US 2013 0096O13A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2013/0096013 A1 Esfandyarpour et al. (43) Pub. Date: Apr. 18, 2013

(54) METHODS AND SYSTEMIS FOR (60) Provisional application No. 61/389,490, filed on Oct. ELECTRONIC SEQUENCING 4, 2010, provisional application No. 61/389,484, filed on Oct. 4, 2010, provisional application No. 61/443, (71) Applicants: Hesaam Esfandyarpour, Los Altos, CA 167, filed on Feb. 15, 2011. (US); Mark Oldham, Emerald hills, CA (US); Eric Nordman, Palo Alto, CA (US) Publication Classification (72) Inventors: Hesaam Esfandyarpour, Los Altos, CA (51) Int. C. (US); Mark Oldham, Emerald hills, CA CI2O I/68 (2006.01) (US); Eric Nordman, Palo Alto, CA HOIL 29/00 (2006.01) (US) (52) U.S. C. CPC ...... CI2O I/6869 (2013.01); HOIL 29/00 (73) Assignee: Genapsys, Inc., Redwood City, CA (US) (2013.01) (21) Appl. No.: 13/632,513 USPC ...... 506/2:435/6.1; 435/287.2: 257/414 (22) Filed: Oct. 1, 2012 ABSTRACT Related U.S. Application Data (57) (63) Continuation of application No. 13/397.581, filed on The present invention provides for methods and systems for Feb. 15, 2012, now abandoned, which is a continua Electronic DNA sequencing, single molecule DNA sequenc tion-in-part of application No. PCT/US2011/054769, ing, and combinations of the above, providing low cost and filed on Oct. 4, 2011. convenient sequencing.

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METHODS AND SYSTEMIS FOR 0005 Thus, a need exists for improved systems and meth ELECTRONIC SEQUENCING ods for extracting, amplifying and sequencing polynucle otides. CROSS-REFERENCE TO RELATED APPLICATIONS SUMMARY OF THE INVENTION 0001. This application is a continuation of U.S. patent 0006. The embodiments described herein relate to systems application Ser. No. 13/397.581 filed Feb. 15, 2012, which is and methods for sequencing polynucleotides. In some a continuation-in-part of PCT/US 11/54769, filed Oct. 4, embodiments the polynucleotides to be sequenced may be 2011, and which claims priority to and benefit of U.S. Provi individual double or single stranded polynucleotides, which sional Application Ser. Nos. 61/389,490 filed Oct. 4, 2010, may be circularized in some embodiments, as opposed to 61/389,484 filed Oct. 4, 2010, 61/443,167 filed Feb. 15, 2011, clonal polynucleotides. and 61/491,081 filed May 27, 2011, each of which is hereby incorporated by reference in its entirety. This application also BRIEF DESCRIPTION OF THE DRAWINGS claims priority to and the benefit of U.S. Provisional Appli 0007 FIG. 1 shows a schematic representation of a cross cation Nos. 61/443,167 filed Feb. 15, 2011 and 61/491,081 section of an under-etched NanoNeedle Sensor. filed May 27, 2011, each of which is hereby incorporated by 0008 FIG. 2 shows a schematic representation of an iso reference in its entirety. metric view of several under-etched NanoNeedle Sensors. 0009 FIG. 3 shows a cross sectional view of a schematic FIELD OF THE INVENTION representation of several NanoNeedle Sensors with attached 0002 The present invention provides for methods and sys polymerase and target DNA. tems for Electronic DNA sequencing, single molecule DNA 0010 FIG. 4 is a photomicrograph of several under-etched sequencing, and combinations of the above, providing low NanoNeedle Sensors. cost and convenient sequencing. 0011 FIG. 5 is a photomicrograph of several interdigi tated under-etched NanoNeedle Sensors. BACKGROUND OF THE INVENTION DETAILED DESCRIPTION OF THE INVENTION 0003 Methods for quick and cost effective DNA sequenc ing (e.g., at high-throughput) remain an important aspect of 0012. As used herein, “bead may mean beads, moieties or advancing personalized medicine and diagnostic testing. particles that are spherical or non-spherical, wherein said Some known systems for DNA sequencing require that DNA beads, moieties or particles may be porous or Solid or a samples be transferred between various Subsystems (e.g., mixture of solid and porous, and can include magnetic beads between the nucleic acid isolation Subsystem and the ampli that are may be paramagnetic, Super-paramagnetic, diamag fication Subsystem), thus resulting in inefficiencies and netic, or ferromagnetic. potential contamination. Some known methods for DNA 0013 As used herein, “bead capture features' may mean sequencing employ optical detection, which can be cumber features that can temporarily hold a single bead in a fixed Some, expensive, and can limit throughput. Other systems position relative to the sensor and can include local magnetic utilize some forms of electronic sensing, but the sensor and structures on the Substrate, depressions which may utilize an sequencing