ПАНЕЛЬ И СПОСОБ ПОЛУЧЕНИЯ ПРОСТРАНСТВЕННОЙ ИНФОРМАЦИИ О НУКЛЕИНОВЫХ КИСЛОТАХ

Изобретение относится к области биотехнологии. Описана группа изобретений, включающая панель нуклеиновых кислот для получения пространственной информации о нуклеиновой кислоте в образце, набор для получения пространственной информации о нуклеиновой кислоте в образце (варианты), способ изготовления панели нуклеиновых кислот для получения пространственной информации о нуклеиновой кислоте в биологическом образце и способ получения пространственной информации о нуклеиновой кислоте в образце (варианты). В одном варианте реализации панель нуклеиновых кислот включает в себя твердую подложку с многочисленными видами последовательностей-носителей, присоединенных к ее поверхности, где каждый вид последовательности-носителя занимает в панели отличное от другого вида положение, где множественные копии последовательности-носителя представляют собой продукт амплификации, образованный посредством амплификации с применением в качестве матрицы последовательности, комплементарной последовательности-носителю ...

VERFAHREN ZUM NACHWEIS VON CYTOSIN-METHYLIERUNGEN

Microarray hybridization assay methods

Methods for detection of a target nucleic acid by forming a cleavage structure with a cleavage resistant probe

RECA ASSISTED DETECTION OF MUTATIONS, SINGLE NUCLEOTIDE POLYMORPHISMS AND SPECIFIC SEQUENCES

A method for detecting a specific sequence, a mutation and/or a polymorphism s, including a single nucleotide polymorphism (SNP), is based on the use of Rec A- like recombinase protein and primer extension (PE) or oligonucleotide ligati on assays (OLA) . RecA coated, specific DNA oligonucleotide probes (RecA filaments) are used for homology searching in duplex DNA. Location of homologous sequences results in the formation of D-loop or double D-loop structures containing a duplex regions comprising the oligonucleotide probe and one strand of the target DNA. In the case of the PE methods, probes are selected to terminate with their 3' end adjacent to the site of mutation or SNP such that a single nucleotide or terminator addition to the primer will be diagnostic of the mutation or SNP. In the case of the OLA methods, sets of oligonucleotide probes (ligation partners) are selected to have adjacent end s terminate at, or adjacent to, the site of mutation or SNP such that ligation is possible only when both ends are correctly base-paired. Successful ligati on is diagnostic of the specific sequence, mutation or SNP. Also disclosed are compositions and kits useful for practicing the foregoing methods.

METHODS AND COMPOSITIONS FOR IDENTIFYING AND TREATING LUPUS

A unique set of genetic variations associated with lupus are provided. Also provided are methods for detecting such genetic variations and for assessing risk of developing lupus as well as for diagnosing and treating lupus.

Method of determining the nucleotide sequence of oligonucleotides and DNA molecules

The present invention relates to a novel method for analyzing nucleic acid sequences based on real-time detection of DNA polymerase-catalyzed incorporation of each of the four nucleotide bases, supplied individually and serially in a microfluidic system, to a reaction cell containing a template system comprising a DNA fragment of unknown sequence and an oligonucleotide primer. Incorporation of a nucleotide base into the template system can be detected by any of a variety of methods including but not limited to fluorescence and chemiluminescence detection. Alternatively, microcalorimetic detection of the heat generated by the incorporation of a nucleotide into the extending template system using thermopile, thermistor and refractive index measurements can be used to detect extension reactions.

Method of determining the nucleotide sequence of oligonucleotides and DNA molecules

The present invention relates to a novel method for analyzing nucleic acid sequences based on real-time detection of DNA polymerase-catalyzed incorporation of each of the four nucleotide bases, supplied individually and serially in a microfluidic system, to a reaction cell containing a template system comprising a DNA fragment of unknown sequence and an oligonucleotide primer. Incorporation of a nucleotide base into the template system can be detected by any of a variety of methods including but not limited to fluorescence and chemiluminescence detection. Alternatively, microcalorimetic detection of the heat generated by the incorporation of a nucleotide into the extending template system using thermopile, thermistor and refractive index measurements can be used to detect extension reactions.

ПАНЕЛЬ И СПОСОБ ПОЛУЧЕНИЯ ПРОСТРАНСТВЕННОЙ ИНФОРМАЦИИ О НУКЛЕИНОВЫХ КИСЛОТАХ

Изобретение относится к области биотехнологии. Описана группа изобретений, включающая панель нуклеиновых кислот для получения пространственной информации о нуклеиновой кислоте в образце, набор для получения пространственной информации о нуклеиновой кислоте в образце (варианты), способ изготовления панели нуклеиновых кислот для получения пространственной информации о нуклеиновой кислоте в биологическом образце и способ получения пространственной информации о нуклеиновой кислоте в образце (варианты). В одном варианте реализации панель нуклеиновых кислот включает в себя твердую подложку с многочисленными видами последовательностей-носителей, присоединенных к ее поверхности, где каждый вид последовательности-носителя занимает в панели отличное от другого вида положение, где множественные копии последовательности-носителя представляют собой продукт амплификации, образованный посредством амплификации с применением в качестве матрицы последовательности, комплементарной последовательности-носителю ...

