AU2016203092B2 - Nano-pcr: methods and devices for nucleic acid amplification and detection - Google Patents

Abstract

Methods and apparatuses are described that provide a breakthrough technology for PCR amplification of nucleic acid sequences. The technology permits PCR to be performed without reliance upon temperature cycling and can be applied at a wide range of ambient temperatures, thus freeing the methods from conventional benchtop thermal cycling devices. The methods allow to exercise precise control when desired during replication and amplification, and enable substantial improvements over conventional PCR in a number of parameters including: improved accuracy and fidelity of replication of normal and problematic sequences (GC rich or tandem repeat sequences), greater sequence length, improved overall reaction yield, lower costs, less overall PCR process time, greater portability, and robustness to various environmental parameters, such as temperature, pH, ionic strengths, and contaminants.

Description

NANQ-PCR: METHODS AND DEVICES FOR. NUCLEIC ACID AMPLIFICATION AND DETECTION

This is a divisional out of Australian patent application no, 2013205814, which itself is divided out of Australian patent application no, 2009208132. which itself is divided out of 2005324505 dated 13 May 2005.

Field of the Invention

The invention .relates to amplification and detection of nucleic acids. In particular embodiments, the invention provides improved methods, devices, and material s for performing the polymerase chain reaction.

Background of the Invention

The polymerase chain reaction (PCR) has become the conventional technique used to amplify specific DNA or ENA sequences. U.S. Patent No. 4,683,202, issued My 28,1987 to Mullis andU.S. Patent No. 4,683,195, issued July 28,1987 to Mullis at ai describe the basic PCR technique. Since the first, disclosure of the PCS. method, it has had a profound effect on the practice of biotechnology and biomedical science. More than a thousand subsequently-issued 118. patents reference one or both of these disclosures.

Typically, the amplificaaos of a DNA sequence is performed by first selecting and obtaining two oligonucleotide primers complesnenisry ends of a target DNA sequence. The primers, apolymemse enzyme, a mixture of the four common nucleotide triphosphates, various salts and buffers ate mixed with the target DNA which is heated above about 90* C to denature the DNA, separating the target double-stranded DNA info single-stranded DNA templates. Annealing (ie. sequence-specific hybridization or binding) of the primers to the ends of the DMA templates is promoted by slowly cooling the reaction mixture to less than about 60s C. The temperature is then raised above about 70* C for a period of replication, a process also known as primer extension. The polymerase reads each DNA template strand in. the 3’ to 5* direction, synthesizing a complementary strand from the ends of the primers in the 55 to 3* direction, This completes one cycle· of DNA amplification, which creates starting material tor a new cycle. With each complete cycle of denahtrarion, primer annealing, and primer extension, the process generates m exponmiiaSy increasing (2M ntanher of copies of fc oagiaalj target DNA sequence. To begin a sow cycle, the reaction mixtura is again heated aboveifo^G to denature tke dcwMe-ssraaded grodnet into smgle»simuded DMA templates; The stops are then repeated.

Tiiis basic PGR mngliPcation scheme., together with variows extensions and modificationSs, enables many different methods forthe maaipidadon and detection of nucleic acids, including for e>mrnple diagnostic and forensic assays, which requite the creation of a threshold amount of DMA if can a small initial sample. PCI. technology is used, for example, In infectious and genetic disease monitoring, DMA and KM A sequencing, gene expression; studies, drag devMqpm^t, andforensic Sngeipriuimg, This has besdme the standard technology for the detection, idsntiReahofoand quandfioaiion of viral and bacterial pathogens. Several PGR» based diagnqstie tests are available for detecting and/or quantifying pathogens, for example, indeding· HfV-l, which causes AiDifo hegadds B and € viruses, which, can cause liyer cancer; banian papillmnarVirus,^ which can canss cervical cancer: .RSY, which is the leading cause is. infants* (Mamydm ptaekonmiis agonorrhoeue, which can lead to pelvic inflammatory disease and infertility In women; eytomegalofoius, which can cause ufe-tm'eatenlng disease in transplant paneuts and other imnmno-compionfesed causes cough and fatiguoinits active state and can irrevemibly damage infected organs. However, despite addressing needs in nxnnerous areas, currwrt PCR and PGR-hased technologies still suSerfeotn S8vemietfosthifoall.imita.uom

MmHatmis ofCmwndomiPCR andPCR'Bm&i Teckn&I@gies

Fideidy: Accuracy on normal sequences limits conventional PCR. For example, Tag, a thennostable polymerase ctanmoily nsed for DMA afopifocfolon, exhibits an error rate of approximately 1 x ! O'4 errors/base pair during PCR. Tils means that the PCR amplification^of ia 400 base pair DMA sequence will randomly ®tro»dn»e ^prtedmately40,000 errors among all molecules In foe PGR product over 20 cycles.

Accuracy on difficult target, sequences (e,g. GC rich or repeat sequences) is an even more significant limitation of conventional PCR and PCR- based technologies. The error rate for conventional polymerase enzymes such as Taq, depends strongly on fee target nucleotide sequence, For example, when the sequence is G+C rich (as seen for example in the 5' regulatory region of the chicken avidin gene), PCR with Taq is oftentimes not a viable process. Likewise, simple repeating sequences, such as txmncleoiide repeats (AGC)n or other tandem repeats (A)n, can increase Taq's error rate to 1.5 x 10-2 errors per repeat sequence. See, Shinde ei al, Nucleic Acids Research, 3 .1:974, For this reason, several patents have been issued for polymerases that have been genetically engineered to have incrementally higher fidelity (i.e. lower error rates). These include Hi-Fidelity and Phusion Polymerases.

Length Limitations: The length of the target sequence to be amplified also limits current PCR techniques. Although a few reports have claimed amplification of sequences up to 1.0 to 20 ktiobases, this is highly unusual and quite difficult in routine practice. Moreover, PCR amplification of long target templates is only possible on a limited set of well-behaved DMA sequences. The practical upper-hunt for fairly routine and cost-effective amplification ofDNA on well-behaved sequences is about 300 to 400 bases in length and is generally reduced for sequences having high G-C content.

Limited Amplification: Current PCR techniques are also limited in the number of amplification cycles that can be carried out in a reaction mixture.

Repeated heating and cooling cycles result in progressive enzyme degradation, which limits the factor by which starting material can be amplified. Conventional PCR amplification can. rarely be extended beyond 30-35 cycles.

Robustness; Conventional PCR typically requires significant volumes of reagents, bulky equipment (eg,, thermal cyclers), substantial human labor (e.g., tedious optimizations), and minimum amounts of starting material, each of which contributes to making conventional PCR a costly and time-consuming process. Current PCR techniques typically take from several hours for normal sequences to several days to weeks for difficult sequences or long template. Conventional PCR requires a sigaificant amount of time to cycle and equilibrate the temperature of the reaction mix. Moreover, time-consuming optimizations can be required in order to reliably amplify targets that are less fern ideal.

Tightly controlled conditions (e.g., temperature, pH, and buffer ingredients) are required for performance of conventional PCR techniques. Additionally, various contiuninants can interfere with PCR amplification by directly inhibiting or interfering with polymerase enzymes used to copy the target DNA or RNA, This further limits the quality of starting material that can be used for amplification and places additional requirements on the level of purity that must be obtained by DNA or RNA extraction techniques before the amplification steps can be reliably performed. The performance environment of conventional PCR is generally limited to laboratories, and is rarely practicable in remote locations, doctor's offices, at the patient’s bedside, or out in die field.

Sensitivity and Specificity of Diagnostics' The sensitivity of PCR-based diagnostic aad forensic kits and assays depends on the overall yield, accuracy, robustness, and target length achievable in a PCR reaction. The above-mentioned limitations in performance parameters of current PCR set limits on the minimum amount of starting DNA or RNA necessary in order to reliably carry out PCR amplification. This, in turn, limits the sensitivity of any pathogen detection system, diagnostic, or forensic kits or assays that rely upon conventional PCR or PCR-based technologies. The specificity of a PCR-based diagnostic, forensic, or pathogen detection system depends critically on the accuracy with which DNA can be amplified and read as well as tea length of the target DNA or RNA that can be reliably amplified and Identified.

For these and other reasons, current generation PCR-based technologies and detection systems are generally limited with respect to overall speed, efficiency, cost-effecdveness, and scope of use. Incremental improvements to conventional PCR methods and devices have been proposed with respect to some of the isolated performance parameters described above. For example, Tso et al discloses a PCR microreactor for amplifying DNA using nucroquantities of sample fluid in IPS. Patent No. 6,613,560, issued September 2, 2003. Alternatives to high temperature DNA denaturation have also been proposed. For example, Purvis disclosed a method of electrochemical denaturation of double-stranded nucleic acid in U.S. Patent No. 6,291,185, issued September 18, 2001. Stanley discloses another method of electrochemical denaturation of nucleic acids in U.S. Patent No. 6,197,508, issued March 6, 2001. Dattagupta et al. have disclosed a method of using primers to displace the DNA strand from the template in U.S. Patent No. 6,214,587, issued April 10, 2001. Mullis, supra, suggested the use of helicase enzymes for separating DNA strands.

In view of the limitations of conventional PCR, and despite the proposal of various incremental improvements, there remains a need in the art for improved methods, devices, and compositions for the amplification, manipulation, sequencing, and detection of nucleic acids. It is an object of the present invention to go some way towards meeting this need and/or to provide the public with a useful choice.

Summary of the Invention

The methods and apparatuses described herein provide a breakthrough technology to perform PCR. The technology described herein also permits PCR to be performed without reliance upon thermal cycling. The technology may be applied at a wide range of ambient temperatures or using controlled temperature. It is possible to exercise precise control when desired during replication and amplification, thereby enabling substantial improvements in a number of performance parameters. Dubbed "Nano-PCR™," this technology introduces a new paradigm in PCR-based detection and amplification of nucleic acids.