flow cell are one-time use disposables, which external magnet, local magnetic structures, Van der Waals Substantially increase the cost to the user, and limits the forces, or gravity as forces that fix the position of a bead. complexity of the sensor which may be cost effectively manu Optionally, the bead may be bound in place using covalent or factured, as it will be thrown out after a single use. Some non-covalent binding. systems utilize amplification methods within the same flow 0014. As used herein, “clonal may mean that substan cell, in which the sequencing is performed, binding the ampli tially all of the populations of a bead or particle are of the fied directly to the flow cell, preventing reuse. Other systems same nucleic acid sequence. In some embodiments there may utilize emulsion PCR, wherein beads and samples are mixed be two populations associated with a single sample DNA into Small emulsions utilizing low concentrations. Due to fragment, as would be desired for “mate pairs.” “paired ends', Poisson distribution, most of the beads and sample do not or other similar methodologies; the populations may be come together in an emulsion with a single bead and a single present in roughly similar numbers on the bead or particle, sample, and are thus lost. The cost of the beads and amplifi and may be randomly distributed over the bead or particle. cation is a Substantial portion of the cost of the sequencing, 0015. As used herein, “confinement may mean when a and most of that cost is thrown away without ever generating molecule generated (such as DNA) at one bead or particle any useful data. The current system enables utilization of stays associated with the same bead or particle so as to Sub virtually all of the sample, thus reducing the cost to the user. stantially maintain the clonal nature of the beads or particles. 0004 Current DNA sequencing systems typically need 0016. As used herein'isolate may mean the prevention of whole genome amplification in order to have sufficient migration, diffusion, flow, or other movement, from one Vir sample, as the sample is very inefficiently utilized. Such tual well to another virtual well as necessary to maintain the whole genome amplification methods typically introduce sig clonal nature of the beads or particles. nificant amounts of bias in amplification in different portions 0017. As used herein, “localized magnetic feature' may of the genome, and require higher levels of coverage to over mean a magnetic feature created on a Substantially planar come said bias. Methods for localizing samples, and reagents substrate to hold individual beads on said substantially planar into a Volume wherein a desired reaction or binding may substrate. occur is another aspect which is envisioned for the system, 0018. As used herein, “localized magnetic field’ may which may eliminate or reduce the need for whole genome mean a magnetic field that Substantially exists in the Volume amplification, and thus reduce the coverage needed. between the north pole of a first magnetic region and the South US 2013/00960 13 A1 Apr. 18, 2013

pole of a second magnetic region or Substantially exists in the nm thickness 105, followed by a conductive p+ silicon layer Volume between the north and South poles of a single mag of 80 nm thickness 106, followed by a silicon oxide layer of netic region. 20 nm thickness 107. The channel may be created after the 0019. As used herein, "nanosensor may mean a sensor structure is fabricated. The structure may be generated such designed to detect beads or particles less than one of 0.1. 1, 5, that an oxide layer or a resist layer covers all sections which 10 or 20 micrometers as measured on the diameter or the long are to be retained in the final structure. A chemical wet etch, axis for non spherical beads or particles. Alternatively, the a plasma etch, or a vaporphase etch may be utilized to remove sensor may be sensitive to moieties associated with said beads the silicon or other similar substrate from under the structure. or particles, or with reaction products or byproducts wherein The conductive tip of the structure may then be exposed using the reaction includes a moiety associated with said bead or an ion milling step. particle. Said moieties may include DNA fragments, hydro 0026. All of the thickness may be varied, as may the mate gen ions, or other ions which are counter ions and thus asso rials. The channel in the substrate may alternatively be fabri ciated with said beads or particles or moieties bound or asso cated using an oxide layer, with a resist layer in the Volume of ciated with said beads or particles. Nanosensors can include the