CENTRIFUGE AND METHOD FOR LOADING A DEVICE

An apparatus includes a robotic system providing movement in three orthogonal directions to an arm operable to receive a pipette tip and to facilitate movement of fluid into and out of the pipette tip. In addition, the apparatus can include a tray for receiving pipette tips, receptacles for receiving tubes, an apparatus for forming an emulsion, a device for forming particles that include copies of the polynucleotide, a device for enriching the particles and an apparatus for loading such particles onto a sensor array. The apparatus can further include receptacles for holding containers of reagent solutions. Optionally, the robot can include a gripper arm in addition to the pipette receiving arm. 1. A centrifuge comprising:a rotor to spin within a plane;a carrier;an upper plate;a first arm pivotally coupled at a first end to the upper plate, a second end of the first arm pivotally coupled to the carrier; anda second arm pivotally coupled to the rotor at a first end, a second end of the second arm pivotally coupled to the first arm at a position on the first arm between the first end and the second end of the first arm;wherein an angle of the carrier relative to the plane changes responsive to position of the upper plate.2. The centrifuge of claim 1 , further comprising an actuator coupled to the upper plate to move the upper plate in a direction normal to the plane.3. The centrifuge of claim 1 , wherein the carrier is configured to receive a sequencing component including two fluid ports.4. The centrifuge of claim 3 , wherein the two fluid ports are to face inward toward an axis of spinning of the rotor.5. The centrifuge of claim 3 , further comprising a fastener to secure the sequencing component to the carrier.6. The centrifuge of claim 5 , wherein the fastener includes elements to engage the two fluid ports of the sequencing component.7. The centrifuge of claim 1 , further comprising a third arm and a fourth arm claim 1 , the third arm pivotally coupled to the ...

METHODS AND APPARATUS FOR MEASURING ANALYTES USING LARGE SCALE FET ARRAYS

Methods and apparatus relating to very large scale FET arrays for analyte measurements. ChemFET (e.g., ISFET) arrays may be fabricated using conventional CMOS processing techniques based on improved FET pixel and array designs that increase measurement sensitivity and accuracy, and at the same time facilitate significantly small pixel sizes and dense arrays. Improved array control techniques provide for rapid data acquisition from large and dense arrays. Such arrays may be employed to detect a presence and/or concentration changes of various analyte types in a wide variety of chemical and/or biological processes. In one example, chemFET arrays facilitate DNA sequencing techniques based on monitoring changes in the concentration of inorganic pyrophosphate (PPi), hydrogen ions, and nucleotide triphosphates. 123.-. (canceled)24. An apparatus comprising:a substrate including an array of sensors;an array of wells disposed over the substrate, a well of the array of wells corresponding to a sensor of the array of sensors; anda cover disposed over the array of wells and defining a flow volume between the array of wells and the cover, the cover defining a fluid inlet and a fluid outlet in fluid communication with the flow volume, the flow volume at a diffuser including a non-flat wall defining a curved boundary of the flow volume, the non-flat wall being the top or the bottom wall, the fluid flow at the diffuser being more restricted in the center of the flow volume than at the edges of the flow volume when viewed in cross-section perpendicular to the fluid flow.25. The apparatus of claim 24 , wherein the non-flat wall defines a bottom boundary of the flow volume.26. The apparatus of claim 24 , wherein the non-flat wall defines a top boundary of the flow volume.27. The apparatus of claim 24 , wherein the sensor is a field effect transistor.28. The apparatus of claim 27 , wherein the field effect transistor is an ion sensitive field effect transistor.29. The apparatus of ...

Methods for Determining Sequence Variants Using Ultra-Deep Sequencing

The claimed invention provides for new sample preparation methods enabling direct sequencing of PCR products using pyrophosphate sequencing techniques. The PCR products may be specific regions of a genome. The techniques provided in this disclosure allows for SNP (single nucleotide polymorphism) detection, classification, and assessment of individual allelic polymorphisms in one individual or a population of individuals. The results may be used for diagnostic and treatment of patients as well as assessment of viral and bacterial population identification.

METHODS AND COMPOSITIONS FOR MULTIPLEX PCR

The present invention provides methods, compositions, kits, systems and apparatus that are useful for multiplex PCR of one or more nucleic acids present in a sample. In particular, various target-specific primers are provided that allow for the selective amplification of one or more target sequences. In one aspect, the invention relates to target-specific primers useful for the selective amplification of one or more target sequences associated with cancer or inherited disease. In some aspects, amplified target sequences obtained using the disclosed methods, kits, systems and apparatuses can be used in various downstream processes including nucleic acid sequencing and used to detect the presence of genetic variants. 1. A method for amplifying a plurality of different target sequences within a sample , comprising:amplifying within a single amplification reaction mixture at least one hundred different target sequences from a sample including a plurality of different target sequences, wherein the amplifying includes contacting at least some portion of the sample with a plurality of target-specific primers, and a polymerase under amplification conditions, thereby producing at least one hundred different amplified target sequences, wherein at least two of the different amplified target sequences are less than 50% complementary to each other and wherein at least one of the plurality of target-specific primers and at least one of the amplified target sequences includes a cleavable group;cleaving a cleavable group of at least one amplified target sequence;ligating at least one adapter to at least one amplified target sequence in a blunt-ended ligation reaction, thereby producing one or more adapter-ligated amplified target sequences, andreamplifying at least one of the adapter-ligated amplified target sequences.2. The method of claim 1 , wherein one or more of the at least one adapter is not substantially complementary to at least one amplified target sequence.3. The method ...

METHODS AND COMPOSITIONS FOR MULTIPLEX PCR

The present invention provides methods, compositions, kits, systems and apparatus that are useful for multiplex PCR of one or more nucleic acids present in a sample. In particular, various target-specific primers are provided that allow for the selective amplification of one or more target sequences. In one aspect, the invention relates to target-specific primers useful for the selective amplification of one or more target sequences associated with cancer or inherited disease. In some aspects, amplified target sequences obtained using the disclosed methods, kits, systems and apparatuses can be used in various downstream processes including nucleic acid sequencing and used to detect the presence of genetic variants. 1. A method for amplifying a plurality of different target sequences within a sample , comprising:a) amplifying within a single amplification reaction mixture at least one hundred different target sequences from a sample including a plurality of different target sequences, wherein the amplifying includes contacting at least some portion of the sample with a plurality of target-specific primers, and a polymerase under amplification conditions, thereby producing at least one hundred different amplified target sequences, wherein at least two of the different amplified target sequences are less than 50% complementary to each other and wherein at least one of the plurality of target-specific primers and at least one of the amplified target sequences includes a cleavable group;b) cleaving a cleavable group of at least one amplified target sequence;c) ligating at least one adapter to at least one amplified target sequence in a blunt-ended ligation reaction, thereby producing one or more adapter-ligated amplified target sequences andd) reamplifying at least one of the adapter-ligated amplified target sequences.2. The method of claim 1 , wherein one or more of the at least one adapter is not substantially complementary to at least one amplified target sequence.3. ...

METHODS AND COMPOSITIONS FOR MULTIPLEX PCR

The present invention provides methods, compositions, kits, systems and apparatus that are useful for multiplex PCR of one or more nucleic acids present in a sample. In particular, various target-specific primers are provided that allow for the selective amplification of one or more target sequences. In one aspect, the invention relates to target-specific primers useful for the selective amplification of one or more target sequences associated with cancer or inherited disease. In some aspects, amplified target sequences obtained using the disclosed methods, kits, systems and apparatuses can be used in various downstream processes including nucleic acid sequencing and used to detect the presence of genetic variants. 110-. (canceled)11. A method for amplifying a plurality of different target sequences within a sample , comprising:a) producing at least one hundred different amplified target sequences by amplifying at least one hundred different target sequences within a single amplification reaction mixture by contacting the at least one hundred different target sequences with a plurality of target-specific primers and a polymerase under amplification conditions, at least one of the plurality of target-specific primers and at least one of the amplified target sequences including a cleavable group, and wherein the amplifying includes no more than one round of target specific selection for at least one of the target sequences to be amplified;b) cleaving a cleavable group of at least one amplified target sequence;c) producing one or more adapter-ligated amplified target sequences by ligating at least one adapter to at least one amplified target sequence in a blunt-ended ligation reaction; andd) reamplifying at least one of the adapter-ligated amplified target sequences using primers.12. The method of claim 11 , wherein one or more of the at least one adapter is not substantially complementary to at least one amplified target sequence.13. The method of claim 11 , wherein ...

METHODS AND COMPOSITIONS FOR MULTIPLEX PCR

The present invention provides methods, compositions, kits, systems and apparatus that are useful for multiplex PCR of one or more nucleic acids present in a sample. In particular, various target-specific primers are provided that allow for the selective amplification of one or more target sequences. In one aspect, the invention relates to target-specific primers useful for the selective amplification of one or more target sequences associated with cancer or inherited disease. In some aspects, amplified target sequences obtained using the disclosed methods, kits, systems and apparatuses can be used in various downstream processes including nucleic acid sequencing and used to detect the presence of genetic variants. 110-. (canceled)11. A method for amplifying a plurality of different target sequences , comprising:producing a plurality of amplified target sequences by amplifying a plurality of different target sequences within a single amplification reaction mixture, wherein the amplifying includes contacting the plurality of different target sequences with a plurality of target-specific primers and a polymerase under amplification conditions and wherein at least one of the target sequences is amplified using no more than one pair of target-specific primers, wherein at least one of the target-specific primers and at least one of the amplified target sequences includes a cleavable group;cleaving the cleavable group of at least one of the amplified target sequences; andproducing at least one adapter-ligated amplified target sequence by ligating at least one adapter to at least one amplified target sequence, wherein the at least one adapter is not complementary to the at least one amplified target sequence.12. The method of claim 11 , wherein at least one member of the group consisting of: an amplified target sequence claim 11 , a target-specific primer claim 11 , and an adapter claim 11 , includes a cleavable group.13. The method of claim 12 , wherein the cleavable ...

METHODS AND COMPOSITIONS FOR MULTIPLEX PCR

The present invention provides methods, compositions, kits, systems and apparatus that are useful for determining copy number variation of one or more nucleic acids present in a sample. In some aspects, the method includes various target-specific primers that allow for the selective amplification of one or more target nucleic acids in the sample. In yet another aspect, the invention relates to determining copy number variation with respect to gene or chromosome representation of a nucleic acid in the sample. In some aspects, the method for determining copy number variation of different target nucleic acids in a sample using the disclosed methods, kits, systems and apparatuses can be used in various downstream processes including diagnosis, predictive therapeutic regimes or other therapeutic purposes. 1. A method for determining copy number variation , comprising amplifying a plurality of different target sequences in a sample , comprising:a) producing a plurality of different amplified target sequences within a single amplification reaction mixture by contacting the plurality of different target sequences with a plurality of target-specific primers and a polymerase under amplification conditions, where at least one of the plurality of target-specific primers and at least one of the amplified target sequences includes a cleavable group, and wherein the amplifying includes no more than one round of target specific selection for at least one of the target sequences to be amplified;b) cleaving a cleavable group from at least one amplified target sequence;c) producing one or more adapter-ligated amplified target sequences, by ligating at least one adapter to at least one amplified target sequence;d) reamplifying the at least one adapter-ligated amplified target sequence using primers;e) sequencing the at least one amplified adaptor-ligated target sequence;f) calculating the number of sequencing reads for the at least one amplified adaptor-ligated target sequence; and,g) ...