Specifically, in a first embodiment, the invention provides a device for amplifying one or more nucleic acid molecules, comprising: one or more fluid channels for directing fluid flow within the device; a means of retaining a sample including one or more nucleic acid molecules within the one or more fluid channels; and a mechanism for applying a variable and controlled amount of tension, that tends to stretch the one or more nucleic acid molecules, to the nucleic acid molecules retained therein during at least one cycle, said cycle including the following operations: (a) optionally when the sample includes a double-stranded nucleic acid, denaturing the double-stranded nucleic acid into a single-stranded nucleic acid target sequence; (b) annealing the primer to its complementary nucleic acid target sequence; and (c) extending the primer to form one or more extension products; and, wherein said device is configured such that at least one of the one or more extension products formed in (c) is used as template strands in a subsequent cycle.

In a second embodiment, the invention provides a device for sequencing one or more double-stranded nucleic acid molecules, comprising: one or more fluid channels for directing fluid flow within the device; means of retaining a sample including said nucleic acid molecules within the one or more fluid channels; and a mechanism for applying a variable and controlled amount of tension, that tends to stretch the nucleic acid molecule, to the nucleic acid molecules retained therein during at least one cycle of said device, said cycle including the following operations: (a) denaturing said double-stranded nucleic acid molecules into singlestranded nucleic acid molecules; (b) hybridizing primers to complementary single-stranded nucleic acid molecules; and (c) extending the hybridized primers to form extension products; and a detector for determining the sequence of a nucleic acid molecule in said sample.

In a third embodiment, the invention provides a method of sequencing a nucleic acid molecule, comprising: (a) providing a sample, including: a single-stranded or double stranded nucleic acid target sequence, a nucleic acid polymerase; a primer complementary to the nucleic acid target sequence; and one or more nucleotides; (b) optionally when the sample includes a double-stranded nucleic acid target sequence, denaturing the double-stranded nucleic acid target sequence into a singlestranded nucleic acid target sequence; (c) annealing the primer to its complementary nucleic acid target sequence; (d) extending the primer to form an extension product; (e) optionally repeating steps (b) through (d) to form further extension products; and (f) determining the sequence of the nucleic acid target sequence, and wherein at least one cycle of the steps (b) through (d) comprises applying tension that tends to stretch the nucleic acid molecule, the nucleic acid target sequence or the primer extension product.

Also described is a device for applying tension to a nucleic acid sequence, comprising: one or more fluid channels for directing fluid flow within he device; a means of retaining nucleic acid molecules within the one or more fluid channels; a means for applying a variable and controlled amount of tension to the nucleic acid molecules retained therein during at least one cycle of amplification; and further comprising one or more chambers configured for nucleic acid amplification, replication or template-driven primer extension, for reacting, storing, or introducing reagents, optionally wherein the reagents include nucleic acid primers, nucleotide triphosphates, and polymerase.

Brief Description of the Drawings FIGS. 1A and IB illustrate exemplary flow charts of PCR methods that do not rely on temperature cycling. FIGS. 2A-2C illustrate exemplary methods of and arrangements of elements of a reaction chamber for applying tension to a DNA strand anchored between opposed surfaces. FIGS, 3A and 3B ilfustete methods of aadiairangemeiits of elements ofn reaction staaber for appl)dUg tmsion: to a IMA strand using optical or magnetic traps.: MG. 4 is an illustration.,of mi exemplary method of and arrangemeni of Gemems of a reaction chamber for applying tension to a DNA strand bound to a polymerase rixed to a substrate in a Jtoid flow. FIGS. 5 A and SB illustrate exemplary methods of ami arrangements of elements of a resetion chamber for applying tension to at one end in a flind Bow* FIGS. 6A~6C illustrate exemplary methods of and axrangeMeuts of elements of a reaction chamber for applying tension to a DNA strand suspended in a fluid velocity gradient. FIGS;, % A and 7B illustrafe schwiatics of periomning a FGB method that does not rely on tempergit^^yeifegvor:&e«g^:;d^tt^«iL.

Befailed Description of Embodiments of the Invention ΉέβηΜοηζ

The terms "nucleic acid,* "pofynu:cleotidε!i,,and "oligonaciieptide” are used herein to include a polymeric form of nueleotides of any Ieng%ineitidingS:W;n0t limited to, ribonucleotides: or deexyribdnucleotides. Them is no/intended distinction la Furfhsrj these terms refer only to the primary struc ture of the molecule. 'Eras, Ip. certain embodiments these terms can iuelude triple-, double» aud riugle-standedDNA* as well as triple», double» and single-stranded SNA. They also Include modifications, such as by methylatios and/or by capphigi and unmodified forms of the polynucleotide. More particularly, the temA ,fnpcte|c acid,” ^olyauo^^/i^^oHgosupleod^,'* mclude poiydeoxyribonueleotides (comafciug 2»dsoxy»D~riboss)} polyribonucleotides (conGimngD-'ribose), any other type of polynucleotide which is an N- or C~ glyeoside/of a purine of pyrimi dine base, and other polymers containing upsmtcleotidie baeMmnsri, In; example, polyamide fe-g-,.peptide.nucleic aeids (BHAs)} aa#;pofytm^ntmo-'<€^mmemiaay:av^kble:l&amp;teAati-Viials,.M&amp; : Camilla, Greg., as Neugene) poipoers, and other 8yn&amp;etie: sgqp^e-s|i©clic nucleic addpolymers promtlng that foe polymers contain mucleobases m a configuration which allows for base pforidg and base stacking, mail ·μ is feuhd in DNA and RNA.

Mused herein, "primer'* refers to a ulngie-siramleii polynuGieotMe: capable of acting as apaint of initiation ofrenfolatefoireeted DMA synthesis under appropriate pondfosna (Le., in tM presence of fear different pneieuside triphosphates and an agent for polymerization, jndi as, DNA or ;EMA polymerase or reverse transorfofo&amp;G in an appropriate buffer and at a suitable tempexuhm The appropriate length of a primer dreads on foe mtesded use of the primer bat typically is at least ? mjcleorides long and, more typfeally mge from tOfo 30 nucleotides in length. Other primers can be somewhat longer such as 30 to 50 nucleotides long· FOR primers are typically shout A0-30 base pairs long and are chosen to be complementary to one strand upstream 0,e>, 5' to 3 s) of foe forget sequence and the opposite strand dowhstreamifhe,, 3 s to 3s ) of foe sequence. The S* ends of foe primers: fofone foe ends :»f foe amplified FOR. product. Primers may contain approximately foe same GC content· as AT content and no long stretches of any one base. Furihennore, foe primers slmuld not contain structures that are suBstantially complefflentary to one another, This msures foat 'forimsr dimet' fermMian or other secondary structure does not occur. Short primer molecules genefoily require, cooler tefoperatefes to form. sufSbiendy stable hybrifoeompM# with foe template. A primer need not reflect the exact sequence of foe fomplfoe but must be sufficiently complen.Kmf.ary to hybrid!?® with a template. 1¾ term "primer site” or "primer binding site" refers to foe segment of the target DMA to which a primer hybridizes. The form "primer:pairK means a set 6f printers including a 5· "upstreanr primer" that hybridizes with foe complement of foe 51 end Of the DMA sequence to be amplified and a 3' “lfe^sbiamprimnf‘--foahh>%idi^'^fofoe'S’ end of foe sequence to be amplified.

As used herein, foe t^l^eohfolementary*' means font one mfoleic acid is identical to, or hybridises seleotively to, another nuclei» aeM molecule, Setectmty of hybridisation exists when hybridisation occurs that is more selective than total lack of specificity. Typically, selective hybridization will occur when tee is at least about 55% identi ty over a stretch of at least 14-25 nucleotides, alternatively at least 65%, at least 75%, or at least 90%. In one alternative embodiment, one nucleic acid hybridizes specifically to the other nucleic acid. See M. Kanehisa, Nucleic Acids Res. 12:203 (1984). A primer that is "perfectly complementary" has a sequence fully complementary across the entire length of the primer and has no mismatches, the primer is typically perfectly complementary to a portion (subsequence) of a target sequence, A "mismatch" refers to a site at which die nucleotide in the primer and the nucleotide in the target nucleic acid with which it is aligned are not complementary. Hie term "substantially complementary" when used in reference to a primer means that a primer is not perfectly complementary to its target sequence; instead, the primer is only sufficiently complementary to hybridize selectively to its respective strand at the desired primer-binding site.

As used herein, a "probe” is a nucleic acid capable of binding to a target nucleic acid of complementary sequence through one or more types of chemical bonds, usually through complementary base pairing, usually through hydrogen bond formation, thus forming a duplex structure. A probe binds or hybridizes to a "probe binding site." A. probe can be labeled with a detectable label to permit facile detection of the probe, particularly once the probe has hybridized to its complementary target A label attached to the probe can include any of a variety of different labels known in the art that can be detected by chemical or physical means, for example. Labels that can be attached to probes include, but are not limited to, radioisotopes, fhiorophores, chroroophores, gold particles, quantum dots, mass labels, electron dense particles, magnetic particles, spin labels, molecules that emit chemiluminescence, eieetroohemieally active molecules, enzymes, cofactors, and enzyme substrates. Probes can vary significantly in size. Some probes are relatively short. Generally, probes are at least 7 to 15 nucleotides in length. Other probes are at least 20, 30 or 40 nucleotides long. Still other probes are somewhat longer, being at least 50,60,70,80,90 nucleotides long. Yet other probes are longer still, and are at least 100,150,200 or more nucleotides long. Probes can fee of say specific length that tails within tire foregoing ranges as well. A “thermophilic DNA polymerase” is a thermostable DNA polymerase enzyme having an optimum temperature at which it functions, which is higher than 40°C. Oftentimes, the optimum temperature for the function of a thennopMlic DNA polymerase ranges from about 50“ C to 80°C, or 60°C to 80°C. These heat stable enzymes were introduced to provide more robustness to the repeated cycles of heating and cooling the enzyme during conventional ΡΟΚΑ “difficult sequence” refers to sequences on which a polymerase enzyme has a tendency to slip, make mistakes or stop working. Examples of difficult sequences include- sequences of several residues (e.g. segments of 6, 9, .12,15, or 30 base pairs or longer) having greater than about 50% G and C base pairs that are called GC-rich sequences, sequences containing tandem repeat segments, polyrepeat sequences such as poly-A. sequences, trinucleotide repeat regions as found in sequences associated certain diseases like Huntington's, and other' such problematic sequences.