channel. The layers of Oxide and conductors may then be “NanoBridge, “NanoNeedle or ISFET sensors. fabricated on top of the oxide and resist, obviating the need to 0020. As used herein, “particle' can mean a non bead under-etch the structure. FIG. 4 Illustrates a single ended moiety such as a molecule, an aggregation of molecules, NanoNeedle array fabricated in a manner similar to that sche molecules bound to a solid particle, or particles, and other matically depicted in FIG. 1. forms known in the art. 0027. As shown in FIG. 2 such a structure may have sen 0021. As used herein, “single phase liquid” is a liquid with sors 201 on both sides of a channel 202 formed in a substrate relatively uniform physical properties throughout, including 203. Polymerase and or target DNA 204 may be attached to Such properties as density, index of refraction, specific grav the active area of the sensor. The sensor itself may be used to ity, and can include aqueous, miscible aqueous and organic electrophoretically and or dielectrophoretically localize the mixtures but does not include non miscible liquids such as oil polymerase and or target DNA to the active area of the sensor. and water. Among the physical properties not considered to The target DNA may be a single double stranded, single potentially cause a liquid to not be considered a single phase stranded DNA target, or a circularized DNA target, or a local liquid include local variations in pH, charge density, and ionic amplification may be done in place on the active area of the concentration or temperature. sensor, as described in PCT/US1 1/54769, which is hereby 0022. As used herein, "Substantially planar shall allow incorporated by reference. FIG. 5 Illustrates an interdigitated Small pedestals, raised sections, holes, depressions, or asper NanoNeedle array fabricated in a manner similar to that sche ity which does not exceed 40 um relative to the local plane of matically depicted in FIG. 2. the device. Variations due to warpage, twist, cupping or other (0028 Nucleotides or probes 205 may be then be provided, planar distortions are not considered to constitute a portion of and a sequencing by synthesis process, or a sequencing by the permitted offset. Protrusions or depressions which are not essential for the uses as described herein but which exceed 40 ligation process may commence. um do not preclude a device from being considered Substan 0029. To improve the sensitivity of either the Nanoneedle tially planar. Fluidic channels and or structures to generate or the Nanobridge, a local amplifier may be provided. The said fluidic channels which have dimensions of greater than amplifier may be either a BJT or an FET. The sensor can be 40 Lum also do not preclude a device from being considered fabricated as a narrow structure, and can be etched under the Substantially planar. structure so that both sides are accessible to changes in pH, or 0023. As used herein, “virtual wells' refers to local elec to changes in conductivity. The Surface of the device may tric field or local magnetic field confinement Zones where the rough, permitting greater Surface area for binding of sample species or set of species of interest, typically DNA or beads, molecules. The surface associated with the electrodes of a generally does not migrate into neighboring “virtual wells' NanoNeedle may be gold or platinum, or may be platinum during a period of time necessary for a desired reaction or black, iridium oxide, or Ppy/PSS to increase the surface area interaction. and the associated double layer capacitance. 0024. In some embodiments, a NanoNeedle, NanoBridge, 0030 Electric concentration of ions may be effected, con ChemFET or ISFET may be fabricated such that the sensor is centrating the DNA, polymerase, primers nucleotides and created on the Surface of a Substrate Such as silicon, fused other reagents as needed to the active area of the NanoNeedle silica, glass or other similar material. In other embodiments, or Nanobridge sensor. Said concentration allow much more the sensor may be fabricated such that it projects vertically or of the sample to be attached or associated with each sensor, horizontally above the substrate, such that the sensor is more mitigating the need for whole genome amplification. accessible to the fluid and reagents. The greater accessibility 0031 FIG.3 describes and illustrates a device and method to fluid and reagents may decrease the time needed for a whereby a single DNA molecule 307 can be sequenced by a sequencing reaction to occur, allow lower concentrations of Nanoneedle biosensor array 300. A polymerase enzyme 306 reagents to be utilized, and increase