METHODS AND COMPOSITIONS FOR MULTIPLEX PCR

The present invention provides methods, compositions, kits, systems and apparatus that are useful for multiplex PCR of one or more nucleic acids present in a sample. In particular, various target-specific primers are provided that allow for the selective amplification of one or more target sequences. In one aspect, the invention relates to target-specific primers useful for the selective amplification of one or more target sequences associated with cancer or inherited disease. In some aspects, amplified target sequences obtained using the disclosed methods, kits, systems and apparatuses can be used in various downstream processes including nucleic acid sequencing and used to detect the presence of genetic variants. 1. A method for amplifying a plurality of different target sequences within a sample , comprising:a) producing at least one hundred different amplified target sequences by amplifying at least one hundred different target sequences within a single amplification reaction mixture by contacting the at least one hundred different target sequences with a plurality of target-specific primers and a polymerase under amplification conditions, at least one of the plurality of target-specific primers and at least one of the amplified target sequences including a cleavable group, and wherein the amplifying includes no more than one round of target specific selection for at least one of the target sequences to be amplified;b) cleaving a cleavable group of at least one amplified target sequence;c) producing one or more adapter-ligated amplified target sequences by ligating at least one adapter to at least one amplified target sequence in a blunt-ended ligation reaction; andd) reamplifying at least one of the adapter-ligated amplified target sequences using primers.2. The method of claim 1 , wherein one or more of the at least one adapter is not substantially complementary to at least one amplified target sequence.3. The method of claim 1 , wherein the reamplifying ...

Bead Emulsion Nucleic Acid Amplification

Disclosed are methods for nucleic acid amplification wherein nucleic acid templates, beads, and amplification reaction solution are emulsified and the nucleic acid templates are amplified to provide clonal copies of the nucleic acid templates attached to the heads. Also disclosed are kits and apparatuses for performing the methods of the invention. 144-. (canceled)45. A method for analyzing nucleic acid sequences comprising:(a) delivering a plurality of molecules of a deoxyribonucleic acid into aqueous microreactors in a water-in-oil emulsion such that a plurality of aqueous microreactors comprise a single molecule of the deoxyribonucleic acid, a single bead capable of hybridizing the deoxyribonucleic acid, and reagents necessary to perform deoxyribonucleic acid amplification;(b) amplifying the deoxyribonucleic acid in the microreactors to form amplified copies of said deoxyribonucleic acid bound to beads in the microreactors;(c) determining the presence of amplified copies of said dexoyribonucleic acid bound to a bead.46. The method of wherein step (c) is accomplished using polymerase chain reaction.47. The method of claim 45 , wherein a majority of the microcarriers include a single molecule of the deoxyribonucleic acid.48. The method of claim 45 , wherein said reagents include a polymerase chain reaction solution further comprising nucleotide triphosphates claim 45 , a thermostable polymerase claim 45 , and a buffer compatible with polymerase chain reaction conditions.49. The method of claim 45 , wherein said emulsion is heat stable.50. The method of claim 45 , wherein amplification is carried out by a method selected from the group consisting of transcription-based amplification claim 45 , rapid amplification of cDNA ends claim 45 , continuous flow amplification claim 45 , and rolling circle amplification.51. The method of claim 45 , performed with at least 50 claim 45 ,000 molecules of deoxyribonucleic acid.52. The method of claim 45 , wherein between at least ...

METHODS AND APPARATUS FOR MEASURING ANALYTES

Methods and apparatus relating to FET arrays for monitoring chemical and/or biological reactions such as nucleic acid sequencing-by-synthesis reactions. Some methods provided herein relate to improving signal (and also signal to noise ratio) from released hydrogen ions during nucleic acid sequencing reactions. 1. An apparatus comprising:an array of sensors, a sensor of the array of sensors including an electrode structure;a structure defining an array of reaction chambers, a reaction chamber of the array of reaction chambers having a sidewall surface and having a lower surface overlying the electrode structure of the sensor; anda buffering inhibitor disposed on at least one of the sidewall surface or the lower surface of the reaction chamber.2. The apparatus of claim 1 , wherein the buffering inhibitor includes a phospholipid.3. The apparatus of claim 2 , wherein the phospholipid includes phosphatidylcholine claim 2 , phosphatidylethanolamine claim 2 , phosphatidylglycerol claim 2 , or phosphatidylserine.4. The apparatus of claim 1 , wherein the buffering inhibitor includes a sulfonic acid surfactant.5. The apparatus of claim 4 , wherein the sulfonic acid surfactant includes poly(ethylene glycol) 4-nonylphenyl 3-sulfopropyl ether (PNSE) claim 4 , poly(styrenesulfonic acid) claim 4 , or a salt thereof.6. The apparatus of claim 1 , wherein the buffering inhibitor includes poly(diallydimethylammonium) claim 1 , tetramethyl ammonium claim 1 , or a salt thereof.7. The apparatus of claim 1 , wherein the buffering inhibitor is covalently bound to the at least one of the sidewall surface or the lower surface of the reaction chamber.8. The apparatus of claim 1 , wherein the buffering inhibitor is non-covalently bound to the at least one of the sidewall surface or the lower surface of the reaction chamber.9. The apparatus of claim 1 , wherein the sensor includes an ion sensitive field effect transistor.10. The apparatus of claim 9 , wherein the ion sensitive field effect ...

Methods and apparatus for measuring analytes

Methods and apparatus relating to FET arrays for monitoring chemical and/or biological reactions such as nucleic acid sequencing-by-synthesis reactions. Some methods provided herein relate to improving signal (and also signal to noise ratio) from released hydrogen ions during nucleic acid sequencing reactions.

METHODS AND APPARATUS FOR MEASURING ANALYTES

Methods and apparatus relating to FET arrays including large FET arrays for monitoring chemical and/or biological reactions such as nucleic acid sequencing-by-synthesis reactions. Some methods provided herein relate to improving signal (and also signal to noise ratio) from released hydrogen ions during nucleic acid sequencing reactions. 1. A method for sequencing a nucleic acid comprising:contacting and incorporating known nucleotides into a plurality of identical nucleic acids in a reaction chamber in contact with or capacitively coupled to an ISFET, wherein the nucleic acids are covalently bound to a single bead in the reaction chamber, anddetecting hydrogen ions released upon nucleotide incorporation in the presence of no or limited buffering activity.2. The method of claim 1 , wherein the ISFET is in an ISFET array that comprises 256 ISFET.3. The method of claim 1 , wherein the reaction chamber comprises a solution having no buffer or low buffer concentration.4. The method of claim 1 , wherein the reaction chamber comprises a solution having a buffering inhibitor.5. The method of claim 1 , wherein the single bead is at least 90% saturated with nucleic acids.6. The method of claim 1 , wherein the nucleic acids are sequencing primers.7. The method of claim 6 , wherein the nucleic acids are hybridized to template nucleic acids.8. The method of claim 6 , wherein the nucleic acids are hybridized to concatemers of identical template nucleic acids.9. The method of claim 1 , wherein the nucleic acids are self-priming template nucleic acids.10154.-. (canceled) This application claims priority under 35 U.S.C. §119(e) to U.S. Provisional Applications 61/196,953, 61/198,222, 61/205,626, filed Oct. 22, 2008, Nov. 4, 2008 and Jan. 22, 2009, respectively, the entire contents of all of which are incorporated by reference.The present disclosure is directed generally to inventive methods and apparatus relating to detection and measurement of one or more analytes including ...

AMPLIFICATION AND ARRAY LOADING APPARATUS AND METHODS

An apparatus includes a robotic system providing movement in three orthogonal directions to an arm operable to receive a pipette tip and to facilitate movement of fluid into and out of the pipette tip. In addition, the apparatus can include a tray for receiving pipette tips, receptacles for receiving tubes, an apparatus for forming an emulsion, a device for forming particles that include copies of the polynucleotide, a device for enriching the particles and an apparatus for loading such particles onto a sensor array. The apparatus can further include receptacles for holding containers of reagent solutions. Optionally, the robot can include a gripper arm in addition to the pipette receiving arm. 1. A centrifuge device comprising:a motor operable to spin in a first direction and in a second direction opposite the first direction;a rotor coupled to a motor, the rotor to spin within a plane in the first direction or the second direction responsive to the motor, the rotor having a recess and an axle projecting from a side of the recess; anda carrier slidably and pivotally coupled to the axle, the carrier including a first tab on a first side and a second tab on a second side, the carrier to slide along the axle and to rotate about the axle out of the plane and engage the rotor with the first tab at a first angle in response to the rotor spinning in the first direction, the carrier to slide along the axle and to rotate about the axle out of the plane and engage the rotor with the second tab at a second angle in response to the rotor spinning in the second direction.2. The centrifuge device of claim 1 , wherein the carrier is configured to receive a sequencing component including two fluid ports.3. The centrifuge device of claim 2 , wherein the two fluid ports are to face inward toward an axis of spinning of the rotor.4. The centrifuge device of claim 2 , further comprising a fastener to secure the sequencing component to the carrier.5. The centrifuge device of claim 4 , ...

METHODS AND APPARATUS FOR MEASURING ANALYTES USING LARGE SCALE FET ARRAYS

Methods and apparatus relating to very large scale FET arrays for analyte measurements. ChemFET (e.g., ISFET) arrays may be fabricated using conventional CMOS processing techniques based on improved FET pixel and array designs that increase measurement sensitivity and accuracy, and at the same time facilitate significantly small pixel sizes and dense arrays. Improved array control techniques provide for rapid data acquisition from large and dense arrays. Such arrays may be employed to detect a presence and/or concentration changes of various analyte types in a wide variety of chemical and/or biological processes. In one example, chemFET arrays facilitate DNA sequencing techniques based on monitoring changes in the concentration of inorganic pyrophosphate (PPi), hydrogen ions, and nucleotide triphosphates. 123-. (canceled)24. An apparatus comprising:a substrate including an array of sensors;an array of wells disposed over the substrate, a well of the array of wells corresponding to a sensor of the array of sensors; anda cover disposed over the array of wells and defining a flow volume between the array of wells and the cover, the cover defining a fluid inlet and a fluid outlet in fluid communication with the flow volume, the cover including a non-flat wall defining a concave boundary of the flow volume, the non-flat wall having greater eccentricity at a first position in proximity to the fluid inlet than at a second position further from the fluid inlet.25. The apparatus of claim 24 , wherein the sensor is a field effect transistor.26. The apparatus of claim 25 , wherein the field effect transistor is an ion sensitive field effect transistor.27. The apparatus of claim 24 , wherein the fluid inlet corresponds with a first corner of the array of wells and the fluid outlet corresponds with a second corner of the array of wells.28. The apparatus of claim 24 , wherein the cover further comprises flow disruptors extending into the flow volume.29. The apparatus of claim 24 ...

METHODS AND COMPOSITIONS FOR MULTIPLEX PCR

The present invention provides methods, compositions, kits, systems and apparatus that are useful for multiplex PCR of one or more nucleic acids present in a sample. In particular, various target-specific primers are provided that allow for the selective amplification of one or more target sequences. In one aspect, the invention relates to target-specific primers useful for the selective amplification of one or more target sequences associated with cancer or inherited disease. In some aspects, amplified target sequences obtained using the disclosed methods, kits, systems and apparatuses can be used in various downstream processes including nucleic acid sequencing and used to detect the presence of genetic variants. 110-. (canceled)11. A method for sequencing a plurality of different target sequences from a sample for detection of mutations associated with cancer , comprising:amplifying within a single amplification reaction mixture a multiplex of different target sequences from a sample including a plurality of different target sequences,wherein the amplifying includes contacting at least some portion of the sample with a plurality of target-specific primers, and a polymerase under amplification conditions, thereby producing a multiplex of different amplified target sequences, wherein at least one of the plurality of target-specific primers and at least one of the produced multiplex of different amplified target sequences includes a cleavable group;cleaving the cleavable group of at least one of the multiplex of different amplified target sequences and forming a cleaved end; andligating at least one adapter to a cleaved end of at least one of the multiplex of different amplified target sequences, thereby producing one or more adapter-ligated amplified target sequences;thereby preparing a library of different adapter-ligated target sequences, wherein the number of different target-specific sequences amplified during the single multiplex amplification reaction is ...