The term “comprising” as used in this specification means “consisting at least in pash of. When interpreting each statement in this specification that includes the term “comprising”, features other than that or those prefaced by the ten» may also be present Related terms such as “comprise” and “comprises” are to be interpreted in the same manner.

Overview of ike Conventional Polymerase Chain Reaction (PCM}

To perform the standard (henna! cycling polymerase chain reaction using a thermophilic (i.e. heat stable) DNA polymerase, one typically executes the following steps: 1) prepare a cocktail containing a FOR buffer, a dNTP mixture, a primer pair, a DNA polymerase, and doubly-deionized water in a tube; 2) add the DNA to be amplified to the tube 3) place the tube in a temperature block of a thermal cyder (e.g Perkin-Elmcr™ 9600 or 9700 FCR Thermal Cycler) 4)

Program the thermal cycler with specific reaction conditions (e.g. a period for thermal denaturatiou of double-stranded DNA by heating to above about 9(TC for about .1 to 2 minutes, a period of annealing by slowly cooling to about 50 to 65s C for 2 min, and. a period for polymerisation, also called primer extension, by heating to about 70 to 75° C for a few minutes) feat are to be repeated for about 25 to 35 cycles. Executing the method produces about, a 2” fold amplification of the starting material, where n is the total number of cycles of amplification that are carried out

While some limitations of conventional PCR stem from flow the conventional technique is typically carried out, several limitations in the performance parameters stem directly or indirectly from the reliance on thermal cycling. Overall reaction yield, amplification efficiency, sensitivity, robustness, and portability are each, for instance, restricted by thermal cycling. The Nano~PCR'w methods overcome not only limitations due to thermal cycling but also several that are inherent to typical implementation of conventional PCR.

Nana-PCK™·

Nano-PCR.™ methods and apparatuses can dramatically extend the detection and amplification capabilities of the polymerase chain reaction by breaking through several limitations imposed by conventional approaches. Table 1 compares typical performance parameters of current PCS. with Hano-PCR™.

Table 1; Performance parameters of current PCR vs. Nano-PCR™

The common denominator of PCR. and FCR-based technologies practiced to date has been the use of thermal cycling to sequentially denature DMA, anneal primers, and then extend primers via a polymerase enzyme. The methods described herein, dubbed Nano-PCR™, and apparatuses for performing those methods utilize the applicati on of controlled amounts of force or stress to the nucleic acid molecules to provide new alternatives to thermal cycling for implementing DMA or KNA amplification. As used herein, applying stress to a nucleic add includes direct and indirect application of force to a nucleic acid that tends to stretch or elongate the nucleic acid. As examples, stress can be applied to a nucleic acid by direct application of mechanical tension, by hydrodynamic stresses in a fluid flow, or electromagnetic fields, whether acting on the nucleic acid molecules themselves and/or on surfaces, substrates, or particles and the like that are bound to tire nucleic acid. In many applications, Nano-PCR™ can break through one or more of the limitations dud have traditionally restricted the performance and scope of conventional PCR.

Cycling of Mechanical Tension

The application of controlled tension to nucleic acids provides not only an alternative to thermal denaiuradon of double-stranded DNA (clsDNA) but also a unique capability to precisely control each step of the PCR process. Nano-PCR™ introduces a new approach to amplification of DNA or RNA by exploiting the effects of precisely controlled forces, such as mechanical, hydrodynamic, or electromagnetic stresses cm the DNA/llNA molecule and/or on the polymerizing enzyme.

Increasing temperature of solution comprising a DNA molecule and increasing stress on a DNA molecule produce analogous results. Thus, a. polyinerase reaction cycle can be initiated by increasing tension applied to &amp; DNA template to above about 65 pN to denature the DNA, A step corresponding to the annealing of primers can be effected by slowly decreasing tension on the DNA template to below about 50 pN to allow primers to anneal to the template. Tension in the DNA template can then be adjusted within about 0 to about 30 pN during extension of tire primer via enzymatic polymerization in order to control the progress, rate and/or accuracy of the replicat ion. As with thermal cycling in conventional PCR, cycling of stress can be repeated in a pre-proginnmed cycle in Nano-PCR™ methods.

In the examples below, various modes by which these methods can he put into practice are· described Hydrodynamic stresses and/or electric fields applied to the nucleic acid molecules, like mechanical tension can be cycled to perform Nano-PCR™ without reliance on thermal cycling. Of course, although Hano-PCR.™ methods can be performed without, any temperature cycling, this is not to say that control of temperature will not be advantageous in some embodiments as more fully discussed below. Figures 1A and IB show exemplary flow diagrams for Nano-FOR™ methods. It will be appreciated that alterations and additions to the basic protocol will be made as appropriate for specific tasks such as sequencing, cloning, mutagenesis, mutation screening, and pathogen detection, etc.

At room temperature and standard buffer conditions, application of tension above about 65 pH to double-stranded DNA can cause denaturation (ie. melting) into single stranded DNA (ssDNA). As used herein, “room temperature” is understood to be a temperature within the normal range of comfortable laboratory temperatures, generally about 20-22° C. A theoretical model of the force- induced melting of DNA at room temperature has been described by iottiia Romtina and Victor A. Bloomfield (“Force-Induced Melting of the DNA Double Helix 1. Thesanodynamic Analysis” Bk>physJ.% 80:882-93,2001* and, tsForce--Induoed Melting of the DNA Double Helix. 2. Effect of Solution Conditions” Biophys J., 80:894-900,2001). Using the equations of Rourlna and Bloomfield as described herein, it is possible for one of ordinary skill to determine a precise level of tension that will melt a pri mer in a manner analogous to conventional mel ting point temperature calculations.

Slowly decreasing the applied tension below about 65 pN in the presence of primer oligonucleotides can permit the selective binding of the primers to template DNA in a manner analogous to slowly cooling denatured DNA below the melting point temperature of a primer in a thermal cycler. Accordingly, during a primer annealing step, tension applied to a nucleic acid template strand can he slowly reduced from an amount that causes dsDNA to melt to an amount that permits primer annealing. It may also be desirable to maintain tension on a DNA strand at a level that substantially inhibits polymerase action, tor example at about 30 pN or greatest belo w about SflpM, until unbound primer is flushed from, the reaction chamber to minmiizeKo^specifie hiMiag and non-specific primer extension.

The application of tension to a nucleic acid template irx the range lorn about 0 to about 30 or 35 plf can be used to slow the rate of polymerase activity, The exact speed, of the enzyme depends oa yarious factors, including the ambient temperaisre or ambient eoneentrariansof polymerase and/or nucleotide diphosphate substrates; Tension greater than ahoet 35--45 pN at room temperature promotes the nfenral proofreading exonuclease atdridy of the poly merase enzyme.

An exemplary embodimmt of a Mano/BCR?*4 nmi&amp;od can comprise: (0 providing a sample; of doidde«stmaded X>NA(d^l|&amp;3 containing &amp; target eeixuenee» one or more oligonncieolide primers, far example a pair of primers compfementarf to the ;33 ends of the tajget sequence and its complement;; at least fern different imdeoside fepho^hafos (be, ATP, CTP, <TTP, TTP); and a DNA polymerase; (b) denatming the dsIMA iafe single-stranded DMA (usDMA) template strands using a non-fkermfily-driven process, tor example by the application of tension snSleient to cause dsDMA. to melt (&amp;g. tension greater than about ilTpM’Bo the dsEiMA' |c) controlling the non-fempally-drivm process to promote hybridization of primers to complementary template strands, for example, Wfeean tension was used to dienature the dsDNA, by reducing dm tension applied tp the ssDNA; to extend fee primers to formdsDNA; andy(e) repeating steps ffed) until a desired; amount of DMA sequence arhjrilflcation is obtained.

The use of a *feu»dhemraliy driven processί, in -S«;:ipeStbds - d«sdiibb&amp; hernia means, for example, :'th^.:dis0KA' denstU^.nh·^ -sot -aobdj^liahed solely ihfebgh an; increase intPnxp««^*K©::hi$oVd the msliingiemperatare of dsDMA, but ratter that a physical or mechanical fores1 is exerted oh the nucleic acid that does not rely on temperature, The npn-feerihally driven; piboess may comprise appiyingliensionto the DMA strand, for example by direcil^lpa^bn of meolMfoai 1¾¾ by Sdld flow, by application of an efectriq field,: and/or by the action of one or more denaturing agents. As described hemin, fe: effect of Such, a force may be affected by temperafere so ::®at it may be desip^Kid^^ control and optionally to modutafo fhe ienmersture; during , one or more st^s of fee methods. A target: sequence to be amplified can be contained in isolated D'NA or in a mixture of nucleic acids and can foe contained on complementary strands of equal or unequal lengths. A method may also Include starting with a composition comprising KMA and producing a DMA template using reverse transcriptase or a similar method A target sequence may alternatively be provided on a single stranded nucleic acid, rendering step (b) unnecessary in the first cycle. The reaction components of step (a) can be combined at tire start of die- procedure or may be introduced separately as needed. Optionally, reaction components oan also be removed from the reaction chamber dining certain steps. For example, nucleoside triphosphates (NTPs) may be introduced during or prior to step (d) and the primers may be introduced during or prior to step (o) and unbound primers may be flashed from fee chamber before the primer extension (replication) step. Further, in various embodiments of the method, tension in the range of about 0-45 pH, about 0-35 pH or about 0 to 20 pN can be applied to template DMA strands during step (d). In such embodiments, tire amount of tension applied to the template strands during step (d) can optionally be varied over time. The amount of tension applied to the template strands in step (d) can varied according to the known or estimated progress of the polymerase in relation to positions of difficult or error prone subsequences, such as G~€ rich segments (eg. segments containing greater than about 50% G»C base pairs, or greater than about 70% G-C base pairs) or the positions of segments containing repeating sequences.