the sensitivity of the may be attached to a sensor 301. A DNA sample with asso sensor by increasing the Surface area associated with the ciated primers may then be caused to enter the volume with active area of the sensor. said polymerase attached sensors, utilizing for example, pres 0025. As shown in FIG. 1, a NanoNeedle sensor structure Sure induced flow, electrophoretic induced flow and or migra 100 may be fabricated with a Silicon substrate 101, and may tion, or similar means. A single molecule from the DNA have a 800 nm deep channel 102 etched in said substrate. A sample 307 may then be bound by a polymerase attached to a silicon oxide layer of 200 nm thickness 103 may be fabricated sensor 301 in a sensor array 300. Additional single DNA on the substrate, followed by a conductive p+ silicon layer of molecules 307 may also be bound by other polymerases 306 80 nm thickness 104, followed by a silicon oxide layer of 30 bound to sensors 301 in the sensor array 300. US 2013/00960 13 A1 Apr. 18, 2013

0032. In one embodiment, one of the four native dNTPs cess determines which dNTPs do not follow the completion 302 is then flowed into the channel volume 304 with the of a previous set of dNTPs, along with information as to the sensors. If the dNTP is complementary to the next base in the length of the incorporation, wherein said length determina sample DNA 307, it may be bound and incorporated. The tion need not be exact. Nanoneedle sensor 301 may then detect the resulting change 0042. Reversibly terminated dNTPs may be utilized as in the local charge of the extended primer DNA, permitting part of the dNTP pools. If reversibly terminated dNTPs are detection of the incorporation event, at each appropriate posi used, all four dNTPs may be provided, wherein one or more tion of the sensor array 300. If the sample has more than one of the dNTPs is a reversibly terminated dNTP base in a row which is complementary to the type of dNTP 0043 Reverse sequencing may be performed, wherein a 302 which has been introduced into the channel volume 304 polymerase with 3' to 5’ exonuclease activity is utilized with with said sensors 301, a second or Subsequent binding and a dNTP pool that is missing at least one dNTP. The poly incorporation of a dNTP302 may be detected by said Nanon merase with 3' to 5’ exonuclease activity will remove bases eedle sensors 301. The dNTPs 302 may then be washed out of back to the next dNTP in the provided dNTP pool, at which the channel volume 304 containing the sensors 301. point equilibrium will be reached, and no further nucleotides 0033. In some embodiments, the nucleotides may be will be removed. The amount of charge present on the DNA, native dNTPs. In other embodiments, the dNTPs may be or change in conductivity due to changes in the counter ions, modified, with charge modifying structures. The charge or an increased quantity of hydroxide with concomitant modifying structures may be associated, bound or conjugated change in pH may be measured during the reaction, or may be to the polyphosphate, and Subsequently cleaved as part of the measured after the completion of the reaction. incorporation process, obviating the need for a separate pro 0044. In a further embodiment, blunt end ligation may be cess to cleave, separate, or remove the charge modifying performed with ligands that have different binding reagents Structure. on the 3' and 5' ends of said ligands. The electrodes of the 0034. In an alternative embodiment, the charge modifying NanoNeedle may be conjugated with the complementary structure may be utilized as a terminator and thus be associ reagents for binding e.g. the 3' end of the ligands may have a ated, bound or conjugated to the 3' position of the Sugar of the thiol group, and one electrode may be fabricated of gold, dNTP and may thus act as a terminator. Detection may occur while the 5' end of the ligands may have a PNA sequence, and as a result of the process of incorporation, or may result from second electrode may have the complement to said PNA cleavage of the charge modifying structure. sequence. The strand of DNA may then be electrophoretically 0035. In other embodiments, the charge modifying struc and or dielectrophoretically concentrated to the area of the ture may be associated, bound or conjugated to the 2' or 4 NanoNeedle, wherein said DNA strand may then bind with positions of the dNTP sugar. In yet further embodiments, the one end associated with one electrode, and the other end charge modifying structure may be associated, bound or con associated with the second electrode of the NanoNeedle. jugated to the base of the nucleotide. The charge modifying