METHODS AND COMPOSITIONS FOR MULTIPLEX PCR

The present invention provides methods, compositions, kits, systems and apparatus that are useful for multiplex PCR of one or more nucleic acids present in a sample. In particular, various target-specific primers are provided that allow for the selective amplification of one or more target sequences. In one aspect, the invention relates to target-specific primers useful for the selective amplification of one or more target sequences associated with cancer or inherited disease. In some aspects, amplified target sequences obtained using the disclosed methods, kits, systems and apparatuses can be used in various downstream processes including nucleic acid sequencing and used to detect the presence of genetic variants. 1. A method of amplifying a plurality of different target nucleic acid molecules comprising:contacting a plurality of different target nucleic acid molecules with a pool of target-specific primers having a cleavable group, and a polymerase under synthesizing conditions, and synthesizing a plurality of different target-specific molecules by extending one or more of the pool of target-specific primers having the cleavable group;digesting a plurality of the different target-specific molecules having the cleavable group of the target-specific primer;ligating at least one adapter to at least one of the plurality of digested different target-specific molecules; andproducing at least one adapter-ligated amplified target sequence, wherein the method includes using no more than two target-specific primers to produce any one of the adapter-ligated amplified target sequences.2. The method of claim 1 , wherein the cleavable group includes a modified nucleobase or modified nucleoside.3. The method of claim 1 , wherein the cleavable group includes an apurinic nucleotide.4. The method of claim 1 , wherein the cleavable group includes methylguanine claim 1 , 8-oxo-guanine claim 1 , xanthine claim 1 , hypoxanthine claim 1 , 5 claim 1 ,6-dihydrouracil claim 1 , uracil ...

METHODS AND COMPOSITIONS FOR MULTIPLEX PCR

The present invention provides methods, compositions, kits, systems and apparatus that are useful for multiplex PCR of one or more nucleic acids present in a sample. In particular, various target-specific primers are provided that allow for the selective amplification of one or more target sequences. In one aspect, the invention relates to target-specific primers useful for the selective amplification of one or more target sequences associated with cancer or inherited disease. In some aspects, amplified target sequences obtained using the disclosed methods, kits, systems and apparatuses can be used in various downstream processes including nucleic acid sequencing and used to detect the presence of genetic variants. 1. A method of generating an amplicon library from genomic DNA comprising:contacting a portion of a genomic DNA with a plurality of different target-specific primers having a cleavable group, and a polymerase under amplification conditions and producing a plurality of different target-specific amplicons by extending one or more of the plurality of target-specific primers having a cleavable group;digesting at least one of the plurality of different target-specific amplicons with a cleaving reagent; andligating an adapter to the end of the at least one digested different target-specific amplicon and producing at least one adapter-ligated target-specific amplicon, wherein the adapter is not complementary to 20 contiguous nucleotides from a 3′ end or 5′ end of the at least one digested different target-specific amplicon.2. The method of claim 1 , wherein the genomic DNA is from 1 ng to 200 ng of total genomic DNA.3. The method of claim 1 , wherein the genomic DNA is from 10 ng to 50 ng of total genomic DNA.4. The method of claim 1 , wherein the genomic DNA is from 1 ng to 10 ng of total genomic DNA.5. The method of claim 1 , wherein the method further includes phosphorylating at least one of the digested different target-specific amplicons at the 5′ end prior ...

Methods and compositions for multiplex pcr

The present invention provides methods, compositions, kits, systems and apparatus that are useful for multiplex PCR of one or more nucleic acids present in a sample. In particular, various target-specific primers are provided that allow for the selective amplification of one or more target sequences. In one aspect, the invention relates to target-specific primers useful for the selective amplification of one or more target sequences associated with cancer or inherited disease. In some aspects, amplified target sequences obtained using the disclosed methods, kits, systems and apparatuses can be used in various downstream processes including nucleic acid sequencing and used to detect the presence of genetic variants.

METHODS AND APPARATUS FOR MEASURING ANALYTES USING LARGE SCALE FET ARRAYS

Methods and apparatus relating to very large scale FET arrays for analyte measurements. ChemFET (e.g., ISFET) arrays may be fabricated using conventional CMOS processing techniques based on improved FET pixel and array designs that increase measurement sensitivity and accuracy, and at the same time facilitate significantly small pixel sizes and dense arrays. Improved array control techniques provide for rapid data acquisition from large and dense arrays. Such arrays may be employed to detect a presence and/or concentration changes of various analyte types in a wide variety of chemical and/or biological processes. In one example, chemFET arrays facilitate DNA sequencing techniques based on monitoring changes in the concentration of inorganic pyrophosphate (PPi), hydrogen ions, and nucleotide triphosphates. 1. A method of using a semiconductor device , the method comprising:flowing a reagent solution uniformly through a flow cell of an apparatus, the apparatus comprising:a substrate including an array of sensors;an array of wells disposed over the substrate, each well capacitively coupled to at least one sensor of the array of sensors, each well of array of wells forming a reaction chamber for a chemical reaction; anda flow cell cover disposed over the array of wells to cover the flow cell and defining a flow volume between the array of wells and the flow cell cover, the cover defining a fluid inlet and a fluid outlet in fluid communication with the flow volume, the flow cell including a diffuser providing uniform fluid flow over the sensor array device; andmeasuring a signal from each sensor of the array of sensors in response to a reaction in a reaction chamber.2. The method of claim 1 , wherein a nucleic acid is disposed within the well and the reagent solution includes a nucleotide claim 1 , wherein measuring the signal includes measuring a byproduct released as a result of nucleotide incorporation during an extension reaction.3. (canceled)4. The method of claim 2 ...

METHODS AND COMPOSITIONS FOR MULTIPLEX PCR

The present invention provides methods, compositions, kits, systems and apparatus that are useful for determining copy number variation of one or more nucleic acids present in a sample. In some aspects, the method includes various target-specific primers that allow for the selective amplification of one or more target nucleic acids in the sample. In yet another aspect, the invention relates to determining copy number variation with respect to gene or chromosome representation of a nucleic acid in the sample. In some aspects, the method for determining copy number variation of different target nucleic acids in a sample using the disclosed methods, kits, systems and apparatuses can be used in various downstream processes including diagnosis, predictive therapeutic regimes or other therapeutic purposes. 1. A method for determining copy number variation , comprising amplifying a plurality of different target sequences in a sample , comprising:a) producing a plurality of different amplified target sequences within a single amplification reaction mixture by contacting the plurality of different target sequences with a plurality of target-specific primers and a polymerase under amplification conditions, where at least one of the plurality of target-specific primers and at least one of the amplified target sequences includes a cleavable group;b) cleaving a cleavable group from at least one of the amplified target sequences;c) producing one or more adapter-ligated amplified target sequences, by ligating at least one adapter to the cleaved end of at least one of the amplified target sequences, wherein the ligating excludes patch oligonucleotides;d) reamplifying at least one of the one or more adapter-ligated amplified target sequences using primers;e) sequencing at least one of the one or more amplified adaptor-ligated target sequences;f) calculating the number of sequencing reads for at least one of the one or more amplified adaptor-ligated target sequence; andg) determining ...

SCAFFOLDED NUCLEIC ACID POLYMER PARTICLES AND METHODS OF MAKING AND USING

The invention provides particle compositions having applications in nucleic acid analysis. Nucleic acid polymer particles of the invention allow polynucleotides to be attached throughout their volumes for higher loading capacities than those achievable solely with surface attachment. In one aspect, nucleic acid polymer particles of the invention comprise polyacrylamide particles with uniform size distributions having low coefficients of variations, which result in reduced particle-to-particle variation in analytical assays. Such particle compositions are used in various amplification reactions to make amplicon libraries from nucleic acid fragment libraries. 1. A method of sequencing a template nucleic acid comprising:{'sup': 4', '3, '(a) disposing a particle in a reaction chamber comprising or capacitively coupled to a field effect transistor, wherein the particle includes a non-nucleosidic polymer network and a plurality of template nucleic acids attached to the non-nucleosidic polymer network through its volume at an average density of at least 6.9×10per μm, the particle having a coefficient of variance of volume of not greater than 15%, the non-nucleosidic polymer network includes a polyacrylamide gel having a total monomer percentage in a range of 3% to 20% and being permeable to proteins having a size in the range of 50 kilodaltons to 200 kilodaltons;'}(b) performing a polymerase extension reaction on a primer annealed to one or more of the plurality of template nucleic acids to incorporate a nucleotide into the primer; and(c) detecting a signal output in the field effect transistor indicative of the incorporation of the nucleotide into the primer.2. The method of claim 1 , further including disposing a plurality of particles into a plurality of reaction chambers in an array of reaction chambers claim 1 , wherein at least one of the reaction chambers of the plurality of reaction chambers includes or is operatively coupled to a field effect transistor.3. The ...

SCAFFOLDED NUCLEIC ACID POLYMER PARTICLES AND METHODS OF MAKING AND USING

The invention provides particle compositions having applications in nucleic acid analysis. Nucleic acid polymer particles of the invention allow polynucleotides to be attached throughout their volumes for higher loading capacities than those achievable solely with surface attachment. In one aspect, nucleic acid polymer particles of the invention comprise polyacrylamide particles with uniform size distributions having low coefficients of variations, which result in reduced particle-to-particle variation in analytical assays. Such particle compositions are used in various amplification reactions to make amplicon libraries from nucleic acid fragment libraries. 1. A method for forming a particle , the method comprising:suspending an emulsion in an initiator-saturated oil-based continuous phase, the emulsion comprising the continuous phase and a disperse phase, the disperse phase including an acrylamide monomer, a bis-acrylamide crosslinker, a surfactant, and an acrydite oligonucleotide in an aqueous solution;heating the emulsion, the acrylamide monomer, bis-acrylamide crosslinker and acrydite oligonucleotide polymerizing to form acrylamide gel particles having a plurality of oligonucleotides attached through a polymer network of the acrylamide gel; andremoving the initiator-saturated oil-based continuous phase; andresuspending the acrylamide gel particles in a buffered aqueous solution,wherein the acylamide gel particles have a coefficient of variance of volume of not greater than 15%, a total monomer percentage of the acrylamide gel particles is in a range of 3% to 20%, and the acrylamide gel particles are permeable to proteins having a size in the range of 50 kilodaltons to 200 kilodaltons.2. The method of claim 1 , further comprising cooling the emulsion following polymerization and prior to removing the continuous phase.3. The method of claim 1 , wherein removing the continuous phase includes centrifuging to form a pellet of the acrylamide gel particles.4. The ...

METHODS AND APPARATUS FOR MEASURING ANALYTES

Methods and apparatus relating to FET arrays for monitoring chemical and/or biological reactions such as nucleic acid sequencing-by-synthesis reactions. Some methods provided herein relate to improving signal (and also signal to noise ratio) from released hydrogen ions during nucleic acid sequencing reactions. 1. (canceled)2. A method of fabricating a chemically-sensitive field effect transistor (chemFET) sensor device , comprising:forming an array of chemFET sensors in a substrate; each chemFET sensor having a floating gate structure defining an array of floating gate structures;forming a plurality of signal lines for each chemFET sensor within one or more of a metal layer below a top metal layer of the floating gate structure;forming an array of wells in a dielectric layer that overlays the array of floating gate structures; each well having sidewalls and a bottom; andforming a passivation layer of between about 200-600 Angstroms on bottom of each well.3. The method of claim 2 , wherein forming the passivation layer includes forming a passivation layer on the sidewalls and bottom of each well.4. The method of claim 2 , wherein forming a plurality of signal lines comprises forming at least one signal line that is separated by the top metal layer by one or more metal layers.5. The method of claim 2 , wherein forming a plurality of signal lines comprises forming at least one signal line that is separated by the top metal layer by at least two metal layers.6. The method of claim 2 , before forming a passivation layer on the sidewalls and bottom of each well claim 2 , further comprising:forming an adhesion layer on the sidewalls and bottom of each well; andforming a passivation layer on the adhesion layer.7. The method of claim 2 , further comprising applying a forming gas anneal to mitigate effects of trapped charge.8. The method of claim 2 , further comprising irradiating the chemically-sensitive transistor with UV radiation. This application is a continuation of U.S ...