Accuracy ofNano~PCR'm over Normal and "Difficult ” Sequences

When tension is applied to the template DMA during the primer extension step, a polymerase can be induced to “reverse direction” and the exonuclease activity of the polymerase can predominate, it will be appreciated that at the atomic scale and over times on the order of a single poiymems&amp;'exonuclease step, the process is stochastic. However, when considered from an average over the time scale of several steps the polymerase is seen to exhibit sustained exonuelease activity when the applied tension is greater than a threshold that can be theoretically predicted tor a given temperature and solution conditions.

By applying a modulated amount of tension to template DNA during the primer extension step in an amount below the threshold at which the exonuclease activity of a polymerase becomes predominant, e.g, below about 35 to 45 pN at room temperature and normal PCR solution conditions, more preferably in the range of about. 10 to 30 pN. Nauo-PCRTM can provide substantially increased accuracy of replicating DNA over conventional PCR, Furthermore, this effect can be achieved over those sub-sequences that are difficult using conventional PCR methods. The amount of tension applied to a template DNA strand can be adjusted in the range of about 0 to 45 pH, about 0 to 35 pN, or about 0 to 20 pN over time during the primer extension off a template that contains a mixture of more and less problematic segments. For example, according to a map of the sequence, tension may be increased as necessary to a level below about 35 pH to promote increased accuracy over difficult regions and then be carefully decreased to permit faster processing over less problematic segments. The length of the template strand is changed during the primer extension. In some variations of the methods, it is possible to adjust the tension on the template in direct response to the changes m length of the template strand so as to calibrate the applied tension precisely according to the progress of the prdymerization reaction and the particular location of the "difficult*1 subsequences.

In embodiments where it is not practical to directly monitor the progression of the polymerase, the position of tire polymer ase can be estimated by multiply ing the elapsed time by the known rate of replication for the polymerase at the applied amount of tension.

Thus Nano-PCR™ permits accurate replication of not only normal target templates but also difficult sequences (e.g. GC rich DNA, tandem repeat, microsatellite or trinucleotide repeat DNA) to be replicated and amplified with substantially increased accuracy relative to conventional PCR. As an example, where one of the highest fidelity polymerases currently available (e.g. Phusion Enzyme) is used in conventional thermally-driven, PCR,. error rates of approximately 4.0 x 1 O'7 errors/base pair are observed in favorable eases, that is, only on well-behaved sequences. By contrast Nano-PCR™ methods as described herein can produce an error rate less than about 1.0 x HP7 errors/base pair, less than 5 ,0 x to4 ermrs/base pair, 1,0 x 10'8 errors/base pair, 5.0 x 10~9 80-018/¾¾¾¾ pair, 1.0 x 10'y errors/basepair, 5.0 x 10*w errors/base pair, or even 1.0 x 1040 errors/base pair. Furthermore the methods described herein can permit the efficient amplification of oligonucleotide fragments having a GC content higher than 50 'percent, 60 percent, 70 percent, 75 percent, or even E0 percent or ES percent.

In certain cases, oligonucleotide fragments contain a section of repeating base pair units at least eight base pairs in length (e,g., AAAAAAAA, GGGCGCGC). The error rate for conventional FOR is increased in such cases, usually approaching 1.0 x 10*2 errors/base pair. The present methods of Nano-FCB.™ provide for the amplification of repeating base pair units with an error rate less than about LOx Iff3 errors/base pair* Under certain conditions, error rates less than 1,0x Iff4 errors/basepair., LOx TO"5 errors/basepair, LOx I O'6 errors/base pair, or 1.0 x l O'7 errors/base pair can be achieved. These low error rates may also he obtained where the repeating base pair unit is at least 1.0 base pairs in length, at least 1.5 base pairs In length, or at least 20 base pans in length. In the methods described herein, such results can also he obtained over microsatellite regions, polymerase slippage regions, and other tandem repeat regions, which are difficult sequences when using conventional PCR methods.

Amplification Efficiency

Where amplification efficiency is defined by the equation N; « NaCl+Yf, arid N) is the number of product copies, N2 is the number of template oligonucleotide copies, n is the number of cycles and Y is the efficiency, efficiencies greater than SO percent are achieved. Under certain conditions, amplification efficiencies greater than 85 percent, 90 percent, 95 percent, 96 percent, 97 percent, 98 percent, or even 99 percent can be achieved. Such -amplification efficiencies can. also be obtained where the GC content of the oligonucleotide fragment is greater than 55 percent, 60 percent, 65 percent, 70 percent or even 75 percent.

Amplification efficiencies greater than 50 percent can be achieved where the GC content of an oligonucleotide .fragment is greater than 50 percent, 55 percent, 60 percent, 65 percent or 70 percent. Efficiencies greater than 60 percent, 70 percent, 80 percent» 90 percent, 95 percent, 97 percent, or even 99 percent can be observed. In conventional PCR, these high GC-rich regions are amplified and sequenced with the addition of various denaturing agents, including but not limited to sodium hydroxide, TMA chloride, TMA oxalate, TMA acetate, TMA hydrogen sulfate, ammonium chloride, bermykUtuethylfeexadccylanunonltmi chloride, HTA bromide, HTA oxalate, betaine monohydrate, PMSO, and formamide, and the like. In certain embodiments of NanoPCR?*1, efficient amplification is also seen using methods as described herein in the absence of such polymerase chain reaction additives. JRobusiness and Adaptability

Conventional PCR .methods generally rely on precise control and cycling of temperature. Further, conventional PCR methods can require additional factors such as various denaturing agents and tedious optimizations. However, the use of tension cycling to drive amplification as described herein enables a much higher degree of precision and control over the PCR process than allowed by thermal cycling alone. Moreover, the methods described herein can function under a wide range of temperature conditions, limited only by factors such as the range of temperatures under which a chosen polymerase can function and the melting point, temperature of the primer/iemplafe bond at a given tension.

Of course, it will be appreciated that temperature can affect the rate and accuracy of polymerase enzymes and the melting of DNA under tension. Generally, the amount of tension required to melt dsDNA is decreased with increasing temperature, As a rough guide, from about to 0 to 20° C, up to about 75 pH can be required to melt dsDNA. At about 6Cf C, the amount of tension required to denature dsDNA can be about 45 pH. The melting tension decreases to about 7 pH at just below the free DNA melting point

Although it is generally not necessary, it may be advantageous to control the temperature during one or more steps of the methods depending on requirements of an individual application. For example, the temperature of the reaction mixture can be maintained at a temperature that optimizes the accuracy, polymerization rate, and/or tension response of a chosen DNA polymerase, that increases or decreases tiie amount of tension required to achieve DNA melting, or that is otherwise advantageous because of the individual device or working environment Unless otherwise desired, the temperature can be generally constant, and fee entire process can be performed at or near normal room temperature. Unless otherwise indicated, the examples herein are described using amounts of tension that will be appropriate at room temperature. One of ordinary skill will readily be able to adjust the tension applied to the DNA template for higher or lower temperatures.

Through application of even small amounts of tension, feemal cycling temperatures no longer impose a limitation on the temperature at which the PCR reaction must be carried out By applying a tension of about 7 to 45 pN, for example, one can decrease the temperature at which double-stranded DNA denatures by up to about 30 degrees C. Adjusting the amount of tension applied to DNA enables performance of PCR at temperatures well below the amount required for denaturation in conventional PCR. This effect permits PCR using low amplitude thermal cycling, For example, a method can comprise a denaiuratxon step in which an amount of force less than about 65 pN is applied in concert with an increase in the temperature of the solution to less than about 90° C, alternatively less than about 80° C.

The methods aid apparatuses described herein may be carried out or operated at temperatures below 90* C. Oligonucleotide denaturation steps, for instance, can be conducted at below 80° C, 70* C, 6if C, 50* C, 40* C, 30* C, or even 25* C. Annealing steps can be conducted below 50* C, 45* C, 40* C, 35* C, 30° C, or even 25* C. Furthermore, polymerization steps can be conducted below 70° C, 60* C, 50* €, 40° C, 30* C, or even 25* C.

Similarly, pH and ionic strength of fee solution in which, fee DNA is immersed can affect the tension-induced melting curves of DNA. Accordingly, by adjusting the levels offeree applied to DNA in the methods, Nano-FCR™ permits using a wider range of pH and ionic strength solution conditions to carry out the PCR process than in conventional PCR. Similarly, all these and additional parameters can affect tension control of primer extension. The methods described herein can be adjusted for and even take advantage of these effects. Thus Nano- PCR™ methods can be more robust to a wider range of temperature, ionic strengths, pH and buffer conditions m general, which means that it can be performed in a wider range of situations, demanding less stringent extraction and. ptniftearion of the starting DNA or KNA material, and can be more resistant to various contaminants and enzyme inhibitors that typically restrict the scope of conventional PCR. hr preferred implementations, the presence of contaminating substances can bo removed by flushing the sample as part of the Nano-PCR,™ process . For example, an unpurified DNA sample containing contaminants can be introduced into a Mauo-PCR™ device, the DNA is retained in the reaction chamber, e.g, by any of the means described herein for retaining DNA for the controlled application of stress. The cojharoinaats are flushed out of the reaction chamber and reagents are flushed in, This can provide substantial robustness to the Nano-PCR™ process, permitting rapid accuracte amplification in environments that are unfavorable to conventional PCR,

Nam~PCR'iM Using Direct Application of Mechanical Force

Nano-PCR*8* methods can be performed utilizing various methods to directly apply mechanical tension to DMA strands as a non-ihermally-drivcn process that can provide for DMA denafiuatiou and/or precise control of the activity of DNA polymerase. There are several different ways to apply tension to a double-stranded oligonucleotide. .For example, DNA strands may be anchored in an array to a movable element, to individually controllable elements, or to particles that can be manipulated.