Polymerase and primer may provided with the DNA strand, structures may act as terminators, preventing the incorpora or may be introduced later. Measurement of incorporation tion of additional dNTPs. events may then result from direct measurement of the imped 0036. The linkage, association or conjugation may be bro ance of the DNA combined with the much larger conductivity ken as a result of a physical process, such as temperature of the counter ions associated with the DNA. change, or may be broken as a result of a chemical process, or 0045. In an alternative embodiment, other molecules and may be as a result of a photochemical reaction. assays may be utilized, most particularly those which allow 0037 Said linkage, association of conjugation may be detection of kinetics of single molecule reactions, such as broken after each nucleotide incorporation, or several nucle other enzymatic reactions. otides may be incorporated, and the the number of nucle 0046. In a yet further embodiment, ligation may be uti otides which were incorporated may be determined as a result lized rather than polymerization. Four pools of probe oligos of measuring the amount of charge which was added as a may be utilized, wherein the first base of each probe in a result of said incorporation(s). single probe pool is the same. The probes may be utilize a 0038 A different dNTP may then be flowed into the sensor reversibly terminated tail, or may have a native tail, such that array Volume, permitting detection of incorporation events. multiple ligations may occur, with concomitant increases in Subsequent cycles of Washing, introduction of each of the signal levels. In a manner similar to the use of multiple dNTPs four dNTPs one at a time, and detection of incorporation and polymerase, more than one pool of oligos (with all probes events permit determination of the different sample DNA starting with a single base) may be combined, again with Sequences. concomitant increase in the number of ligations and signal 0039. In an alternative embodiment, non-natural dNTPs levels. The second strand may be removed and a new primer may be utilized. introduced wherein the length of said primer may be shorter 0040. In yet another embodiment, all four nucleotides may or longer than the length of the previous primers. be present, and determination as to which nucleotide is incor 0047. In order to permit repeated measurements of the porated may determined by observation of the kinetics asso same DNA sample, the DNA sample may be circularized, ciated with the incorporation reaction. whilst the polymerase may be a strand displacing polymerase, 0041. In a further embodiment, two or three nucleotides at or may be a polymerase with 5' to 3' exonuclease activity. a time may be utilized, allowing the addition of multiple bases Thus the DNA sample may be repeatedly sequenced by at a time, and a correspondingly large signal. After complet allowing the primer extension reaction to continue for many ing the extension of the primer, with associated data collec cycles completely around the circular DNA sample. In a tion, the extended primer may be melted off, new primer distinct advantage over a system which utilizes detection of added, and the process of extension may be performed again fluorophores, the system of the current invention can utilize using a different order of combinations of dNTPs. This pro the full capability of the read length of the polymerase, unhin US 2013/00960 13 A1 Apr. 18, 2013

dered by having the read length reduced by phototoxicity. In and/or schematics described indicate certain events, and/or Some embodiments, a strand displacing enzyme may be uti flow patterns, and/or chemical reactions occur in a certain lized, thus generating an increase in charge and associated order, the ordering of certain events and/or flow patterns counter ions. In other embodiments a polymerase with 5' to 3' and/or chemical reactions may be modified. While the exonuclease activity may be utilized, allowing net charge to embodiments have been particularly shown and described, it remain the same, while generating protons and or hydroxide will be understood that various changes in form and or detail ions, which may be measured as an increase in conductivity, may be made. or may be measured as a result of the ions interaction with the 0054 Although various embodiments have been surface of an ISFET, ChemFET, or NanoBridge sensor. described as having particular features and/or combinations 0048. The polymerase bound to the sensor may be a highly of components, other embodiments are possible having a processive polymerase, permitting more bases to be incorpo