extension product formed in step (c) is used as a template strand in a subsequent cycle.
Activatable complexing primer immobilization of extension products with tension-assisted cyclic amplification
Denaturing a double-stranded nucleic acid to produce single-stranded template strands, annealing primers to the template strands, extending the primers by a template-strand-driven polymerase to produce extension products thereby forming double-stranded nucleic acid molecules, and repeating steps (a) through (c) to amplify, wherein at least one extension product in step (c) is used as a template strand in a subsequent cycle, 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.
Activatable complexing primers with tension during cycle steps and extension-product template reuse
Denaturing a double-stranded nucleic acid to produce single-stranded template strands, annealing primers to the template strands, extending the primers by a template-strand-driven polymerase to produce extension products thereby forming double-stranded nucleic acid molecules, and repeating steps (a) through (c) to amplify, wherein at least one extension product in step (c) is used as a template strand in a subsequent cycle of steps (a) through (c), wherein at least one of steps (a) through (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).
Exponential copy increase with tension-assisted cyclic amplification
Denaturing a double-stranded nucleic acid to produce single-stranded template strands, annealing primers to the template strands, extending the primers by a template-strand-driven polymerase to produce extension products thereby forming double-stranded nucleic acid molecules, and repeating steps (a) through (c) to amplify wherein at least one double-stranded nucleic acid molecule produced in step (c) in a cycle is denatured in step (a) in a subsequent cycle so that at least one extension product formed in step (c) is used as a template strand in a subsequent cycle, 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 repeating steps (a) through (c) for n times, where n is an integer greater than 1.
Tension-controlled annealing stringency with extension-product template reuse
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 the nucleic acid template strand or to the primer to control the stringency of annealing of the primer to the 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.
Lowering extension error rates by tension-assisted primer extension with extension-product template reuse
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 the nucleic acid template strand or to the primer to lower the error rates of extending the 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.
GC-rich primer annealing stringency controlled by adjustably applied tension with template reuse
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 the GC-rich nucleic acid template strand or to the primer to control the stringency of annealing of the primer to the 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.
Lowering extension error rates on GC-rich templates using adjustably applied tension with template reuse
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 the GC-rich nucleic acid template strand or to the primer to lower the error rates of extending the primer on the 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.
Improving amplification fidelity by tension-assisted cyclic amplification with extension-product template reuse
Repeating steps of denaturing a double-stranded nucleic acid to produce single-stranded template strands, annealing primers to the template strands, extending the primers by a template-strand-driven polymerase to produce extension products thereby forming double-stranded nucleic acid molecules, and repeating steps (a) through (c) to amplify, wherein at least one double-stranded nucleic acid molecule produced in step (c) in a cycle is denatured in step (a) in a subsequent cycle so that at least one extension product formed in step (c) is used as a template strand in a subsequent cycle of steps (a) through (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.
Mechanical, hydrodynamic, or electromagnetic tension during extension with extension-product template reuse and optional final separation
Contracting one or more template strands of single-stranded nucleic acid with one or more oligonucleotide primers complementary to a portion of the template strands, annealing at least one primer to the complementary portion of the template strands, contacting the template strands with a nucleic acid polymerase and at least four different nucleoside triphosphates, extending the annealed primer while applying mechanical tension, hydrodynamic tension, or electromagnetic tension to the primer or the template strands thereby forming one or more extension products bound to the template strands, separating the extension products from the template strands, and repeating steps (a) through (e) to amplify wherein at least one extension product in step (e) is used as template strands in a subsequent cycle, and wherein for the last cycle, step (e) is optional.
Hydrodynamic-tension-assisted cyclic amplification with optional final separation and extension-product template reuse
Contacting one or more template strands of single-stranded nucleic acid with one or more oligonucleotide primers complementary to a portion of the template strands, annealing at least one primer to the complementary portion, contacting the template strands with a nucleic acid polymerase and at least four different nucleoside triphosphates, extending the annealed primer thereby forming one or more extension products bound to the template strands, separating the extension products from the template strands, and repeating steps (a) through (e) to amplify wherein at least one extension product in step (e) is used as template strands in a subsequent cycle, wherein for the last cycle, step (e) is optional, and wherein at least one of steps (a) through (e) comprises applying hydrodynamic tension to the template strands.
Cycle-by-cycle varying tension that tends to stretch nucleic acid in isothermal amplification with optional final separation and extension-product template reuse
Contacting single-stranded nucleic acid template strands with complementary oligonucleotide primers, annealing at least one primer to the complementary portion, contacting the template strands with a nucleic acid polymerase and at least four different nucleoside triphosphates, extending the annealed primer thereby forming extension products bound to the template strands, separating extension products from template strands, repeating steps (a) through (e) to amplify wherein at least one extension product in step (e) is used as template strands in a subsequent cycle and 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) through (e), and wherein each step (a) through (e) is isothermal.
Across the independent claims, the core coverage requires cyclic nucleic acid amplification in which denaturation, primer annealing, and/or primer extension are associated with tension that tends to stretch nucleic acid, and where extension products (or formed dsDNA molecules) are reused as template strands in subsequent cycles. Several independent claims further narrow the tension modality (mechanical, hydrodynamic, electromagnetic), specify isothermal operation, or require adjustably applied tension to control annealing stringency, lower error rates, and improve fidelity, including for GC-rich templates and in connection with activatable primers using complexing groups for immobilizing extension products.
Stated Advantages
Lowering the error rates of extending a primer by a nucleic acid polymerase.
Improving fidelity of nucleic acid amplification.
Controlling the stringency of annealing of a primer to a nucleic acid template strand.
Controlling the stringency of annealing of a primer to a GC-rich nucleic acid template strand.
Lowering the error rates of extending a primer on a GC-rich template strand.
Documented Applications
Pathogen detection using probing for pathogen-associated nucleotide sequences, including selection among named viruses and microorganisms such as Mycobacterium tuberculosis, HIV-1, Hepatitis B Virus, Hepatitis C Virus, Adenovirus, Cytomegalovirus, Epstein-Barr Virus, and additional listed viruses and microorganisms.
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