Using opposed coated surfaces:

As an example, the process can be performed using nucleic acids anchored to opposed coated surfaces, generally as illustrated in Figures 2A-2C: Coated surfaces are prepared by attaching a first complexing molecule (e.g., streptavidin) 201,207, which can be the same or different for each surface, to two substrate surfaces 203, 209. The coated substrate surfaces are arranged in opposition to one another at a suitable distance apart. Double-straaded nucleic acids 205, where both ends of one stand comprise a completing molecule that Is complementary to the first completing molecule (e,g., biotin) which, can recognize and bind to the first completing molecule, are immobilized onto the coated surfaces. Force can be applied to the ends of the immobilized nucleic acids by increasing the distance between the coated surfaces or by lateral translation of one or both surfaces. For example, one or both substrates may be a movable dement or comprise a movable element, such as a piezoeleetle element Tension sufficient to cause dsDNA to melt (e.g. greater than about 65 pN at room temperature) can be applied to the nucleic acids, producing anchored strands and freed strands. Both the anchored and the freed strands can be replicated using appropriate primers and polymerase. Preferably, the freed strands can he flushed away and optionally collected so that only the anchored strands will be replicated. The position of the opposed surfaces can be controlled dur ing replication to modulate the amount of tension applied to the anchored template strands. The cycle can be repeated as desired.

In variations of a device for performing file method using opposed coated surfaces 215, 217, one or both surfaces can also be arranged to form an array of individually movable elements 219, each of which may be individually addressed by a control circuit driven by a programmable processor as illustrated in Figure 2C. Such a control circuit can include a feedback charnel that reports three and/or displacement parameters to the processor. Printing or lithography techniques can be used to pattern sites for anchoring molecules on a siif&amp;ee. A device for performing the method can also comprise a channel for introducing reagents to the chamber or channel comprising the coated surfaces and apparatus for delivering (and optionally storing) reagents separately or in combination and for collecting reaction products. A wide variety of suitable methods of anchoring a nucleic acid to a surface are known, including bid not limited to covalent bonding, antigen-antibody, and siTqjtavidin* biotin.

Arrangements of fluid flow can be utilized to orient and extend DMA strands between opposed surfaces. For example^ as illustrated in Figure 2B, DMA may be anchored at one end to a surface 213 having passages for fluid flow distributed between the anchoring locations. Flowing fluid though these passages can be used to orient and extend DHA strands more or less uniformly in. a desired direction, for example towards an. opposed surface 211 or array of movable elements, which may-have passages distributed between anchoring surfaces to receive the fluid flow.

Thus, a method may comprise anchoring 1>NA strands to a first surface, flowing fluid through openings in the first surface towards and through openings in a second surface opposed to the first surface, and anchoring DHA. strands oriented in the fluid flow to the second surface.

To increase the number of anch ored strands with each cycle, aetivatable primers can he used in replicating the attached strands. “Aetivatable primers” comprise chemical moieties that can be acti vated by chemical or physical methods. These “aetivatable groups” are inert until activated, for example by photoactivation using a laser at an appropriate wavelength. Many different aetivatable chemical groups are known in the art, which can be converted into or unblock functional eompSexing groups, in a variation of the method, aetivatable primers are allowed to anneal to the anchored angle-stranded nucleic acids. Primer extension and fragment replication is performed The resulting double-stranded nucleic acids are denatured through the application of tension to the template strand. The free copy strands will then comprise the aetivatable groups of the primers. Activation allows the copy strands to become immobilized on the opposing coating surfaces. This cycle can be repeated until a desired degree of amplification is obtained. When desired, anchored nucleic acids can be released, tor example by the use of a restriction enzyme that recognizes a sequence near an anchored end of the nucleic acid or that has been introduced into the end of the copied nucleic add by the primers.

Using optical or magnetic traps.'

Another way to directly apply tension to DMA can utilize optical or magnetic tweezers or other traps to manipulate particles to which the DMA is anchored. An optical tweezers traps particles with forces generated by optical intensity gradients. Dielectric particles polarized by the Sight's electric field are drawn up the gradients to the brightest point. Reflecting, absorbing and low-dielectric particles, by contrast, are driven by radiation pressure to the darkest, point Optically generated farces strong enough to form a feree-dimensional trap can he obtained by bringing a laser beam with an appropriately shaped wavefront to a tight focus with a high numerical aperture lens. Figure 3A illustrates a DNA strand extended between heads 301 tapped at the focus of a laser beam 303. ft is possible to manipulate large numbers of particles using an array 307 of optical tweeters as illustrated in Figure 3B. Commercially available optical tweezers arrays include those produced by Airyx, Inc. Another implementation of as array of optical tweezers, see E. R. Dufrssse and D. G. Grier, Rev. Sei instr. 69:1974 (1993); and, U. S. Patent No. 6,055,106 (2000). An optica! tweezers array can comprise about 10*, 104,10*, 10*, or more pairs of optical or magnetic tweezers.

Amplification using optical or magnetic tweezers can generally be per.fenn.ed as follows: A nucleic acid is anchored to appropriate particles at each end in a fluid medium. The particles may be adapted to he .manipulated using optical, or magnetic traps. Tension sufficient to denature a dsDNA, for example greater than about 65 pN. is applied to the oligonucleotide through the application of force (e,g., optical or magnetic) to tire particles, resulting in the denaftsraiion of the nucleic acid. The tension can be reduced in die presence of primers to allow fee prime»' and nucleic acid to anneal Polymerization by DNA polymerase can be initiated by further relaxing the tension. To repeat the cycle, tension can be increased snob that the resulting double-stranded nucleic acids are denatured. In a variation, a nucleic acid can be anchored at one end to ahead that is trapped in a fluid flow, for example by a magnetic field. Fluid flow rate can be used to control tension on the nucleic acid.

It is possible to begin from a single target molecule and sequentially populate an array wife copy strands. Copy strands can be anchored to new heads rising activatable primers. New beads can be brought into proximity with fee copied strands. Alternatively, heads having prsrimniobilized primers can be brought into proximity with the copied strands in conjunction with fee denaiuration step. Manipulation of fee beads in this fashion optionally may be automatically controlled by a programmable processor.

Nana~PC&amp;'m Using Hydrodynamic Stress

Naao-PCR™ methods can be performed ufiltsang the application of tension to DMA by hydrodynamic stress in controlled fluid flow. Methods using this approach can he performed in a mierofMdic device, which can be a bencfafcop device or alternatively can be reduced to a portable size such as may be incorporated in a handheld device. Naao-FCR™ methods utilizing the application of tension to DNA by hydrodynamic stress in controlled fluid flow can be performed using any arrangement that provides for a controlled rate of fluid flow.

Using anchored DNA polymerase: A method o f performing a Nano-PGR*** method using polymerase anchored to a surface in a device can comprise the following steps. Polymerase is immobilized on a surface that is arranged such that fluid can be flowed over the surface at a controlled ra te. For example, a surface in. a. channel or chamber that has been coated with a .first complexing moiety can be used to immobilize a DNA polymerase that has been modified to comprise a second complexing moiety. Exemplary complexing moieties include antigen-antibody, histidine to Ni-NTA, or biotin- sireptavidin pairs, Target dsDNA is denatured in the presence of primers, for example dsDNA and primers can be subjected to a flow rate such that a force sufficient to cause dsDNA to melt (e.g. greater than about 65 pN) is applied to the dsDNA, Polymems^hiieieotide/primer complexes can be allowed to form by reducing the flow rate. Primer extension mid fragment replication can be promoted through a Anther reduction of flow rate. Double-stranded nucleic acid products comprising template and copy strands can. be denatured through the application of an increased flow rate, and the cycle can be repeated until &amp; desired degree of amplification is obtained. In such a method, polymerase may be immobilized in a microchamel, for example a mierochauuel in a microfluidie “lab on a chip” type device.

In variations of a device for perforating the method using polymerase anchored on one or more coated surfaces, such a device can also comprise a channel for introducing reagents to a reaction chamber or channel comprising the coated surfaces and apparatus for delivering and optionally storing reagents separately ox in combination and for collecting reaction products.

Msdteg'Of liftogr^l^ techniques knows, m the art can be used to pattern sites for $sckp«ag: teolscutes. Such a device will comprise an apparatus for creating aud conteollteg Said Sew 1¾ the reaction chamber ter channel:, Any suitable method for mathig: aqd: CQO.troiHng Suid Sow can bo ossdincfodlng eieoirodyMnrtc methods, pomps: and syringe. apparatuses. Reagent solutions optiontelf can be recycled through the reaction chamber,

Figure 4 illustmtes an approach in which polymerase 401 is anchored to a. substrate 407, S>r example by strepteyilin tendingunddbe like. '.DMA strands 403 are pemrihed to bmd to the aochered pclypierase. Donteteted fluldflowdffi passed over teesuhste&amp;te: 407 causes application of sketching force on the DMA strands in the ferm: of h^mdynamic stress, Mpo4?pR:omb® earned out using sneh. an application ofikse according tp:-tftq;|«oe^i .^^pqill^teated in Figured as described abpye.

Using mmhored DMA stmids in a cmiraifedfitud flow. Another way to apply tension to nucleic acids ia«Ql^.'itemdbih^Mclitec:;^ds in a complied Sold flow. The process can generally be performed as SdldwSt Nucleic acids comprislng5 at one end:. a first complexmg moiety that recognizes and can bind to a second completing moiety coated on .a-snrikee, are allowed to become immobilized on tee coated surface, Fluid is Sowed over tee surface such that a force sufficient to cause dsDNA to melt (e.g. greater tea» about 65 pN) is applied to anchored double-stranded nucleic acids, which respite in strand separation. DMA polymerase and primers, optionally primers comptemg achvatable groups, are flushed over the aurihee at a reduced flow rate. A reduced or stopped flow rate allows termadon of poI^OTS^dKgohucisPtide/pPid^' complexes. Printer extension and teagmeni replication can be promoted in the presence of MTFd feongh a further reduedon or stoppage of flow rate. After replication, the flow rate can ^,incfease?4'^^g««^g the resulting dsDMA fo tension such that the dsDNA, is denatured. If nctmtabb primers are used, the extended primers can be activated, primers sa&amp; b« alfowitebtteM^ can repeated until a desimd degme of ampliSeaflon is obitened. In such a method, polymerase may be immobilized on a surface in a microchannel, for example a mierocharme! in a microfluidic “lab on a chip” type device.