combination of any features and/or components as discussed rated then might occur with a less processive polymerase. The above. polymerase may be phi29, T4, F-530, B104, or other highly 1-12. (canceled) processive polymerases. The polymerase may be modified, so 13. A method for sequencing DNA comprising: that it has reduced or no 3' to 5’ exonuclease activity, or the a) contacting DNA proximate to nanosensors in a nanosen polymerase may have no or little 3' to 5’ exonuclease activity Sor array with a fluid containing one or more of sequenc in its native form. Similarly, any 5' to 3' exonuclease activity ing reagents, primers, polymerase and nucleotides, may be modified so that it is reduced or virtually eliminated. wherein the nucleotides are modified nucleotides with a The polymerase and or DNA may be directly bound to or near charge modifying structure; the sensor, or may be bound through a linker. b) sequencing said DNA by detecting a nucleotide incor 0049. In some embodiments the sensor combines pH sens poration event with the nanosensors, the nanosensors ing with electrochemistry detection as a result of the incor detecting a change in associated electrical, chemical, or poration of a reversibly reducible layer which may be fabri thermal signals; cated above the previous sensor design. Such sensors are c) washing said nanosensor array to remove at least one of available from Senova Systems. During a sequencing cycle, a sequencing reagents, primers, polymerase, nucleotides, reducing reaction will occurifabase has been incorporated in and DNA; and the bead associated with a sensor. The level of reduction can d) repeating steps a, b, c, and d to obtain the sequence be measured, and after the completion of the sequencing information. cycle, a Voltage can be impressed on the sensor, causing an 14. The method of claim 13, wherein the charge modifying oxidation of the Surface, returning it to its original state, structure is a terminator, and is associated, bound, or conju whereupon it can be utilized for the next sequencing cycle. gated to a 3' position of the nucleotide. 0050. In yet another embodiment, the attached DNA mol 15. The method of claim 13, wherein the charge modifying ecule may have a hairpin primer, wherein a portion of the structure is a terminator, and is associated, bound, or conju hairpin primer has a restriction site. Subsequently, after gated to a 2' or 4 position of the nucleotide. completion of the primer extension and associated determi 16. The method of claim 13, wherein the charge modifying nation of the sample DNA sequence, the restriction site may structure is a terminator, and is associated, bound, or conju be cleaved by an appropriate endonuclease enzyme or nicking gated to the base of the nucleotide. enzyme, and the extended primer may be melted off by 17. The method of claim 13, wherein the nucleotides are changing one of the temperature or pH of the Solution in non-natural nucleotides. which the sample DNA is solvated. The sample may then be 18. The method of claim 13, wherein the nucleotides are re-sequenced restoring the temperature or pH of the Solution reversibly terminated nucleotides. in which the sample DNA is solvated to the conditions appro 19. The method of claim 13, wherein the nanosensor is at priate for primer extension, including appropriate concentra least one of a pH sensor, ISFET, ChemFET, NanoBridge, and tions of nucleotides and cations. In an alternative embodi NanoNeedle. ment, a strand displacing enzyme, or an enzyme with 5' to 3' 20. A method for reverse sequencing of DNA comprising: exonuclease activity may be utilized, obviating the need to a) contacting DNA proximate to nanosensors in a nanosen remove the second strand. Sor array with one or more of sequencing reagents, prim 0051. In a further embodiment, a linkage may be provided ers, polymerase and pools of nucleotides, wherein the which may be chemically cleaved, obviating the need for polymerase has 3' to 5’ exonuclease activity and the enzymatic cleavage. nucleotide pools are missing at least one nucleotide; 0052. In other embodiments, a primer may be provided b) sequencing said DNA by detecting a nucleotide incor which has a nick site. In still further embodiments, multiple poration event with the nanosensors, the nanosensors adjacent primers may be provided, obviating the need for a detecting a change in associated electrical, chemical, or nicking endonuclease. The primers may be complementary to thermal signals; a ligated primer, or may be complementary to a targeted c) washing said nanosensor array to remove at least one of section of DNA. The sequencing primers may comprise all or