PDMS Membrane-Confined Nucleic Acid and Antibody/Antigen-Functionalized Microlength Tube Capture Elements, and Systems Employing Them

A microfluidic device comprising a microfluidic channel network sealed on one side by a membrane sheet, the sheet having PDMS defining at least the surface sealing the channel, the membrane sheet on its opposite side sealing one side of a pneumatic channel, the pneumatic channel arranged to enable pneumatic deflection of a deflectable portion of the membrane sheet into contact with an opposed surface to control flow in a channel of the network, the membrane sheet confining in a channel of the network at least one micro-particle, micro-length tube or glass nano reactor, functionalized with a capture agent, that has been inserted into that channel. A microfluidic device having a microfluidic channel containing at least two micro-particles, micro-length tubes or glass nano reactors, one functionalized with nucleic acid and another with antibody or antigen. A microfluidic device having a microfluidic channel containing at least one micro-length tube or glass nano reactor functionalized to capture nucleic acid, the device constructed to enable recovery of the nucleic acid captured by the device.

Bead Emulsion Nucleic Acid Amplification

Disclosed are methods for nucleic acid amplification wherein nucleic acid templates, beads, and amplification reaction solution are emulsified and the nucleic acid templates are amplified to provide clonal copies of the nucleic acid templates attached to the beads. Also disclosed are kits and apparatuses for performing the methods of the invention. 144-. (canceled)45. A method of enriching for sequestered populations of amplified nucleic acid molecules , comprising:distributing a solution comprising a plurality of beads and a plurality of species of template nucleic acid molecules into a plurality of aqueous emulsion droplets in a continuous oil phase, wherein a first subset of the droplets comprise one or more of the beads and one or more of the species of template nucleic acid molecule compartmentalized therein, and a second subset of the droplets comprise one or more of the beads without any of the species of template nucleic acid molecules compartmentalized therein;amplifying the species of template nucleic acid molecules within the first subset of the droplets, wherein a plurality of nucleic acid molecules complementary to the species of template nucleic acid molecules are immobilized to the one or more beads within the first subset of the droplets;breaking the aqueous emulsion droplets to release the beads of the first and second subsets;attaching one or more of the beads of the first subset comprising the immobilized complementary copies to one or more enrichment beads, wherein the beads of the first subset are attached to the one or more enrichment beads by hybridizing a capture primer to a portion of one or more of the immobilized complementary nucleic acid molecules and the capture primer is thereafter coupled to the enrichment beads;isolating the one or more enrichment beads away from the beads of the second subset; andseparating the enrichment beads from the beads of the first subset by separating the capture primers from the immobilized complementary ...

BEAD EMULSION NUCLEIC ACID AMPLIFICATION

Disclosed are methods for nucleic acid amplification wherein nucleic acid templates, beads, and amplification reaction solution are emulsified and the nucleic acid templates are amplified to provide clonal copies of the nucleic acid templates attached to the beads. Also disclosed are kits and apparatuses for performing the methods of the invention. 144-. (canceled)45. A method of enriching for sequestered populations of amplified nucleic acid molecules , comprising:distributing a solution comprising a plurality of beads and a plurality of species of template nucleic acid molecules into a plurality of aqueous emulsion droplets in a continuous oil phase, wherein a first subset of the droplets comprise one or more of the beads and one or more of the species of template nucleic acid molecule compartmentalized therein, and a second subset of the droplets comprise one or more of the beads without any of the species of template nucleic acid molecules compartmentalized therein;amplifying the species of template nucleic acid molecules within the first subset of the droplets, wherein a plurality of nucleic acid molecules complementary to the species of template nucleic acid molecules are immobilized to the one or more beads within the first subset of the droplets;breaking the aqueous emulsion droplets to release the beads of the first and second subsets;attaching one or more of the beads of the first subset comprising the immobilized complementary copies to one or more enrichment beads, wherein the beads of the first subset are attached to the one or more enrichment beads by hybridizing a capture primer to a portion of one or more of the immobilized complementary nucleic acid molecules and the capture primer is thereafter coupled to the enrichment beads;isolating the one or more enrichment beads away from the beads of the second subset; andseparating the enrichment beads from the beads of the first subset by separating the capture primers from the immobilized complementary ...

Peristaltic pumping of fluids and associated methods, systems, and devices

Embodiments described herein generally relate to apparatuses, cartridges, and pumps for peristaltic pumping of fluids and associated methods, systems, and devices. The pumping of fluids is, in certain cases, an important aspect of a variety of applications, such as bioanalytical applications (e.g., biological sample analysis, sequencing, identification). The inventive features described herein may, in some embodiments, provide an ability to pump fluids in ways that combine certain advantages of robotic fluid handling systems (e.g., automation, programmability, configurability, flexibility) with certain advantages of microfluidics (e.g., small fluid volumes with high fluid resolution, precision, monolithic consumables, limiting of the wetting of components to consumables).

PERISTALTIC PUMPING OF FLUIDS FOR BIOANALYTICAL APPLICATIONS AND ASSOCIATED METHODS, SYSTEMS, AND DEVICES

Embodiments described herein generally relate to apparatuses, cartridges, and pumps for peristaltic pumping of fluids and associated methods, systems, and devices. The pumping of fluids is, in certain cases, an important aspect of a variety of applications, such as bioanalytical applications (e.g., biological sample analysis, sequencing, identification). The inventive features described herein may, in some embodiments, provide an ability to pump fluids in ways that combine certain advantages of robotic fluid handling systems (e.g., automation, programmability, configurability, flexibility) with certain advantages of microfluidics (e.g., small fluid volumes with high fluid resolution, precision, monolithic consumables, limiting of the wetting of components to consumables). 1. An apparatus comprising a roller and a crank-and-rocker mechanism connected to the roller , for performing at least one of the following on a sample: preparing the sample for analysis , analyzing the sample , and sequencing at least a portion of the sample.2. The apparatus of claim 1 , for preparing the sample for analysis.3. The apparatus of claim 1 , for analyzing the sample.4. The apparatus of claim 1 , for sequencing at least a portion of the sample.5. The apparatus of claim 1 , wherein the sequencing comprises nucleic acid sequencing.6. The apparatus of claim 1 , wherein the sequencing comprises deoxyribonucleic acid (DNA) sequencing.7. (canceled)8. The apparatus of claim 1 , wherein the sequencing comprises peptide sequencing.9. The apparatus of claim 1 , wherein the sequencing comprises protein sequencing.10. The apparatus of claim 1 , wherein one or more of preparing the sample for analysis claim 1 , analyzing the sample claim 1 , and sequencing at least a portion of the sample comprises performing a polymerase chain reaction (PCR) claim 1 , cell culturing claim 1 , performing emulsion-based assays claim 1 , performing array-based diagnostics claim 1 , and/or performing reagent ...

Systems and methods for sample preparation

Methods and devices for isolating or enriching target molecules from a sample are provided herein. In some embodiments, methods and devices further involve detection, analysis and/or sequencing of a target molecule.

CENTRIFUGE AND METHOD FOR LOADING A DEVICE

A sample preparation apparatus includes a robotic system providing movement in three orthogonal directions to an arm operable to receive a pipette tip and to facilitate movement of fluid into and out of the pipette tip. Optionally, the robot can include a gripper arm in addition to the pipette receiving arm. In addition, the sample preparation apparatus can include a tray for receiving pipette tips, receptacles for receiving tubes, an apparatus for forming an emulsion, a device for forming particles that include copies of the polynucleotide, a device for enriching the particles, as well as a centrifuge for loading such particles onto a sensor array. The sample preparation apparatus can further include receptacles for holding containers of reagent solutions. 1. (canceled)2. A method for automatically performing sample preparation on an integrated apparatus , comprising:preparing a particle sample in a first location of the integrated apparatus; wherein the particle sample includes a mixture of polynucleotide-enhanced particles and of particles lacking a polynucleotide;forming a polynucleotide-enhanced particle sample in a second location of the integrated apparatus by enriching the particle sample, andloading a sequencing device with the polynucleotide-enhanced particle sample in a third location of the integrated apparatus.3. The method of claim 2 , wherein preparing the particle sample comprises forming the polynucleotide-enhanced particles by amplifying the particle sample.4. The method of claim 3 , wherein forming the polynucleotide-enhanced particles comprises forming at least one target polynucleotide bound to a particle.5. The method of claim 4 , wherein forming at least one target polynucleotide bound to a particle includes using a polymerase chain reaction (PCR).6. The method of claim 4 , wherein forming at least one target polynucleotide bound to a particle includes using a recombinase polymerase amplification (RPA).7. The method of claim 3 , wherein ...

METHODS AND APPARATUS FOR MEASURING ANALYTES USING LARGE SCALE FET ARRAYS

Methods and apparatus relating to very large scale FET arrays for analyte measurements. ChemFET (e.g., ISFET) arrays may be fabricated using conventional CMOS processing techniques based on improved FET pixel and array designs that increase measurement sensitivity and accuracy, and at the same time facilitate significantly small pixel sizes and dense arrays. Improved array control techniques provide for rapid data acquisition from large and dense arrays. Such arrays may be employed to detect a presence and/or concentration changes of various analyte types in a wide variety of chemical and/or biological processes. In one example, chemFET arrays facilitate DNA sequencing techniques based on monitoring changes in the concentration of inorganic pyrophosphate (PPi), hydrogen ions, and nucleotide triphosphates. 123.-. (canceled)24. A method of making a bead array , the method comprising:{'sup': '3', 'contacting a substrate having a surface comprising discrete reaction sites with a solution including a population of different beads, the surface includes 10of the discrete reaction sites; and'}applying centrifugal force to the substrate or the solution, or both, such that at least a subpopulation of the population of different beads are randomly associated with the discrete reaction sites.25. The method of claim 24 , wherein the surface of the substrate includes 10of the discrete reaction sites.26. The method of claim 25 , wherein the surface of the substrate includes 10of the discrete reaction sites.27. The method of claim 24 , wherein a center distance between adjacent reaction sites of the reaction sites is in a range of 1 micrometers to 9 micrometers.28. The method of claim 24 , wherein a ratio of the diameter of a reaction site of the discrete reaction sites to a diameter of a bead of the population of different beads is at least 0.7.29. The method of claim 24 , wherein a ratio of the height to the width or diameter of a reaction site of the discrete reaction sites is ...

Systems and methods for sample preparation

Methods and devices for isolating or enriching target molecules from a sample are provided herein. In some embodiments, methods and devices further involve detection, analysis and/or sequencing of a target molecule.

METHODS AND DEVICES FOR SEQUENCING

Methods and devices for enriching and analyzing target molecules from a sample are provided herein. In some embodiments, methods and devices involve enrichment of target molecules (e.g., using SCODA) and subsequent detection and analysis using sequencing. 1. A device for analyzing a target molecule from a biological sample , the device comprising an automated sample preparation module connected to a sequencing module ,wherein the automated sample preparation module comprises a cartridge housing that is configured to receive a removable cartridge.2. The device of claim 1 , wherein the removable cartridge is a single-use cartridge or a multi-use cartridge.3. The device of claim 1 , wherein the removable cartridge is configured to receive the biological sample claim 1 , optionally wherein the removable cartridge further comprises the biological sample.4. The device of claim 1 , wherein the automated sample preparation module is directly connected or indirectly connected to the sequencing module.5. The device of claim 1 , wherein the cartridge comprises one or more microfluidic channels configured to contain and/or transport a fluid used in a sample preparation process.6. The device of claim 1 , wherein the cartridge comprises one or more affinity matrices claim 1 , wherein each affinity matrix comprises an immobilized capture probe that has a binding affinity for the target molecule.7. The device of claim 1 , wherein the biological sample is a blood claim 1 , saliva claim 1 , sputum claim 1 , feces claim 1 , urine or buccal swab sample.8. The device of claim 1 , wherein the target molecule is a target nucleic acid.9. (canceled)10. The device of claim 6 , wherein the immobilized capture probe is an oligonucleotide capture probe claim 6 , and wherein the oligonucleotide capture probe comprises a sequence that is at least partially complementary to a target nucleic acid.11. The device of claim 10 , wherein the oligonucleotide capture probe comprises a sequence that is at ...