Figure 5 illustrates the stretching of DNA in a fluid flow by hydrodynamic stress where DNA stands SOS are anchored to a substrate 507 through anchoring molecules 501. Fluid flowing in direction 509 extends and stretches the DNA m a controlled manner as a function of the fluid flow velocity. Figure 5B illustrates a variation in which DNA strands 503 are anchored by binding molecules 50 i to a plurality of substrate structures 505 such that fluid can flow between the structures at a controlled rate. It will be appreciated that there are a large number of other variation® that can be used to achieve a similar result.

In variations of a device for performing the method using nucleic acid anchored on one or more coated surfaces, such a device can also comprise a channel for introducing reagents to a reaction chamber or channel comprising the coated surfaces and apparatus for delivering and optionally storing reagents separately or in combination and for collecting reaction products. Mating or lithography techniques known in the art can be used to pattern sites for anchoring molecules. Such a device can comprise an apparatus for creating and controlling fluid flow in the reaction chamber or channel. Any suitable method for creating and controlling fluid flow can he used. For example flow can be provided by means of a pump or can be electrostatically dri ven. Reagent solutions can optionally be recycled through the reaction chamber.

Stretching DNA in a velocity gradient and using hydrodynamic focusing: An alternative approach to applying tension using fluid flow can be used in combination with the above methods, or may form the basis of a distinct method. DNA can be stretched in a fluid that has a velocity gradient Various exemplary arrangements for hydrodynamic focusing and counter propagating elongations! flows are illustrated hi Figures 6A-6C.

For example, Wong et al, reviewed the basis of several such techniques and described a method of hydrodynamic focusing (Wong et al.» “Deformation of DNA molecules by hydrodynamic focusing.” J. Fluid Mech. 497:55-65. 2003). In hydrodynamic focusing, illustratedby Figure 6A, two streams of buffer 607 flowing at &amp; relatively high rate converge in a microchannel 605 with a center stream that is introduced at a low flow rate. The converging streams accelerate the center stream without substantially mixing. The result Is a region of flow having a strong velocity gradient in the flow direction, DNA 601 in this gradient is stretched to an extended state. By increasing the flo w rates of the converging streams even more. It will he possible to deimlure dsDNA such that ssDNA emerges from the microchanneL This permits delivering ssDNA to a reaction chamber, for example where polymerase has been anchored.

Stagnation flow car housed to trap and apply tension to nucleic acids without the need for any anchoring. Perkins et a!, described elongation of DMA in a planar elongation flow apparatus (‘‘Single Polymer Dynamics in an Elongation Flow” Science* 276:2016-21., 1997). In Perkins' apparatus, fluid is flowed 623 if out opposing directions into a T-shaped junction 625 such as .illustrated in Figure 6C, At the center of the junction, a stagnation point 629 is established. Outside of this point, a fluid velocity gradient is established. DMA 601 can become trapped at the stagnation point, being pulled equally in opposite directions by the velocity gradient. Alternative armgemeats such as channel 615 illustrated in Figure 6B can include offset jets 617 of fluid entering a channel 615, or flowing buffer in opposing directions across a slot in which nucleic acid resides.

Nano-PCR™ Methods Using Cycling of Applied Electric Fields

It is possible to apply force to DNA strands in Nano-PCR™ methods through the use of electric and magnetic fields, There are a variety of ways that this can be accomplished. For example, electric fields can be used to indirectly apply force to DNA by driving fluid flow in a nrierofhndie device. As described above, fluid flow can be used to apply hydrodynamic stress to DNA, tor example, DNA anchored to a surface and/or to a particle, or bound to a DNA polymerase that is anchored to a surface. Electrophoretic forces can also apply force directly to DNA strands,

Electric fields may also be used to manipulate DMA strands bound to conductive particles. Accordingly, Nano-PCRTM methods can be performed where denatnration, annealing, and/or primer extension steps are controlled by a nonthermally-driven process wherein one or both ends of a DMA strand is bound to a conductive particle, e.g. gold nanoparticles or the like, which can be manipulated by electric fields to apply tension to the DMA strand. Where one end of a DMA molecule is attached to a conductive particle, tire other end can be anchored to a surface in a reaction chamber in a device. Such methods may utilize achvatable primers as described herein to anchor DMA strands produced in each cycle.

Exemplary Applications (>fNano-PCR’m Methods

Maao-PCR™ methods can be employed in kits and systems for pathogen and bioweapon detection. Examples of such pathogens include, without limitation: Adeno-associated Virus (AAV), Adenovirus, Cytomegalovirus (CMV), Epstein· Barr Virus, Bnteiovirus, Hepatitis A Virus (HAY), Hepatitis B Virus (HBV), Hcpafitis C Vims (HCV), Human Herpes Virus Type 6 (HEY-6). Human Immunodeficiency Vims Type 1 (HIV-1), Human Immunodeficiency Vims Type 2 (HIV-2), Herpes Simplex Virus Type .1 and Type 2 (HSV-1 and HSV-2), Human X-Cell Lymphotropio Virus Type I and Type II (HTLV-I and ΗΤΧΥ-Π), Mycobacterium tuberculosis, Mycoplasma, Parwwirus Β-19» Respiratory Synedtial Vims (RSV) and Porcine Endogenous Refomrus (PBRV). Mano-PCR?** methods can be used for detection of any pathogen in any environment because of the enhancements in sensitivity, accuracy and robustness these methods can provide.

The detection and identification of a particular pathogen using conventional PCR-b&amp;sed diagnostics generally requires that the pathogenic organism or its polynucleotide be present in a biological fluid (e.g. blood, saliva, etc.) at a certain threshold eoneerdmfion, The lower detection limit of Mycobacterium tuberculosis, for example, has been reported, as 7.5 x 1G3 otganisms/mL HCV KMA is detectable in a range from 100 to 1000 RNA molecuies/mi. Slum has reported that the polymerase chain reaction detects around S7.5 percent of proven Mycobacterium mbercuiosis-cfmtdmng nodules. That corresponds to a false-negative rate for detection of 12,5 percent. In preferred embodiments Mano-PCR™ methods can be used to detect pathogens such as tire above with false-negative rates typically less percent, 10 percent., 5 percent, 2,5 percent, 1 percent, 0,5 Ipemeat, 0.25 percent, or 0.1 percent,

Furnhetmore, bhnm-PCRffy can be performed such that it is not limited to about 30-15 cycles cf amplification as eoimentionalPCF. generally: is. This is because of degradation of polymerase after repeated cycles of heating above the :PNA aieliing temperahxre. Im contrast, Nano-PCRTM iaethods cap optionally eotriprise 40, 50, 60, A$:$feap-P€I$** oaa'^.pKrfcamed in an isothermal nrnnner, op using ioW^i^Et^i^aperitare^dolalio^ ^aaO-PGSf^ can be repeated for aiaay cycles, limited only by tire lifetime of the enzyme (e.g. at rooiai teinperature),;

Thus :Haiio~PCRT^; methods can be used to, amplify amounts of starting·. pmtedal (either organisms or their DNA. or RNA) that are substantially less than omountsTequiredlby conventional PGR, Hano-PGR™ mdbsdfe.· can be used to: detect aadreilaliy amplify as itie-.a8:a:Mt3t|le::«a;Qi«idule $$$&amp; orRNA, draatalically decreasing the ihlsmnegstrve rasa and providing increased semhfyify of as much as 100%, For pathogen such as those exemplified above, organisms pr polymrmleGiides cap be detected at amcentxations lower than, 1O00 organisms ox pofyaucleotidesdai 100 organisms otpciyaudeofidesfinlj 50 orgaaisars or polymuoleatides/ml, 25 Organisms or pefyaiublesHdes/nil, 10 organisms or pOiytMcIeotldes/nu, 5 organisms or pdyauoleoftde^aal, or even as little as 1 organism or polynucleotldG ml.

An Osemplary variation of the method can fee used for detecting the presence or absence of at least one specific DHA sequence or disfinguishing between two difBrsntDH’AJequeaces in a sample, In D^pap. be amplified: as described abooe, 1'he method caaifhrlbarmoaipnse: contacting t he amplified DMA with, a probe or probes (e,,g,s an ofigonncleoilde amaplsnientary to die segoence tabs detected that also comprises a detectable moiety, such as a ftnoresoent: label}·,; and,? detectmgAhetbet fbe specifie®MA sequence is in the sample by obsemng the presence or absence of the probe bound to the amplified DNA,, or distinguishing between two different sequences by detecting which of a plaraiity of probes is bound to the amplified DpA,

Another variation of the method carrbe used for amplif&amp;aiioK ffiid/or detection of a sequence encoded on ENA*: The target sequence can be encoded 0¾ "m isolafod KNA or on KKA in a mixtniO of nucleic acids. The method can comprise: isolating BKA from, a. sample (n.gv, :^^reartse^- basscripiion thereby obtaining: a corresponding cS»lii^;-a^:aas|()ii^!gg'ilt&amp; target sequence as described abp^, Inofeiiaethods can Sjrihet comprise de tecting the presence of a specific sequence % the sampleas described above.