sequencing reagents, primers, polymerase, pools of part of primers used for clonal generation viaan amplification nucleotides, and DNA; and reaction, or may comprise regions which are not used as part d) repeating steps a, b, c, and d to obtain the sequence of the primers for amplification, or may comprise both information. regions used for primers in an amplification reaction, and a 21. The method of claim 20, wherein the nanosensor is at region which is not used for amplification reaction. least one of a pH sensor, ISFET, ChemFET, NanoBridge, and 0053 While various embodiments have been described NanoNeedle. above, it should be understood that they have been presented 22. A method for sequencing DNA using ligation compris by way of example only, and not limitation. Where methods 1ng: US 2013/00960 13 A1 Apr. 18, 2013

a) contacting DNA proximate to nanosensors in a nanosen 39. The method of claim 37, wherein the restriction site is Sor array with one or more of sequencing reagents, prim cleaved and extended. ers, DNA ligase and pools of probe oligonucleotides; 40. The method of claim 37, wherein the restriction site is b) sequencing said DNA by detecting a nucleotide incor cleaved by at least one of a endonuclease enzyme and a poration event with the nanosensors, the nanosensors nicking enzyme. detecting a change in associated electrical, chemical, or 41. The method of claim 37, wherein the primer is melted thermal signals; off by one of pH and temperature. c) washing said nanosensor array to remove at least one of 42. The method of claim 37, further comprising the use of sequencing reagents, primers, ligase, pools of probe oli at least one of a strand displacing enzyme and an enzyme with gonucleotides, and DNA; and 5' to 3' endonuclease activity. d) repeating steps a, b, c, and d to obtain the sequence 43. The method of claim 37, further comprising the use of information. a chemically cleavable linkage associated with the primer. 23. The method of claim 22, wherein there are four pools of 44. The method of claim 37, wherein the nanosensor is at probe oligonucleotides and the first base of each probe in a least one of a pH sensor, ISFET, ChemFET, NanoBridge, and single probe pool is the same. NanoNeedle. 24. The method of claim 22, wherein the probes utilize one 45. A method for sequencing polynucleotides, comprising: or more of a reversibly terminated tail and a native tail. contacting sequencing reaction components with a nanosen 25. The method of claim 22, wherein more than one pool of sor etched under so that both sides of the nanosensor are probe oligonucleotides may be combined. accessible to sense a change in electrical, chemical, or ther 26. The method of claim 22, wherein the nanosensor is at mal signals associated with a nucleotide incorporation event. least one of a pH sensor, ISFET, ChemFET, NanoBridge, and 46. A method of sequencing a single polynucleotide mol NanoNeedle. ecule using a nanosensor in a nanosensor array, comprising 27. A method for polynucleotide sequencing, comprising: attaching a polymerase enzyme to the nanosensor, binding a) circularizing a DNA template and immobilizing the the polynucleotide molecule and associated primers to said DNA template proximate to a sensor, polymerase enzyme, and measuring a change in associated b) contacting the DNA template with a sequencing primer electrical, chemical, or thermal signals upon incorporation of for extension, one or more nucleotide triphosphates for a nucleotide to the polynucleotide molecule. incorporation, and a strand-displacing polymerase 47. A method for preparing DNA for sequencing compris under conditions suitable for extending the sequencing 1ng: primer through the DNA template multiple times, and a) performing blunt end ligation on a DNA molecule with c) detecting the sequence of nucleotide incorporation by ligands that have different binding reagents on the 3' and sensing a change in associated electrical, chemical, or 5' end of said ligands; thermal signals. b) subsequently binding said DNA molecule to electrodes 28. The method of claim 27, wherein the one or more of a nanosensor; polynucleotides are DNA, c) contacting the DNA molecule with a sequencing primer 29. The method of claim 27, further providing a nick site on for extension, one or more nucleotide triphosphates for the polynucleotides. incorporation, and a strand-displacing polymerase 30. The method of claim 27, wherein the polynucleotides under conditions suitable for extending the sequencing are bound to at least one of near a nanosensor, near a nanosen primer through the DNA template multiple times; and Sor through a linker, and to a polymerase. d) detecting the sequence of nucleotide incorporation by 31. The method of claim 27, wherein the polymerases are sensing a change in associated electrical, chemical, or polymerases with 5' to 3' exonuclease activity. thermal signals. 