Nucleic acid probe

Methods for detection of genetic disorders

Rapid analysis of variations in a genome

METHOD OF DETERMINING THE NUCLEOTIDE SEQUENCE OF OLIGONUCLEOTIDES AND DNA MOLECULES

The present invention relates to a novel method for analyzing nucleic acid sequences based on real-time detection of DNA polymerase-catalyzed incorporation of each of the four nucleotide bases, supplied individually and serially in a microfluidic system, to a reaction cell containing a template system comprising a DNA fragment of unknown sequence and an oligonucleotide primer. Incorporation of a nucleotide base into the template system can be detected by any of a variety of methods including but not limited to fluorescence and chemiluminescence detection. Alternatively, microcalorimetic detection of the heat generated by the incorporation of a nucleotide into the extending template system using thermopile, thermistor and refractive index measurements can be used to detect extension reactions.

Methods and reagents for molecular barcoding

THAT TURNS OUT THE RELEASE OF NUCLEOTIDES DURING A REACTION STEERS

NUCLEIC ACID SEQUENCING METHOD AND SYSTEM EMPLOYING ENHANCED DETECTION OF NUCLEOTIDE-SPECIFIC TERNARY COMPLEX FORMATION

Provided are methods and systems for detecting formation of nucleotide- specific ternary complexes comprising a DNA polymerase, a nucleic acid, and a nucleotide complementary to the templated base of the primed template nucleic acid. The methods and systems facilitate determination of the next correct nucleotide without requiring chemical incorporation of the nucleotide into the primer. This advantageously improves signal-to-noise ratios and increases the quality of results obtainable in a sequencing-by-binding protocol, and enables extended read lengths. These results can even be achieved in procedures employing unlabeled, native nucleotides.

METHODS FOR THE ANALYSIS OF CIRCULATING MICROPARTICLES

Reagents and methods for the analysis of cell free biomolecules (e.g. cell free nucleic acid molecules and cell free polypeptides) of circulating microparticles (i.e. microparticles originating from blood) are provided. The methods comprise analysing a sample that comprises a circulating microparticle or a sample derived from a circulating microparticle. The methods include methods of measuring at least two linked signals, each signal corresponding to the presence, absence and/or level of a biomolecule of a circulating microparticle. The methods also include methods of determining the presence, absence and/or level of a biomolecule of a ciruclatling microparticle using a barcoded affinity probe. In certain methods both nucleic acid biomolecules and non- nucleic acid biomolecules of a circulatling microparticle are analysed together. Reagents for use in the methods are also provided.

METHODS AND COMPOSITIONS FOR IDENTIFYING LIGANDS ON ARRAYS USING INDEXES AND BARCODES

Some embodiments provided herein include methods and compositions for the detection of target ligands on an array. In some embodiments, a capture probe specifically binds to a target ligand from a sample, the location of a bead comprising the capture probe in an array is determined, and the bead is decoded to identify the capture probe and the sample. In some embodiments, a barcode is indicative of a capture probe attached to a bead; and an index is indicative of a subpopulation of beads. In some embodiments, the barcode and the index are determined by sequencing.

METHOD FOR THE DETECTION OF CYTOSINE METHYLATIONS

Disclosed is a method for detecting 5-methylcytosine in genomic DNA samples. First, genomic DNA from a DNA sample is chemically reacted with a reagent, 5-methylcytosine and cytosine reacting differently. The pre-treated DNA is then ampified with primers from a different sequence using a polymerase. In the following step, the amplified genomic DNA is hybridised to an oligonucleotide array and PCR products are obtained which must be provided with an identifying mark. Alternatively, the PCR products can be extended in a Primer Extension Reaction, the extension products also being provided with an identifying mark. The last step involves examining the extended oligonucleotides for the presence of the identifying mark.

METHODS FOR DETECTION OF A TARGET NUCLEIC ACID BY FORMING A CLEAVAGE STRUCTURE WITH A CLEAVAGE RESISTANT PROBE

The invention relates to compositions and methods for generating a signal indicative of the presence of a target nucleic acid in a sample utilizing a cleavage resistant probe.