METHODS AND DEVICES FOR SEQUENCING

Methods and devices for enriching and analyzing target molecules from a sample are provided herein. In some embodiments, methods and devices involve enrichment of target molecules (e.g., using SCODA) and subsequent detection and analysis using sequencing. 1. A device for analyzing a target molecule from a biological sample , the device comprising an automated sample preparation module connected to a sequencing module ,wherein the automated sample preparation module comprises a cartridge housing that is configured to receive a removable cartridge.2. The device of claim 1 , wherein the removable cartridge is a single-use cartridge or a multi-use cartridge.3. The device of claim 1 , wherein the removable cartridge is configured to receive the biological sample claim 1 , optionally wherein the removable cartridge further comprises the biological sample.4. The device of claim 1 , wherein the automated sample preparation module is directly connected or indirectly connected to the sequencing module.5. The device of claim 1 , wherein the cartridge comprises one or more microfluidic channels configured to contain and/or transport a fluid used in a sample preparation process.6. The device of claim 1 , wherein the cartridge comprises one or more affinity matrices claim 1 , wherein each affinity matrix comprises an immobilized capture probe that has a binding affinity for the target molecule.7. The device of claim 1 , wherein the biological sample is a blood claim 1 , saliva claim 1 , sputum claim 1 , feces claim 1 , urine or buccal swab sample.8. The device of claim 1 , wherein the target molecule is a target nucleic acid.9. (canceled)10. The device of claim 6 , wherein the immobilized capture probe is an oligonucleotide capture probe claim 6 , and wherein the oligonucleotide capture probe comprises a sequence that is at least partially complementary to a target nucleic acid.11. The device of claim 10 , wherein the oligonucleotide capture probe comprises a sequence that is at ...

SCAFFOLDED NUCLEIC ACID POLYMER PARTICLES AND METHODS OF MAKING AND USING

The invention provides particle compositions having applications in nucleic acid analysis. Nucleic acid polymer particles of the invention allow polynucleotides to be attached throughout their volumes for higher loading capacities than those achievable solely with surface attachment. In one aspect, nucleic acid polymer particles of the invention comprise polyacrylamide particles with uniform size distributions having low coefficients of variations, which result in reduced particle-to-particle variation in analytical assays. Such particle compositions are used in various amplification reactions to make amplicon libraries from nucleic acid fragment libraries. 1. A method of sequencing a template nucleic acid comprising:{'sup': 4', '3, '(a) disposing a particle in a reaction chamber comprising or capacitively coupled to a field effect transistor, wherein the particle includes a non-nucleosidic polymer network and a plurality of template nucleic acids attached to the non-nucleosidic polymer network through its volume at an average density of at least 6.9×10per μm, the particle having a coefficient of variance of volume of not greater than 15%, the non-nucleosidic polymer network includes a polyacrylamide gel having a total monomer percentage in a range of 3% to 20% and being permeable to proteins having a size in the range of 50 kilodaltons to 200 kilodaltons;'}(b) performing a polymerase extension reaction on a primer annealed to one or more of the plurality of template nucleic acids to incorporate a nucleotide into the primer; and(c) detecting a signal output in the field effect transistor indicative of the incorporation of the nucleotide into the primer.2. The method of claim 1 , further including disposing a plurality of particles into a plurality of reaction chambers in an array of reaction chambers claim 1 , wherein at least one of the reaction chambers of the plurality of reaction chambers includes or is operatively coupled to a field effect transistor.3. The ...

Scaffolded nucleic acid polymer particles and methods of making and using

The invention provides particle compositions having applications in nucleic acid analysis. Nucleic acid polymer particles of the invention allow polynucleotides to be attached throughout their volumes for higher loading capacities than those achievable solely with surface attachment. In one aspect, nucleic acid polymer particles of the invention comprise polyacrylamide particles with uniform size distributions having low coefficients of variations, which result in reduced particle-to-particle variation in analytical assays. Such particle compositions are used in various amplification reactions to make amplicon libraries from nucleic acid fragment libraries.

MULTIPLEX TRANSCRIPTOME ANALYSIS

In some embodiments, the disclosure relates generally to methods, compositions, systems, apparatuses and kits comprising a multiplex nucleic acid amplification reaction that employs a plurality (e.g., hundreds, thousands, tens-of-thousands or hundreds-of-thousands) of different target-specific primer pairs that enable substantially simultaneous amplification of a plurality of different target sequences-of-interest in a single reaction mixture. In some embodiments, the multiplex nucleic acid amplification reaction generates a plurality of amplicons having sequences derived from a sample containing RNA or DNA, including whole transcriptome or genomic samples. In some embodiments, the sequences and abundances of at least some of the plurality of amplicons are characterized, optionally simultaneously or through a single assay, by suitable detection methods, including sequencing or other procedures known in the art. 1. A method for detecting a plurality of polynucleotides in a sample , comprising:a) contacting, within a single reaction mixture, a plurality of target-specific primer pairs with a plurality of target polynucleotides derived from the sample, under nucleic acid hybridization conditions such that different target-specific primer pairs hybridize to different target polynucleotides, wherein the different target-specific primer pairs include at least one cleavable group;b) extending the target-specific primer pairs to form a plurality of amplicons which contain a sequence derived from a target polynucleotide and a primer-derived sequence which includes the at least one cleavable group;c) cleaving the at least one cleavable group of the plurality of amplicons to generate a plurality of cleaved amplicons; andd) joining one or both ends of the plurality of cleaved amplicons to a Y-shaped adaptor to form a plurality of adaptor-joined amplicons.2. The method of claim 1 , further comprising: (e) detecting the adaptor-joined amplicons.3. The method of claim 2 , wherein ...

BEAD EMULSION NUCLEIC ACID AMPLIFICATION

Disclosed are methods for nucleic acid amplification wherein nucleic acid templates, beads, and amplification reaction solution are emulsified and the nucleic acid templates are amplified to provide clonal copies of the nucleic acid templates attached to the beads. Also disclosed are kits and apparatuses for performing the methods of the invention. 144.-. (canceled)45. A composition comprising a population of nucleic acid template-carrying beads each comprising a plurality of species of template nucleic acid molecules , wherein each bead comprises more than 10 ,000 amplified copies of the species of template nucleic acid molecules attached thereto , wherein the population of beads are suspended in a microreactor comprising a water-in-oil emulsion , and wherein a plurality of the microreactors includes only one bead.46. The composition of claim 45 , wherein the attachment of each of the amplified copies of the species of template nucleic acid molecules to the bead is mediated by chemical groups or oligonucleotides that are bound to the surface of the bead.47. The composition of claim 46 , wherein the attachment of each of the amplified copies of the species of template nucleic acid molecules is a covalent chemical attachment.48. The composition of claim 46 , wherein the attachment of each of the amplified copies of the species of template nucleic acid molecules is a non-covalent attachment.49. The composition of claim 45 , wherein each bead comprises a single nucleic acid species.50. The composition of claim 45 , wherein the beads are made from a material selected from the group consisting of cellulose claim 45 , cellulose derivatives claim 45 , acrylic resins claim 45 , glass claim 45 , silica gels claim 45 , polystyrene claim 45 , gelatin claim 45 , polyvinyl pyrrolidone claim 45 , co-polymers of vinyl and acrylamide claim 45 , polystyrene cross-linked with divinylbenzene claim 45 , polyacrylamide latex gels claim 45 , polystyrene claim 45 , dextran claim 45 , ...

METHODS AND COMPOSITIONS FOR MULTIPLEX PCR

The present invention provides methods, compositions, kits, systems and apparatus that are useful for multiplex PCR of one or more nucleic acids present in a sample. In particular, various target-specific primers are provided that allow for the selective amplification of one or more target sequences. In one aspect, the invention relates to target-specific primers useful for the selective amplification of one or more target sequences associated with cancer or inherited disease. In some aspects, amplified target sequences obtained using the disclosed methods, kits, systems and apparatuses can be used in various downstream processes including nucleic acid sequencing and used to detect the presence of genetic variants. 1. A method for amplifying a plurality of different target sequences within a sample , comprising: cleaving a cleavable group of at least one amplified target sequence;', 'ligating at least one adapter to at least one amplified target sequence in a blunt-ended ligation reaction, thereby producing one or more adapter-ligated amplified target sequences, and', 'reamplifying at least one of the adapter-ligated amplified target sequences., 'amplifying within a single amplification reaction mixture at least one hundred different target sequences from a sample including a plurality of different target sequences, wherein the amplifying includes contacting at least some portion of the sample with a plurality of target-specific primers, and a polymerase under amplification conditions, thereby producing at least one hundred different amplified target sequences, wherein at least two of the different amplified target sequences are less than 50% complementary to each other and wherein at least one of the plurality of target-specific primers and at least one of the amplified target sequences includes a cleavable group;'}2. The method of claim 1 , wherein one or more of the at least one adapter is not substantially complementary to at least one amplified target sequence. ...

METHODS AND COMPOSITIONS FOR MULTIPLEX PCR

The present invention provides methods, compositions, kits, systems and apparatus that are useful for multiplex PCR of one or more nucleic acids present in a sample. In particular, various target-specific primers are provided that allow for the selective amplification of one or more target sequences. In one aspect, the invention relates to target-specific primers useful for the selective amplification of one or more target sequences associated with cancer or inherited disease. In some aspects, amplified target sequences obtained using the disclosed methods, kits, systems and apparatuses can be used in various downstream processes including nucleic acid sequencing and used to detect the presence of genetic variants. 1. A method for amplifying a plurality of different target sequences within a sample , comprising: cleaving a cleavable group of at least one amplified target sequence;', 'ligating at least one adapter to at least one amplified target sequence in a blunt-ended ligation reaction, thereby producing one or more adapter-ligated amplified target sequences, and', 'reamplifying at least one of the adapter-ligated amplified target sequences., 'amplifying within a single amplification reaction mixture at least one hundred different target sequences from a sample including a plurality of different target sequences, wherein the amplifying includes contacting at least some portion of the sample with a plurality of target-specific primers, and a polymerase under amplification conditions, thereby producing at least one hundred different amplified target sequences, wherein at least two of the different amplified target sequences are less than 50% complementary to each other and wherein at least one of the plurality of target-specific primers and at least one of the amplified target sequences includes a cleavable group;'}2. The method of claim 1 , wherein one or more of the at least one adapter is not substantially complementary to at least one amplified target sequence. ...

SCAFFOLDED NUCLEIC ACID POLYMER PARTICLES AND METHODS OF MAKING AND USING

The invention provides particle compositions having applications in nucleic acid analysis. Nucleic acid polymer particles of the invention allow polynucleotides to be attached throughout their volumes for higher loading capacities than those achievable solely with surface attachment. In one aspect, nucleic acid polymer particles of the invention comprise polyacrylamide particles with uniform size distributions having low coefficients of variations, which result in reduced particle-to-particle variation in analytical assays. Such particle compositions are used in various amplification reactions to make amplicon libraries from nucleic acid fragment libraries. 1. A method of sequencing a template nucleic acid comprising:(a) disposing a particle in a reaction confinement region associated with an electronic-based sensor, wherein the particle includes a non-nucleosidic polymer network and a plurality of template nucleic acids attached to the non-nucleosidic polymer network, the particle having a coefficient of variance of volume of not greater than 15%, the non-nucleosidic polymer network including polymers selected from the agarose, polyoxybutylene, diethylacrylamide, polyoxyethylene, polyacrylamide, polyoxypropylene, N,N-polydimethylacrylamide, poly(N-isopropylacrylamide), polyvinylpyrrolidone, or poly-N-hydroxyacrylamide, the polymer being permeable to proteins having a size in the range of 50 kilodaltons to 200 kilodaltons;(b) performing a polymerase extension reaction on a primer annealed to one or more of the plurality of template nucleic acids to incorporate a nucleotide into the primer; and(c) detecting a signal output in the field effect transistor indicative of the incorporation of the nucleotide into the primer.2. The method of claim 1 , further including disposing a plurality of particles into a plurality of reaction confinement regions in an array of reaction confinement regions claim 1 , wherein each of the reaction confinement regions of the plurality of ...