32. The method of claim 27, wherein the polymerases are 48. The method of claim 47, further comprising one or highly processive polymerases with reduced exonuclease more of electrophoretically and dielectrophoretically con activity. centrating the DNA near the nanosensor. 33. The method of claim 27, further providing a nick site on 49. The method of claim 47, wherein the nanosensor is a the primers. nanoneedle. 34. The method of claim 27, further providing multiple 50. The method of claim 47, wherein the ends of the ligands adjacent primers. have at least one of a thiol group and a PNA sequence. 35. The method of claim 27, further comprising melting off 51. An apparatus comprising: the extended primers by one of temperature or pH changing a) a base layer, CaS. b) a plurality of nanosensors in an array for binding with 36. The method of claim 27, wherein the nanosensors are at polynucleotides in a fluid sample, each nanosensor hav least one of a pH sensor, ISFET, ChemFET, NanoBridge, and ing a structure with conductors exposed to said fluid NanoNeedle. Sample comprising: 37. A method for sequencing and re-sequencing one or c) a first oxide layer on the substrate; more polynucleotides comprising: d) a first conductive layer on the first oxide layer; a) providing a hairpin primer with a restriction site on a e) a second oxide layer on the first conductive layer; portion of the polynucleotide; and f) a second conductive layer on the second oxide layer, b) sequencing the polynucleotide by using a nanosensor to g) a third oxide layer on the second conductive layer, sense a change in associated electrical, chemical, or h) a plurality of array channels for flowing fluid sample in thermal signals. and out of said array, wherein the nanosensors are in 38. The method of claim 37, wherein the polynucleotide is proximity to said channel and said channel is optionally DNA. etched under the structure; and US 2013/00960 13 A1 Apr. 18, 2013

i) a detection means coupled to each nanosensor and con iv. an oxide layer optionally covering a portion of the figured and arranged to sense a change in associated first or second conductive layers that are exposed to electrical, chemical, or thermal signals. said fluid sample: 52. The apparatus of claim 51, further comprising having V. a plurality of array channels for flowing fluid sample in and out of said array, wherein the nanosensors are the nanosensors on both sides of a channel in said array. in proximity to said channel; and 53. The apparatus of claim 51, wherein said channel is vi. a detection means coupled to each nanosensor and created by removing a portion of the base or oxide layer configured and arranged to sense a change in associ through at least one of a chemical wet etch, a plasma etch, or ated electrical, chemical, or thermal signals. a vapor phase. 57. The apparatus of claim 56, wherein a reversibly reduc 54. The apparatus of claim 51, wherein a reversibly reduc ible layer is located on the nanosensor for the detection of a ible layer is located on the nanosensor for the detection of a nucleotide incorporation event. nucleotide incorporation event. 58. The apparatus of claim 56 wherein a voltage is 55. The apparatus of claim 51, wherein a voltage is impressed on the nanosensor to return it to its original State. impressed on the nanosensor to return it to its original State. 59. A system comprising: a) a nanosensor array with associated reaction chambers 56. An apparatus comprising: configured for at least one of clonal amplification and a) a base layer; sequencing of polynucleotides, optionally configured b) a plurality of nanosensors in an array for binding with for both clonal amplification and sequencing in a polynucleotides in a fluid sample, each nanosensor hav sequential manner in the same reaction chamber; ing a structure with coaxial conductors exposed to said b) DNA proximate to the nanosensors and in contact with fluid sample comprising: one or more of sequencing reagents, primers, DNA poly i.a first oxide layer in the substrate; merase and nucleotides; and ii. a first conductive layer located in the substrate, in c) wherein the nanosensors are configured to detect a contact with the first oxide layer; nucleotide incorporation event by detecting a change in iii. a second conductive layer located in the Substrate in associated electrical, chemical, or thermal signals. contact with the first oxide layer; k k k k k