Another variation of the method can: ;be used for sequencing atENA, Snob a method nan comprise opdonaiiy :gmp!%mg 'ifit'ISltA as described above and sequencing the DNA. Sequencing thefMA CaAeomprise £d) providing a sample of dsDHA conhdning a target oerpreace, the sample being mv!ded: pid:ibor paiaiM reactions,: a primer eomplememary to the 3* end of the target sequence:; at least four different nucleoside iriphcsphates (i,c. ATP, QTP,·©®, TT^^.-pro^ding,a.4life^*4. d; decay nucleoside triphosphate fiMNTP) seltsc^sd.'fesfin'amemg' (MMfpaSCTP, d:dGTFs and ddTTP optionally labeled'·with a detectable ehemfoal moiety such as a fluorescent moiety, and a ENA polymerase in each parallel reaction; (b) denaturing the daEHA into ssEHAJempkte strandsusing a nomthenmlly-driven process, for example by the applicationof tension sufficient to cause dsENA. to mekfmg. greater than about dS pN) to the dsDNA; (e) promote hybridisation of primers to complementary template strands, tor example, where tension was nsed:|p'4eaai^::the'i^b^s.|yiedti^g/%ei&amp;insioa applied to the ssENA; (φ pemutdng the ENAp to extend the primers to form dsENA; (e) optionally repeating step© (b- d) natil a desired amount of DNA sequence ampiifieafcnis obtained, and defermhnng fe sequmce by detecting the length o f eaeh miolbotide prodneed irr tbe foaofion of by detecting the boss specific Spprescent moiety Or some 'Other base-speeiSe signal asin various: single moleeuie aeqnenoing schemes,

Nmo-PCM^SevUes

There are: tnahy dilferent: device types and configurations one canitse to perform non-therxnally-driven polymerase chain reactions as described hesrehr. One such device is a microflmdic device, where the flow rate within microSuidic channels on. the device is controllable and variable, in preferred embodiments, a device will have a reaction chamber, which can be a channel, an arrangement of channels, or an enclosed space. The reaction chamber will generally comprise a means of retaining nucleic acids and a means of applying stress or tension to the nucleic acids retained therein. Thus, arrangements designed to cany out any of the methods described herein can be envisioned comprising a combination of channels and enclosed spaces having disposed therein particles capable of binding nucleic acids or eomplexing molecules capable of securing their complementary completing molecules, surfaces having completing molecules, movable elements, channels for directing fluid flow and generating a fluid velocity gradient, pumps, valves, membranes, and the like. The chamber can comprise an optically transparent window, for example, if optical tnicromatiipulators are to be used The devices can be manufactured as microfluidic devices which may be incorporated into handheld units. If desired, NanoPCR™ can be performed in solution volumes of less than about a microliter, for example about 50-1000 »L, preferably about .100-500 nL,

As an example, Figure 7A illustrates a possible configuration of a device in which reagents can be introduced through inlets 701,707 and 715. One or more storage chambers 70S, 711 can be provided to contain prepared buffers, dideoxy nucleotide triphosphates, polymerase, and Ore like. Valves 703, 709,717 and 718 may comprise one or mote fluid gates arranged to control, fluid flow at junctions between channels. Reaction chamber 715 may be arranged to permit controlled application of tension to nucleic acid molecules therein, for example as illustrated in Figures 2-6. A channel 721 and pump 723 are optionally provided to permit recycling and controlled flow of regents through chamber 715. Pump 723 may operate by any appropriate mechanism recognized in the art, for example peristaltic pumping, pumping by use of one or more bellows or pistons, by electromotive force, and variations or combinations of such devices and the like. Where .recycling is not desired, flow may be controlled within chamber 715 or externally, for example by syringes attached at inlet and/or outlets 701 and 719. An example of a microfluidic device utilizing a circular, or roughly circular, channel configuration is illustrated by

Figure 7B. Mete 751 permit introduction of reagents either direct l y to a charnel feeding reaction, channel 763 or into one or more storage chambers 753,754 for later use. Valves 755,757, arid 759 control flow into and out of the reaction channel. Pumps 761 may operate to control fluid velocity in channel 763 by peristaltic action, for example by deflection of one or more valve gates into channel 763 in a sequentially controlled manner, electromotive force, or any other means recognised in the microfluidics art For example, a device may be constructed using valves and a peristaltic pumping arrangement that comprise structures constructed of elastomeric material that can foe deflected into the channels of the device in a controlled sequence to control flow, such as described in published PCX application WO/02081729.

The operation of the device illustrated in Figure 7B can be further understood through a description of its operation during a nomihetmally-driven polymerase chain reaction, in the specific instance of nucleic acid amplification reactions, a sample containing or potentially containing a target nucleic acid is introduced into loop 763 through an inlet 751. In some examples, one or more walls of the loop 763 have been prepared for anchoring polymerase or nucleotides as illustrated in Figures 4-5. Alternatively, the loop may be arranged to create fluid velocity gradients, counter propagating fluid flow, and die like by utilizing additional inlets or rotating surfaces such as illustrated in Figure 6. Other reagents necessary to conduct the amplification reaction are similarly introduced through the inlets. Typical reagents Include a primer or primers (e.g,, forward and reverse primers) that specifically hybridize to the target nucleic acid, the four deoxynueteoside triphosphates (i.e., dATP, dTTP, dGTP and dCTF), a polymerase, a buffer and various cofactors required by the polymerase flag-, metal ion).

Following introduction of the sample and necessary amplification reagents into loop 763, the resulting solution is circulated under the action of pumps 761. By varying the rate of pump action, one can control the solution circulation/tlow rate. A flow rate resulting m application of about 65 pN of force to the target nucleic add is established, which denatures it, The flowrate is decreased such that the fores applied to the target nucleic acid is in a range from about 30 pH to 60 pH, This allows formation of polymerase/nocleic add/primer complexes. Primer extension is initiated by further reducing the flow rate to a value corresponding to less than 30 pH of applied force. Upon completion of primer extension, the flow rate is again increased to denature the resulting double-tended nucleic add. The recited steps are repeated until a desired quantity of target nucleic add is obtained. One can access the amplified target nucleic acid by flushing solution through outlet 765 by opening valve 759,

An apparatus for conducting Nano-PCR™ methods can comprise a programmable control device that can individually address and control elements of the reaction device and may also include sensors and feedback circuits so that the control device can monitor, analyze, and if desired can adjust reaction parameters, such as applied stress and template extension.

Example 1; Method and Device Using Opposing Coated Surfaces A pair of streptevidin-coated surfaces are prepared according to standard methods, ( Sahanayagam, Smith, and Cantor. ‘Oligonucleotide immobilization on micropattemed streptavidin surfaces,” Nucleic Acids Res, 2000, VoL 28, No. 8 pp. i-iv ) Biotinylated dsDNA (biotinylation at both ends of one tend) is added to the surfaces, which immobilizes the dsDNA between the surfaces. Jeffrey M.Rotlienherg andMeir Wilchek. p-Diaaobenzoyi-biocytin: a new biotinylating reagent for DMA Nucleic Acids Research Volume 16 Number 14 1988) By adjusting the concentration of the template that is applied to the surface, the surface density of the DNA molecules can be controlled. At mom temperature, greater than about 65 pN of tension is applied to tire dsDNA by increasing the distance between the coated surfaces. This denatures the dsDNA, leaving only target ssDNA tor amplification. The bound DNA is contacted with primers comprising caged biotin groups, while fee. distance between the surfaces is reduced so that between 30 pN and 60 pN is applied to the immobilized ssDNA. The primers are allowed to anneal to the target DNA, and DNA polymerase and nucleotides are added to the resulting complex. Primer extension is initiated by further reducing fee applied tension to <30 pN. Once primer extension is complete, a force >65 pN is applied to the resulting duplex by increasing the distance between the surfaces. This application of force denatures replica nucleotide strand tom its template. The replica strands containing caged biotin moieties are photoactivated and allowed to bind to the stosptavidin-coated, opposing surfaces. The above-recited steps are repeated until a desired degree of amplification is obtained tor the target nucleotide.

Caged biotin reagents can be purchased from commercial vendors such as Molecular Probes or Pierce. For example, a derivative of biotin with a photoactivatahle nitrobenzyl group (MeNPQC-bioiin) exists in a form well-suited for easy linkage to biomolecules and surfaces. (Firrung MC, Huang CY, A general method for the spatially defined immobilization of biomolecuies on glass surfaces using "caged" biotin. Bioconjug Chera. 1996 May~Jo«;7(3):317-21).

Example 2: Method and Device Using Immobilized Polymerase A streptavidin-eoated .microcbaunel surface is prepared according to standard methods. Sahanayagam. Smith, and Cantor. ‘Oligonucleotide immobilization on miempatleroed streptavldin surfaces.” Nucleic Acids Res. 2000, Vol 28, No. 8 pp. i~iv) Biotinylated DMA polymerase is flushed into the mierochannel and incubated to allow surface saturation. Commercial kits for the biotinylation of enzymes are available, for example, from Pierce Labs. Unbound enzyme is flushed out of the nhcrochannel and target nucleotide (e.g., ssDNA, SNA) and primers are flushed in at a chamber flow rate that applies >60 pN offeree on the nucleotide. The polymerase/nucleotide/primer complex is allowed to form by reducing the flow rate such that a force between 30 pN and 60 pN is applied to the nucleotide. Primer extension is allowed to occur by further reducing the chamber flow rate to <30 pN- Once primer extension is complete, a force >65 pN is applied to the resulting duplex by increasing flow rate. This application of feree denatures replica nucleotide strand from its template. The denatured strands are allowed to cycle through the microflaidic chamber until polymerase binding occurs, and the above-recited steps are repeated until a desired degree of amplification is obtained for the target nucleotide.