TERMINAL FUNCTIONALIZATION OF TARGET MOLECULES FOR SEQUENCING

Methods and devices for preparing target molecules (e.g., target nucleic acids or target proteins) from a biological sample are provided herein. In some embodiments, methods and devices involve sample lysis, sample fragmentation, enrichment of target molecule(s), and/or functionalization of target molecule(s). 2. The device of claim 1 , wherein the biological sample is a single cell claim 1 , mammalian cell tissue claim 1 , animal sample claim 1 , fungal sample claim 1 , plant sample claim 1 , blood sample claim 1 , saliva sample claim 1 , sputum sample claim 1 , fecal sample claim 1 , urine sample claim 1 , buccal swab sample claim 1 , amniotic sample claim 1 , seminal sample claim 1 , synovial sample claim 1 , spinal sample claim 1 , or pleural fluid sample.3. The device of claim 1 , wherein the one or more target molecules are nucleic acids or proteins.4. The device of claim 1 , wherein the one or more microfluidic channels are configured to contain and/or transport fluid(s) and/or reagent(s).5. The device of claim 1 , wherein the functionalization cartridge comprises a first chamber comprising reagents that covalently modify a moiety Mof the one or more target molecules claim 1 , or of one or more fragments thereof claim 1 , to a modified moiety M.6. The device of claim 5 , wherein the reagents are non-enzymatic.7. The device of claim 5 , wherein the covalent modification is regiospecific.8. The device of claim 1 , wherein the portion of the one or more target molecules claim 1 , or of the one or more fragments thereof claim 1 , is a C-terminal carboxylate group or a C-terminal amino group.9. The device of claim 5 , wherein the reagents comprise buffers claim 5 , salts claim 5 , organic compounds claim 5 , acids claim 5 , and/or bases.10. The device of claim 5 , wherein the portion of the one or more target molecules claim 5 , or of the one or more fragments thereof claim 5 , is a C-terminal amino group claim 5 , and the covalent modification is diazo transfer. ...

Bead Emulsion Nucleic Acid Amplification

Disclosed are methods for nucleic acid amplification wherein nucleic acid templates, beads, and amplification reaction solution are emulsified and the nucleic acid templates are amplified to provide clonal copies of the nucleic acid templates attached to the beads. Also disclosed are kits and apparatuses for performing the methods of the invention. 144-. (canceled)45. A method of enriching for sequestered populations of amplified nucleic acid molecules , comprising:distributing a solution comprising a plurality of beads and a plurality of species of template nucleic acid molecules into a plurality of aqueous emulsion droplets in a continuous oil phase, wherein a first subset of the droplets comprise one or more of the beads and one or more of the species of template nucleic acid molecule compartmentalized therein, and a second subset of the droplets comprise one or more of the beads without any of the species of template nucleic acid molecules compartmentalized therein;amplifying the species of template nucleic acid molecules within the first subset of the droplets, wherein a plurality of nucleic acid molecules complementary to the species of template nucleic acid molecules are immobilized to the one or more beads within the first subset of the droplets;breaking the aqueous emulsion droplets to release the beads of the first and second sub sets;attaching one or more of the beads of the first subset comprising the immobilized complementary copies to one or more enrichment beads, wherein the beads of the first subset are attached to the one or more enrichment beads by hybridizing a capture primer to a portion of one or more of the immobilized complementary nucleic acid molecules and the capture primer is thereafter coupled to the enrichment beads;isolating the one or more enrichment beads away from the beads of the second subset; andseparating the enrichment beads from the beads of the first subset by separating the capture primers from the immobilized complementary ...

METHODS AND COMPOSITIONS FOR MULTIPLEX PCR

The present invention provides methods, compositions, kits, systems and apparatus that are useful for multiplex PCR of one or more nucleic acids present in a sample. In particular, various target-specific primers are provided that allow for the selective amplification of one or more target sequences. In one aspect, the invention relates to target-specific primers useful for the selective amplification of one or more target sequences associated with cancer or inherited disease. In some aspects, amplified target sequences obtained using the disclosed methods, kits, systems and apparatuses can be used in various downstream processes including nucleic acid sequencing and used to detect the presence of genetic variants. 1. A method of amplifying a plurality of different target nucleic acid molecules comprising:a) contacting a plurality of different target nucleic acid molecules with a pool of target-specific primers having a cleavable group, and a polymerase under synthesizing conditions, and synthesizing a plurality of different target-specific molecules by extending one or more of the pool of target-specific primers having the cleavable group;b) digesting a plurality of the different target-specific molecules having the cleavable group of the target-specific primer;c) ligating at least one adapter to at least one of the plurality of digested different target-specific molecules; andd) producing at least one adapter-ligated amplified target sequence, wherein the method includes using no more than two target-specific primers to produce any one of the adapter-ligated amplified target sequences.2. The method of claim 1 , wherein the cleavable group includes methylguanine claim 1 , 8-oxo-guanine claim 1 , xanthine claim 1 , hypoxanthine claim 1 , 5 claim 1 ,6-dihydrouracil claim 1 , uracil claim 1 , 5-methylcytosine claim 1 , thymine-dimer claim 1 , 7-methylguanosine claim 1 , 8-oxo-deoxyguanosine claim 1 , xanthosine claim 1 , inosine claim 1 , dihydrouridine claim 1 , ...

METHODS AND COMPOSITIONS FOR MULTIPLEX PCR

The present invention provides methods, compositions, kits, systems and apparatus that are useful for multiplex PCR of one or more nucleic acids present in a sample. In particular, various target-specific primers are provided that allow for the selective amplification of one or more target sequences. In one aspect, the invention relates to target-specific primers useful for the selective amplification of one or more target sequences associated with cancer or inherited disease. In some aspects, amplified target sequences obtained using the disclosed methods, kits, systems and apparatuses can be used in various downstream processes including nucleic acid sequencing and used to detect the presence of genetic variants. 1. A method of generating an amplicon library from genomic DNA comprising:a) contacting a portion of a genomic DNA with a plurality of different target-specific primers having a cleavable group, and a polymerase under amplification conditions and producing a plurality of different target-specific amplicons by extending one or more of the plurality of target-specific primers having a cleavable group;b) digesting at least one of the plurality of different target-specific amplicons with a cleaving reagent; andc) ligating an adapter to the end of the at least one digested different target-specific amplicon and producing at least one adapter-ligated target-specific amplicon, wherein the adapter is not complementary to 20 contiguous nucleotides from a 3′ end or 5′ end of the at least one digested different target-specific amplicon.2. The method of claim 1 , wherein the cleavable group includes methylguanine claim 1 , 8-oxo-guanine claim 1 , xanthine claim 1 , hypoxanthine claim 1 , 5 claim 1 ,6-dihydrouracil claim 1 , uracil claim 1 , 5-methylcytosine claim 1 , thymine-dimer claim 1 , 7-methylguanosine claim 1 , 8-oxo-deoxyguanosine claim 1 , xanthosine claim 1 , inosine claim 1 , dihydrouridine claim 1 , bromodeoxyuridine claim 1 , uridine or 5-methylcytidine. ...

PDMS Membrane-Confined Nucleic Acid and Antibody/Antigen-Functionalized Microlength Tube Capture Elements, and Systems Employing Them, and Methods of Their Use

A method for performing a combined protein and nucleic acid assay on a target captured by a capture agent, includes providing a microfluidic device having a microfluidic channel network having at least one microfluidic channel, the channel arranged to receive fluid, the device having at least two micro-particles disposed in fixed position in the channel, the micro-particles being functionalized with a capture agent for the assay, one of the micro-particles in the channel being functionalized with an antibody or antigen capture agent and another of the micro-particles being functionalized with a nucleic acid capture agent. In some embodiments, the network may have at least two microfluidic channels, each channel of the two channels arranged to receive portions of the same fluid and to be fluidicly isolatable from each other, the device having at least two micro-particles disposed in fixed position in the network channels, the micro-particles being functionalized with a capture agent, one of the micro-particles in one of the channels being functionalized with an antibody or antigen capture agent and another of the micro-particles in another of the channels being functionalized with a nucleic acid capture agent. The method may also include detecting both protein and nucleic acid present in an input sample using the respectively functionalized micro particles. In some embodiments, the micro particles may be micro-length tubes or glass nano reactors. 1. A method for performing a combined protein and nucleic acid assay , comprising:providing a microfluidic device having a microfluidic channel network having at least one microfluidic channel, the channel arranged to receive fluid, the device having at least two micro-particles disposed in fixed position in the channel, the micro-particles being functionalized with a capture agent for the assay, one of the micro-particles in the channel being functionalized with an antibody or antigen capture agent and another of the micro- ...

Methods and compositions for multiplex pcr

The present invention provides methods, compositions, kits, systems and apparatus that are useful for multiplex PCR of one or more nucleic acids present in a sample. In particular, various target-specific primers are provided that allow for the selective amplification of one or more target sequences. In one aspect, the invention relates to target-specific primers useful for the selective amplification of one or more target sequences associated with cancer or inherited disease. In some aspects, amplified target sequences obtained using the disclosed methods, kits, systems and apparatuses can be used in various downstream processes including nucleic acid sequencing and used to detect the presence of genetic variants.

MULTIPLEX TRANSCRIPTOME ANALYSIS

In some embodiments, the disclosure relates generally to methods, compositions, systems, apparatuses and kits comprising a multiplex nucleic acid amplification reaction that employs a plurality (e.g., hundreds, thousands, tens-of-thousands or hundreds-of-thousands) of different target-specific primer pairs that enable substantially simultaneous amplification of a plurality of different target sequences-of-interest in a single reaction mixture. In some embodiments, the multiplex nucleic acid amplification reaction generates a plurality of amplicons having sequences derived from a sample containing RNA or DNA, including whole transcriptome or genomic samples. In some embodiments, the sequences and abundances of at least some of the plurality of amplicons are characterized, optionally simultaneously or through a single assay, by suitable detection methods, including sequencing or other procedures known in the art. 1. A method for detecting a plurality of polynucleotides in a sample , comprising:a) contacting, within a single reaction mixture, a plurality of target-specific primer pairs with a plurality of target polynucleotides derived from a sample, under nucleic acid hybridization conditions such that different target-specific primer pairs hybridize to different target polynucleotides;b) extending the target-specific primer pairs in a template-dependent fashion and forming a plurality of amplicons which contain a sequence derived from a target polynucleotide and a primer-derived sequence; andc) detecting the amplicons.2. The method of claim 1 , wherein the sample includes RNA or cDNA derived from one or more cells.3. The method of claim 1 , wherein the plurality of target-specific primer pairs are non-tailed primer pairs.4. The method of claim 1 , wherein at least one primer in the plurality of target-specific primer pairs includes at least one cleavable group.5. The method of claim 4 , wherein the primer-derived sequence in step (b) includes at least one cleavable ...

Methods of amplifying and sequencing nucleic acids

An apparatus and method for performing rapid DNA sequencing, such as genomic sequencing, is provided herein. The method includes the steps of preparing a sample DNA for genomic sequencing, amplifying the prepared DNA in a representative manner, and performing multiple sequencing reaction on the amplified DNA with only one primer hybridization step.

Methods of amplifying and sequencing nucleic acids

An apparatus and method for performing rapid DNA sequencing, such as genomic sequencing, is provided herein. The method includes the steps of preparing a sample DNA for genomic sequencing, amplifying the prepared DNA in a representative manner, and performing multiple sequencing reaction on the amplified DNA with only one primer hybridization step.

Methods of amplifying and sequencing nucleic acids

An apparatus and method for performing rapid DNA sequencing, such as genomic sequencing, is provided herein. The method includes the steps of preparing a sample DNA for genomic sequencing, amplifying the prepared DNA in a representative manner, and performing multiple sequencing reaction on the amplified DNA with only one primer hybridization step.

Methods of amplifying and sequencing nucleic acids

An apparatus and method for performing rapid DNA sequencing, such as genomic sequencing, is provided herein. The method includes the steps of preparing a sample DNA for genomic sequencing, amplifying the prepared DNA in a representative manner, and performing multiple sequencing reaction on the amplified DNA with only one primer hybridization step.

Methods of amplifying and sequencing nucleic acids

An apparatus and method for performing rapid DNA sequencing, such as genomic sequencing, is provided herein. The method includes the steps of preparing a sample DNA for genomic sequencing, amplifying the prepared DNA in a representative manner, and performing multiple sequencing reaction on the amplified DNA with only one primer hybridization step.

Methods of amplifying and sequencing nucleic acids

An apparatus and method for performing rapid DNA sequencing, such as genomic sequencing, is provided herein. The method includes the steps of preparing a sample DNA for genomic sequencing, amplifying the prepared DNA in a representative manner, and performing multiple sequencing reaction on the amplified DNA with only one primer hybridization step.

Methods of amplifying and sequencing nucleic acids

An apparatus and method for performing rapid DNA sequencing, such as genomic sequencing, is provided herein. The method includes the steps of preparing a sample DNA for genomic sequencing, amplifying the prepared DNA in a representative manner, and performing multiple sequencing reaction on the amplified DNA with only one primer hybridization step.

Methods of amplifying and sequencing nucleic acids

An apparatus and method for performing rapid DNA sequencing, such as genomic sequencing, is provided herein. The method includes the steps of preparing a sample DNA for genomic sequencing, amplifying the prepared DNA in a representative manner, and performing multiple sequencing reaction on the amplified DNA with only one primer hybridization step.

Double ended sequencing

This invention relates to methods of sequencing DNA. More specifically, this invention relates to methods of sequencing both the sense and antisense strands of DNA through the use of blocked and unblocked sequencing primers. In brief, these methods include the steps of annealing an unblocked primer to a first strand of nucleic acid; annealing a second blocked primer to a second strand of nucleic acid; elongating the nucleic acid along the first strand with a polymerase; terminating the first sequencing primer; deblocking the second primer; and elongating the nucleic acid along the second strand.

Nucleic acid amplification with continuous flow emulsion

Embodiments of the present invention are directed to methods and devices/systems for amplifying genetic material and may include providing a water-in-oil emulsion in a continuous flow. The emulsion may include a plurality of water droplets comprising microreactors. Each of the plurality of microreactors may include a single bead capable of capturing a nucleic acid template, a single species nucleic acid template and sufficient reagents to amplify the copy number of the nucleic acid template. The method also includes flowing the emulsion across a first temperature zone and a second lower temperature zone to thermally process the microreactors to amplify the nucleic acid template by polymerase chain reaction.

Nucleic acid amplification with continuous flow emulsion

Embodiments of the present invention are directed to methods and devices/systems for amplifying genetic material and may include providing a water-in-oil emulsion in a continuous flow. The emulsion may include a plurality of water droplets comprising microreactors. Each of the plurality of microreactors may include a single bead capable of capturing a nucleic acid template, a single species nucleic acid template and sufficient reagents to amplify the copy number of the nucleic acid template. The method also includes flowing the emulsion across a first temperature zone and a second lower temperature zone to thermally process the microreactors to amplify the nucleic acid template by polymerase chain reaction.

Methods and compositions for multiplex pcr

The present invention provides methods, compositions, kits, systems and apparatus that are useful for multiplex PCR of one or more nucleic acids present in a sample. In particular, various target-specific primers are provided that allow for the selective amplification of one or more target sequences. In one aspect, the invention relates to target-specific primers useful for the selective amplification of one or more target sequences associated with cancer or inherited disease. In some aspects, amplified target sequences obtained using the disclosed methods, kits, systems and apparatuses can be used in various downstream processes including nucleic acid sequencing and used to detect the presence of genetic variants.

Methods and compositions for multiplex pcr

The present invention provides methods, compositions, kits, systems and apparatus that are useful for multiplex PCR of one or more nucleic acids present in a sample. In particular, various target-specific primers are provided that allow for the selective amplification of one or more target sequences. In one aspect, the invention relates to target-specific primers useful for the selective amplification of one or more target sequences associated with cancer or inherited disease. In some aspects, amplified target sequences obtained using the disclosed methods, kits, systems and apparatuses can be used in various downstream processes including nucleic acid sequencing and used to detect the presence of genetic variants.

Methods and compositions for multiplex pcr

The present invention provides methods, compositions, kits, systems and apparatus that are useful for determining copy number variation of one or more nucleic acids present in a sample. In some aspects, the method includes various target-specific primers that allow for the selective amplification of one or more target nucleic acids in the sample. In yet another aspect, the invention relates to determining copy number variation with respect to gene or chromosome representation of a nucleic acid in the sample. In some aspects, the method for determining copy number variation of different target nucleic acids in a sample using the disclosed methods, kits, systems and apparatuses can be used in various downstream processes including diagnosis, predictive therapeutic regimes or other therapeutic purposes.

Methods and compositions for multiplex pcr

The present invention provides methods, compositions, kits, systems and apparatus that are useful for multiplex PCR of one or more nucleic acids present in a sample. In particular, various target-specific primers are provided that allow for the selective amplification of one or more target sequences. In one aspect, the invention relates to target-specific primers useful for the selective amplification of one or more target sequences associated with cancer or inherited disease. In some aspects, amplified target sequences obtained using the disclosed methods, kits, systems and apparatuses can be used in various downstream processes including nucleic acid sequencing and used to detect the presence of genetic variants.

Methods and compositions for multiplex PCR

The present invention provides methods, compositions, kits, systems and apparatus that are useful for multiplex PCR of one or more nucleic acids present in a sample. In particular, various target-specific primers are provided that allow for the selective amplification of one or more target sequences. In one aspect, the invention relates to target-specific primers useful for the selective amplification of one or more target sequences associated with cancer or inherited disease. In some aspects, amplified target sequences obtained using the disclosed methods, kits, systems and apparatuses can be used in various downstream processes including nucleic acid sequencing and used to detect the presence of genetic variants.

Methods and compositions for multiplex PCR

The present invention provides methods, compositions, kits, systems and apparatus that are useful for multiplex PCR of one or more nucleic acids present in a sample. In particular, various target-specific primers are provided that allow for the selective amplification of one or more target sequences. In one aspect, the invention relates to target-specific primers useful for the selective amplification of one or more target sequences associated with cancer or inherited disease. In some aspects, amplified target sequences obtained using the disclosed methods, kits, systems and apparatuses can be used in various downstream processes including nucleic acid sequencing and used to detect the presence of genetic variants.

PDMS membrane-confined nucleic acid and antibody/antigen-functionalized microlength tube capture elements, and systems employing them, and methods of their use

A microfluidic device comprising a microfluidic channel network sealed on one side by a membrane sheet, the sheet having PDMS defining at least the surface sealing the channel, the membrane sheet on its opposite side sealing one side of a pneumatic channel, the pneumatic channel arranged to enable pneumatic deflection of a deflectable portion of the membrane sheet into contact with an opposed surface to control flow in a channel of the network, the membrane sheet confining in a channel of the network at least one micro-particle, micro-length tube or glass nano reactor, functionalized with a capture agent, that has been inserted into that channel. A microfluidic device having a microfluidic channel containing at least two micro-particles, micro-length tubes or glass nano reactors, one functionalized with nucleic acid and another with antibody or antigen. A microfluidic device having a microfluidic channel containing at least one micro-length tube or glass nano reactor functionalized to capture nucleic acid, the device constructed to enable recovery of the nucleic acid captured by the device.

Methods and apparatus for measuring analytes

Methods and apparatus relating to FET arrays including large FET arrays for monitoring chemical and/or biological reactions such as nucleic acid sequencing-by-synthesis reactions. Some methods provided herein relate to improving signal (and also signal to noise ratio) from released hydrogen ions during nucleic acid sequencing reactions.

Bead emulsion nucleic acid amplification

Nucleic acid amplification with continuous flow emulsion

Embodiments of the present invention are directed to methods and devices/systems for amplifying genetic material and may include providing a water-in-oil emulsion in a continuous flow. The emulsion may include a plurality of water droplets comprising microreactors. Each of the plurality of microreactors may include a single bead capable of capturing a nucleic acid template, a single species nucleic acid template and sufficient reagents to amplify the copy number of the nucleic acid template. The method also includes flowing the emulsion across a first temperature zone and a second lower temperature zone to thermally process the microreactors to amplify the nucleic acid template by polymerase chain reaction.

Methods for sequencing individual nucleic acids under tension

The invention provides apparatuses and methods of use thereof for sequencing nucleic acids subjected to a force, and thus considered under tension. The methods may employ but are not dependent upon incorporation of extrinsically detectably labeled nucleotides.

Scaffolded nucleic acid polymer particles and methods of making and using

The invention provides particle compositions having applications in nucleic acid analysis. Nucleic acid polymer particles of the invention allow polynucleotides to be attached throughout their volumes for higher loading capacities than those achievable solely with surface attachment. In one aspect, nucleic acid polymer particles of the invention comprise polyacrylamide particles with uniform size distributions having low coefficients of variations, which result in reduced particle-to-particle variation in analytical assays. Such particle compositions are used in various amplification reactions to make amplicon libraries from nucleic acid fragment libraries.

Methods and apparatus for measuring analytes using large scale fet arrays

Methods and apparatus relating to very large scale FET arrays for analyte measurements. ChemFET (e.g., ISFET) arrays may be fabricated using conventional CMOS processing techniques based on improved FET pixel and array designs that increase measurement sensitivity and accuracy, and at the same time facilitate significantly small pixel sizes and dense arrays. Improved array control techniques provide for rapid data acquisition from large and dense arrays. Such arrays may be employed to detect a presence and/or concentration changes of various analyte types in a wide variety of chemical and/or biological processes. In one example, chemFET arrays facilitate DNA sequencing techniques based on monitoring changes in the concentration of inorganic pyrophosphate (PPi), hydrogen ions, and nucleotide triphosphates.

Scaffolded nucleic acid polymer particles and methods of making and using

Methods and apparatus for measuring analytes using large scale FET arrays

Methods and apparatus relating to very large scale FET arrays for analyte measurements. ChemFET (e.g., ISFET) arrays may be fabricated using conventional CMOS processing techniques based on improved FET pixel and array designs that increase measurement sensitivity and accuracy, and at the same time facilitate significantly small pixel sizes and dense arrays. Improved array control techniques provide for rapid data acquisition from large and dense arrays. Such arrays may be employed to detect a presence and/or concentration changes of various analyte types in a wide variety of chemical and/or biological processes. In one example, chemFET arrays facilitate DNA sequencing techniques based on monitoring changes in the concentration of inorganic pyrophosphate (PPi), hydrogen ions, and nucleotide triphosphates.

Methods and apparatus for measuring analytes using large scale FET arrays

Methods and apparatus relating to very large scale FET arrays for analyte measurements. ChemFET (e.g., ISFET) arrays may be fabricated using conventional CMOS processing techniques based on improved FET pixel and array designs that increase measurement sensitivity and accuracy, and at the same time facilitate significantly small pixel sizes and dense arrays. Improved array control techniques provide for rapid data acquisition from large and dense arrays. Such arrays may be employed to detect a presence and/or concentration changes of various analyte types in a wide variety of chemical and/or biological processes. In one example, chemFET arrays facilitate DNA sequencing techniques based on monitoring changes in the concentration of inorganic pyrophosphate (PPi), hydrogen ions, and nucleotide triphosphates.