Example 3: Method and Device Using DNA Immobilization A sfeeplavidm-coatedimciOctecBel sutfhce is prepared according to standard methods. (Sabanayaganx, Smith, and Cantor. “Oligonucleotide immobilization on micropatierned streptavidi» surfaces.” Nucleic Acids Res. 2000, Vol. 28, No. 8 pp. i~iv. Biotinylated dsDNA (biotinylation at one end of one strand) is flushed into the mierochannel and incubated to allow surface binding. (Jeffrey M..Relhenbergaid Meir Wilchek. p-Diazobenzoyl-biocytin: anew biotinylating reagent tor DNA Nucleic Acids Research Volume 16 Number 14 1988 ) A chamber flow rate that applies a force >65 pN to the bound dsDNA is established. This denatures the dsDNA, leaving only target saDNA for amplification. DNA polymerase, nucleotides, aid caged biotinylated primers are flushed into the mierochannel. Caged biotin reagents can be purchased from commercial vendors such as Molecular Probes or Pierce. For example, a derivative of biotin with a photoactivstable nitrobenayl group (MeNPOC-biotm) exists in a form well-suited for easy linkage to biomolecules and surfaces. (Pirrmig MC, Huang CY. A general method for the spatially defined immobilization of biomolecules on glass surfaces using "caged” biotin. Bioconjug C'hem. 1996 May~Jun;?<3):317-21). The chamber flow rate is decreased such that between 30 pN and 60 pN is applied to the bound >ss DNA. The primers are allowed to anneal to the target DNA. and file resulting compounds are allowed to complex to DNA polymerase. Primer extension is allowed to occur by further reducing the chamber flow rate to <30 pN. Once primer extension is complete, a force >65 pN is applied to the resulting duplex by increasing flow rate. This application of force denatures replica nucleotide strand from its template. The replica strands containing caged biotin moieties arc photoactivaied and allowed to bind to the streptavidin-coatod mierochannel surface. The above-recited steps are repeated until a desired degree of amplification is obtained for the target nucleotide.

Example 4: Method and Device Using Optical Tweezers A double-stranded DNA complex is immobilized between polystyrene beads in an appropriate medium at ambient temperature. {"Overstretching B-DNA: the Elastic 'Response of Individual Double Stranded and Single Stranded DNA Molecules" by Steven B. Smith, Yujia Cui, aad Carlos Bustamante Science ¢1996} vol 271, pp. 795-799) A stretching three of approximately 65 pN is applied to the DNA through the use of optical tweezers. (Rouzma, I,, and V. A. Bloomfield. 2001b, Force-induced melting of the DNA double helix 2, Effect of solution conditions. Biophys. J. 80:894-900) The force results in DMA densturation.

Primers are added to the medium, and the stretching force is reduced to less than 60 pN. This allows the primers to anneal to the denatured, single-stranded DNA, DNA polymerase and nucleotides are added to the medium, and highly accurate replication is initiated by reducing the stretching force to between 0 and 30 pN.

After replication is complete, a stretching force of approximately 65 pN is applied to each of the double-stranded DNA complexes, resulting in. the release of single-stranded DNA molecules. This can be scaled up by using an array of manipulators. For example, an array such as the optical trap arrays made by Airyx, Inc, can be used.

While the invention has been described in detail with reference to particular embodiments thereof, it will be apparent to one skilled in the art that various changes can be made, and equivalents employed, without departing from the scope o f the invention, s in this specification where reference has5been made to patent specifications, other external documents, or other sources of information, this is generally for the purpose of providing a context for discussing the features of the invention. Unless specifically stated otherwise, reference to such external documents is not to be construed as an admission that such documents, or such sources of information, in any jurisdiction, are prior art, or toon part of the common general knowledge in the art. in the description in this specification reference may be made to subject matter which is not within the scope of the claims of the current application. That subject matter should be readily identifiable by a person skilled in the art and may assist in patting into practice the invention as defined in the claims of this application.

Claims (29)

1. THE CLAIMS DEFINING THE INVENTION ARE AS FOLLOWS:

   1. A device for amplifying one or more nucleic acid molecules, comprising: one or more fluid channels for directing fluid flow within the device; a means of retaining a sample including one or more nucleic acid molecules within the one or more fluid channels; and a mechanism for applying a variable and controlled amount of tension, that tends to stretch the one or more nucleic acid molecules, to the nucleic acid molecules retained therein during at least one cycle, said cycle including the following operations: (a) optionally when the sample includes a double-stranded nucleic acid, denaturing the double-stranded nucleic acid into a single-stranded nucleic acid target sequence; (b) annealing a primer to its complementary nucleic acid target sequence; and (c) extending the primer to form one or more extension products; and, wherein said device is configured such that at least one of the one or more extension products formed in (c) is used as template strands in a subsequent cycle.
2. 2. The device of claim 1, wherein the mechanism for applying tension to the nucleic acid molecules retained in the device comprises first and second surfaces with means for anchoring nucleic acid molecules thereon, and further wherein said first and second surfaces are configured for moving relative to each other.
3. 3. The device of claim 1, wherein the mechanism for applying tension to the nucleic acid molecules comprises at least one surface with means for anchoring nucleic acid molecules thereon, the device further comprising a mechanism for providing a controlled and variable fluid flow over said nucleic acid molecules.
4. 4. The device of claim 3, wherein at least one surface with means for anchoring nucleic acid molecules thereon further comprises passages for fluid flow distributed between the means for anchoring said nucleic acid molecules.
5. 5. The device of claim 1 wherein the mechanism is further configured to provide at least one of a controlled and variable fluid flow over said nucleic acid molecules or a velocity gradient in laminar fluid flow over said nucleic acid molecules.
6. 6. The device of claim 1, wherein the mechanism for applying tension to the nucleic acid molecules retained in the device comprises fluid flow channels configured to provide a velocity gradient in laminar fluid flow, a stagnation point within a fluid flow, counter propagating fluid flows, or a combination of these.
7. 7. The device of claim 1, wherein the mechanism for applying tension to the nucleic acid molecules retained in the device comprises an array of optical, electrical, or magnetic manipulators configured to manipulate particles bound to the nucleic acid molecules.
8. 8. The device of claim 1 wherein the nucleic acid molecules are double-stranded nucleic acid molecules, wherein the device is configured to apply tension for denaturing the double-stranded nucleic acid molecules.
9. 9. The device of Claim 1, wherein the means for retaining the nucleic acid molecules includes anchoring means, and the means for applying variable and controlled tension includes an array of movable individually controlled elements or particles that can be manipulated.
10. 10. The device of Claim 9, wherein the movable individually controlled elements are piezoelectric elements.
11. 11. The device of claim 1, wherein the mechanism for applying tension to the nucleic acid molecules comprises at least one surface having a nucleic acid polymerase attached thereto, the device further comprising a mechanism for providing a controlled and variable fluid flow over said nucleic acid molecules.
12. 12. The device of Claim 1, wherein the mechanism for applying tension to the nucleic acid molecules retained in the device comprises mechanism for applying electric fields to drive fluid flow within the device or mechanism for applying electric fields to the nucleic acid molecules retained in the device.
13. 13. The device of Claim 12, wherein electric field is applied to manipulate conductive particles having the nucleic acid molecules attached thereto.
14. 14. A device for sequencing one or more double-stranded nucleic acid molecules, comprising: one or more fluid channels for directing fluid flow within the device; means of retaining a sample including said nucleic acid molecules within the one or more fluid channels; and a mechanism for applying a variable and controlled amount of tension, that tends to stretch the nucleic acid molecule, to the nucleic acid molecules retained therein during at least one cycle of said device, said cycle including the following operations: (a) denaturing said double-stranded nucleic acid molecules into single-stranded nucleic acid molecules; (b) hybridizing primers to complementary single-stranded nucleic acid molecules; and (c) extending the hybridized primers to form extension products; and a detector for determining the sequence of a nucleic acid molecule in said sample.
15. 15. The device of Claim 14, further including in said cycle the operation of detecting the length of the extension product.
16. 16. The device of Claim 14, wherein said sample includes dideoxy nucleoside triphosphates optionally labeled by base-specific fluorescent moieties, and said cycle further includes the operation of detecting the base-specific fluorescent moieties.
17. 17. The device of Claim 14, wherein said one or more double-stranded nucleic acid molecules are amplified prior to performing said at least one cycle.
18. 18. The device of Claim 14, wherein the mechanism for applying tension to the nucleic acid molecules retained in the device comprises mechanism for applying electric fields to drive fluid flow within the device or mechanism for applying electric fields to the nucleic acid molecules retained in the device.
19. 19. The device of claim 1 or 14, wherein the mechanism for applying tension to the nucleic acid molecules retained in the device comprises one or more of: fluid channels creating a velocity gradient; fluid channels for hydrodynamic focusing, fluid channels for counterpropagating flow; and fluid channels for T-shaped junction.
20. 20. The device of Claim 1 or 14, wherein the tension applied is selected from mechanical tension, hydrodynamic tension, or electromagnetic tension, or a combination thereof.
21. 21. A method of sequencing a nucleic acid molecule, comprising: (a) providing a sample, including: a single-stranded or double stranded nucleic acid target sequence, a nucleic acid polymerase; a primer complementary to the nucleic acid target sequence; and one or more nucleotides; (b) optionally when the sample includes a double-stranded nucleic acid target sequence, denaturing the double-stranded nucleic acid target sequence into a single-stranded nucleic acid target sequence; (c) annealing the primer to its complementary nucleic acid target sequence; (d) extending the primer to form an extension product; (e) optionally repeating steps (b) through (d) to form further extension products; and (f) determining the sequence of the nucleic acid target sequence, and wherein at least one cycle of the steps (b) through (d) comprises applying tension that tends to stretch the nucleic acid molecule, the nucleic acid target sequence or the primer extension product.
22. 22. The method of Claim 21, wherein the nucleic acid target sequence is an RNA, a double-stranded DNA or a single-stranded DNA.
23. 23. The method of Claim 21, wherein determining the sequence of the nucleic acid target sequence is performed by detecting length of the extension product.
24. 24. The method of Claim 21, wherein the one or more nucleotides are optionally labeled by at least one detectable chemical moiety.
25. 25. The method of Claim 21, 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.
26. 26. The method of Claim 21, wherein the nucleic acid target sequence is amplified prior to performing steps (a) through (e).
27. 27. The method of Claim 21, 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.
28. 28. The method of Claim 21, wherein tension that tends to stretch the nucleic acid is applied to at least one of the nucleic acid target sequence and the primer to induce proofreading exonuclease activity, thereby improving the fidelity of nucleic acid sequencing.
29. 29. The method of Claim 21, wherein said sample includes polynucleotides, and said device is effective to detect said polynucleotides are detected at concentrations lower than 100 organisms or polynucleotides/ml, lower than 50 polynucleotides/ml, lower than 25 polynucleotides/ml, lower than 10 polynucleotides/ml or lower than 5 polynucleotides/ml.