Methods of amplifying and sequencing nucleic acids

An apparatus and method for performing rapid DNA sequencing, such as genomic sequencing, is provided herein. The method includes the steps of preparing a sample DNA for genomic sequencing, amplifying the prepared DNA in a representative manner, and performing multiple sequencing reaction on the amplified DNA with only one primer hybridization step.

Amplification of nucleic acids in pearl emulsion

Un metodo para enriquecer un amplicón, que comprende: (a) distribuir una solución que comprende una pluralidad de perlas y una pluralidad de especies de moleculas de acido nucleico de molde, en una pluralidad de gotitas de emulsión acuosa en una fase oleosa termoestable; en el que un primer subconjunto de las gotitas comprenden una o mas de las perlas y una o mas de las especies de acido nucleico de molde encapsuladas en ellas, y un segundo subconjunto de las gotitas comprenden una o mas de las perlas sin ninguna de las especies de moleculas de acido nucleico de molde encapsuladas en ellas; (b) amplificar las especies de moleculas de acido nucleico de molde del primer subconjunto de gotitas, en el que una o mas perlas del primer subconjunto de gotitas acuosas tienen un amplicón inmovilizado en ellas; (c) romper las gotitas de emulsión acuosa para liberar las perlas del primer y el segundo subconjuntos; (d) hibridar con el amplicón inmovilizado en las perlas un cebador que es complementario del extremo 3' del amplicón; y o bien: (e) (i) formar un complejo de perla con amplicón inmovilizado y perla de enriquecimiento, en el que la perla de enriquecimiento y el cebador hibridado forman un par de captura-diana, y en el que las perlas de enriquecimiento pueden ser aisladas bajo una condición selectiva; y (f) (i) aplicar la condición selectiva para aislar el complejo de perla con amplicón inmovilizado y perla de enriquecimiento, desde las perlas que no tienen amplicón unido; o bien (e) (ii) exponer las perlas con amplicón inmovilizado a una o mas superficies de unión que comprenden restos de captura, cuyos restos de captura forman pares de captura-diana con el cebador hibridado de las perlas con amplicón inmovilizado, y separar las perlas con amplicón inmovilizado desde las perlas que no tienen amplicón unido. A method for enriching an amplicon, comprising: (a) distributing a solution comprising a plurality of beads and a plurality of species of template nucleic acid ...

Methods of Making Libraries of Nucleic Acids Using Porous Particles

The invention provides particle compositions having applications in nucleic acid analysis. Nucleic acid polymer particles of the invention allow polynucleotides to be attached throughout their volumes for higher loading capacities than those achievable solely with surface attachment. In one aspect, nucleic acid polymer particles of the invention comprise polyacrylamide particles with uniform size distributions having low coefficients of variations, which result in reduced particle-to-particle variation in analytical assays. Such particle compositions are used in various amplification reactions to make amplicon libraries from nucleic acid fragment libraries.

Bead emulsion nucleic acid amplification

Disclosed are methods for nucleic acid amplification wherein nucleic acid templates, beads, and amplification reaction solution are emulsified and the nucleic acid templates are amplified to provide clonal copies of the nucleic acid templates attached to the beads. Also disclosed are kits and apparatuses for performing the methods of the invention.

Nucleic acid amplification with continuous flow emulsion

Embodiments of the present invention are directed to methods and devices/systems for amplifying genetic material and may include providing a water-in-oil emulsion in a continuous flow. The emulsion may include a plurality of water droplets comprising microreactors. Each of the plurality of microreactors may include a single bead capable of capturing a nucleic acid template, a single species nucleic acid template and sufficient reagents to amplify the copy number of the nucleic acid template. The method also includes flowing the emulsion across a first temperature zone and a second lower temperature zone to thermally process the microreactors to amplify the nucleic acid template by polymerase chain reaction.

Universal adaptor for sequencing

Methods and compositions for preparing nucleic acid libraries for nucleic acid sequencing are provided. In some embodiments, disclosed herein is a universal nucleic acid adaptor and methods of using same.

Method for preparing single-stranded dna libraries

This invention relates to methods of generating single stranded DNA libraries for use in amplification and sequencing reactions. In various aspects, the disclosed methods include: fragmenting DNA; polishing the fragments' ends; lighting the fragments to universal adapters; performing strand displacement and extension of the nicked fragments; purifying the double-stranded libation products; capturing the double-stranded libation products onto a solid support; and isolating single stranded DNA library fragments, and binding these fragments to another solid support.

Bead emulsion nucleic acid amplification

Disclosed are methods for nucleic acid amplification wherein nucleic acid templates, beads, and amplification reaction solution are emulsified and the nucleic acid templates are amplified to provide clonal copies of the nucleic acid templates attached to the beads. Also disclosed are kits and apparatuses for performing the methods of the invention.

Systems and methods for sample preparation

Methods and devices for isolating or enriching target molecules from a sample using lysis, fragmentation and affinity purification, e.g., using scodaphoresis, are provided herein. In particular embodiments, a device for enriching a target molecule from a biological sample is characterized in that the device comprises an automated sample preparation module comprising a cartridge housing that is configured to receive a removable cartridge. In some embodiments, methods and devices further involve detection, analysis and/or sequencing of a target molecule.

Methods of amplifying and sequencing nucleic acids

An apparatus and method for performing rapid DNA sequencing, such as genomic sequencing, is provided herein. The method includes the steps of preparing a sample DNA for genomic sequencing, amplifying the prepared DNA in a representative manner, and performing multiple sequencing reaction on the amplified DNA with only one primer hybridization step.

Methods and compositions for multiplex pcr

Methods and compositions for multiplex PCR

The present invention provides methods, compositions, kits, systems and apparatus that are useful for multiplex PCR of one or more nucleic acids present in a sample. In particular, various target-specific primers are provided that allow for the selective amplification of one or more target sequences. In one aspect, the invention relates to target-specific primers useful for the selective amplification of one or more target sequences associated with cancer or inherited disease. In some aspects, amplified target sequences obtained using the disclosed methods, kits, systems and apparatuses can be used in various downstream processes including nucleic acid sequencing and used to detect the presence of genetic variants.

Methods for determining sequence variants using ultra-deep sequencing

The claimed invention provides for new sample preparation methods enabling direct sequencing of PCR products using pyrophosphate sequencing techniques. The PCR products may be specific regions of a genome. The techniques provided in this disclosure allows for SNP (single nucleotide polymorphism) detection, classification, and assessment of individual allelic polymorphisms in one individual or a population of individuals. The results may be used for diagnostic and treatment of patients as well as assessment of viral and bacterial population identification.

Scaffolded nucleic acid polymer particles and methods of making and using

Peristaltic pumping of fluids and associated methods, systems, and devices

Embodiments described herein generally relate to apparatuses, cartridges, and pumps for peristaltic pumping of fluids and associated methods, systems, and devices. The pumping of fluids is, in certain cases, an important aspect of a variety of applications, such as bioanalytical applications (e.g., biological sample analysis, sequencing, identification). The inventive features described herein may, in some embodiments, provide an ability to pump fluids in ways that combine certain advantages of robotic fluid handling systems (e.g., automation, programmability, configurability, flexibility) with certain advantages of microfluidics (e.g., small fluid volumes with high fluid resolution, precision, monolithic consumables, limiting of the wetting of components to consumables).

Bead emulsion nucleic acid amplification

Disclosed are methods for nucleic acid amplification wherein nucleic acid templates, beads, and amplification reaction solution are emulsified and the nucleic acid templates are amplified to provide clonal copies of the nucleic acid templates attached to the beads. Also disclosed are kits and apparatuses for performing the methods of the invention.

Methods for Sequencing Individual Nucleic Acids Under Tension

The invention provides apparatuses and methods of use thereof for sequencing nucleic acids subjected to a force, and thus considered under tension. The methods may employ but are not dependent upon incorporation of extrinsically detectably labeled nucleotides.

Methods for determining sequence variants using ultra-deep sequencing

The claimed invention provides for new sample preparation methods enabling direct sequencing of PCR products using pyrophosphate sequencing techniques. The PCR products may be specific regions of a genome. The techniques provided in this disclosure allows for SNP (single nucleotide polymorphism) detection, classification, and assessment of individual allelic polymorphisms in one individual or a population of individuals. The results may be used for diagnostic and treatment of patients as well as assessment of viral and bacterial population identification.

A carton forming and filling machine including a carton transporting carrier

1,144,115. Packaging; endless conveyers. FMC. CORP. 2 June, 1967 [8 July, 1966], No. 25469/67. Headings B8A and B8C. An apparatus for holding cartons in a fully erected position while they are filled and sealed, has carriers 20 with two adjacent walls and a gate 26 that pivots between a horizontal open position and an upright position to clamp the carton, the carriers being mounted on a conveyer 22. The cartons are introduced two at a time into two adjacent carriers by transfer mechanisms 28, 30 (no details given). Attached to a stationary post 25a are cam means, (92), Fig. 2 (not shown) to move arms 80 by rods 81, 82 away from a stop 89 into the path of a trip lever 64. Contact with the lever closes the gates 26 to hold the cartons erect. Two cam means, arranged at different heights to operate the rods 81, 82 which are of different lengths, allow two gates to be closed simultaneously. The trip lever 64 is connected to an overcentre device Fig. 9 (not shown). When a carton is not present in the carrier, the gate 26 rests on the edge of the wall 50. A device, Fig. 12, for ejecting a filled, sealed carton C from a carrier 20 comprises a triangular shaped cam 120 (shown in plan) that opens the gates in succession, and deflecting fingers 122 each having a roller 124 that pushes a carton C clear of a projection 52 enabling stationary guides 114, 116 to urge it on to the discharge conveyer 34.

Methods and compositions for multiplex PCR

The present invention provides methods, compositions, kits, systems and apparatus that are useful for multiplex PCR of one or more nucleic acids present in a sample. In particular, various target-specific primers are provided that allow for the selective amplification of one or more target sequences. In one aspect, the invention relates to target-specific primers useful for the selective amplification of one or more target sequences associated with cancer or inherited disease. In some aspects, amplified target sequences obtained using the disclosed methods, kits, systems and apparatuses can be used in various downstream processes including nucleic acid sequencing and used to detect the presence of genetic variants.

Scaffolded nucleic acid polymer particles and methods of making and using

The invention provides particle compositions having applications in nucleic acid analysis. Nucleic acid polymer particles of the invention allow polynucleotides to be attached throughout their volumes for higher loading capacities than those achievable solely with surface attachment. In one aspect, nucleic acid polymer particles of the invention comprise polyacrylamide particles with uniform size distributions having low coefficients of variations, which result in reduced particle-to-particle variation in analytical assays. Such particle compositions are used in various amplification reactions to make amplicon libraries from nucleic acid fragment libraries.

Double ended sequencing

This invention relates to methods of sequencing DNA. More specifically, this invention relates to methods of sequencing both the sense and antisense strands of DNA through the use of blocked and unblocked sequencing primers. In brief, these methods include the steps of annealing an unblocked primer to a first strand of nucleic acid; annealing a second blocked primer to a second strand of nucleic acid; elongating the nucleic acid along the first strand with a polymerase; terminating the first sequencing primer; deblocking the second primer; and elongating the nucleic acid along the second strand.

Methods and devices using cartridges for sequencing

Methods and devices for enriching and analyzing target molecules from a sample are provided herein. In some embodiments, methods and devices using cartridges involve enrichment of target molecules (e.g., using SCODA) and detection and analysis using sequencing. The method may employ lysing of cells, fragmentation of target in the sample preparation module and enriching the target in the module and moving the target to the sequencing module.

Double ended sequencing