AGE PREDICTING METHOD USING DNA METHYLATION IN SALIVA

The present invention relates to an age predicting method using DNA methylation in saliva. More specifically, the present invention relates to a method of predicting age of a test object by measuring a methylation level (a ratio of DNA methylation and non-methylation) with respect to a CpG marker of a target gene in a saliva sample which is originated from a test object. Age of a test object can be predicted by using an age predicting method according to the present invention, measuring a DNA methylation level in a saliva sample which is originated from a test object, and analyzing a DNA methylation ratio. In particular, since kinds of CpG markers which are to be analyzed are minimized to seven types and an error range of measured age predicting figures is reduced in an age predicting method according to the present invention in comparison with a conventional age predicting method which analyzes methylation with respect to 70 or more CpG markers in a human body fluid sample or an age predicting method which uses 3 CpG markers in saliva but has low accuracy, age of a test object can be quickly and accurately measured.

métodos e composições para identificar ligantes em arranjos com o uso de índices e códigos de barras

Algumas modalidades aqui apresentadas incluem métodos e composições para a detecção de ligantes-alvo em um arranjo. Em algumas modalidades, uma sonda de captura se liga especificamente a um ligante-alvo a partir de uma amostra, o local de uma microesfera que compreende a sonda de captura em um arranjo é determinado, e a microesfera é decodificada para identificar a sonda de captura e a amostra. Em algumas modalidades, um código de barras é indicativo de uma sonda de captura fixada a uma microesfera; e um índice é indicativo de uma subpopulação de microesferas. Em algumas modalidades, o código de barras e o índice são determinados por sequenciamento. Some modalities presented here include methods and compositions for detecting target ligands in a arrangement. In some modalities, a capture probe turns on specifically to a target ligand from a sample, the site of a microsphere comprising the capture probe in an array is determined, and the microsphere is decoded to identify the probe of capture and the sample. In some modes, a barcode is indicative of a capture probe attached to a microsphere; and an index is indicative of a subpopulation of microspheres. In some modalities, barcode and index are determined by sequencing.

DNA sequencing by synthesis using Raman and infrared spectroscopy detection

This invention provides nucleoside triphosphate analogs having the structure: wherein B is a base and is adenine, guanine, cytosine, uracil or thymine, wherein R″ is an OH or an H, and wherein R′ is azidomethyl, a hydrocarbyl, or a substituted hydrocarbyl, and which has a Raman spectroscopy peak with wavenumber from 2000 cm−1to 2300 cm−1or a Fourier transform-infrared spectroscopy spectroscopy peak with wavenumber from 2000 cm−1to 2300 cm−1, and also to methods of DNA sequencing and SNP detection.

СПОСОБЫ И КОМПОЗИЦИИ ДЛЯ ДИАГНОСТИКИ И ЛЕЧЕНИЯ ВОЛЧАНКИ

1. Способ оценки наличия риска развития волчанки у субъекта, где способ включает детекцию в биологическом образце, полученном от указанного субъекта, наличия генетического признака, указывающего на риск развития волчанки, где указанный генетический признак включает набор из одного или нескольких SNP, выбранных из любых SNP, указанных на фигурах 1-17 и в таблицах 1-10. ! 2. Способ по п.1, где указанный набор SNP содержит приблизительно 1-10, 10-20, 20-30, 30-40 или 40-50 SNP, выбранных из любых SNP, указанных на фигурах 1-17 и в таблицах 1-10. ! 3. Способ по п.1, где указанный набор SNP содержит 2 или более SNP, 3 или более SNP, 4 или более SNP, 5 или более SNP, 6 или более SNP, 7 или более SNP, 8 или более SNP, 9 или более SNP, 10 или более SNP, 11 или более SNP, 12 или более SNP, 13 или более SNP, 14 или более SNP, 15 или более SNP, 16 или более SNP, 17 или более SNP, 18 или более SNP, 19 или более SNP, или 20 или более SNP, выбранных из любых SNP, указанных на фигурах 1-17 и в таблицах 1-10. ! 4. Способ по п.1, где указанный набор SNP включает 1-19 SNP, выбранных из таблицы 6. ! 5. Способ по п.1, где указанный набор SNP включает SNP BLK, выбранный из любого из SNP BLK, указанных в таблицах 7-10. ! 6. Способ по п.1, где указанный набор SNP включает SNP ITGAM, выбранный из любого из SNP ITGAM, указанных в таблицах 7-10. ! 7. Способ по п.6, где указанный набор SNP дополнительно включает SNP BLK, выбранный из любого из SNP BLK, указанных в таблицах 7-10. ! 8. Способ по п.1, где указанный набор SNP включает один или несколько SNP, выбранных из следующей группы SNP: rs2187668, rs10488631, rs7574865, rs9888739, rs13277113, rs2431697, rs6568431, rs10489265, rs2476601, rs2269368, rs1801274, rs4963128, rs5754217, rs6445975, rs3129860, rs10516487, rs6889239, rs2391592 и rs2177770. ! 9. Способ диагностики волчанки у субъекта, где способ включает детекцию в биологическом образце, полученном от ук РОССИЙСКАЯ ФЕДЕРАЦИЯ (19) RU (11) 2009 147 281 (13) A (51) МПК C12Q 1/68 (2006.01 ...

Nucleic acid detection method

PROCEDURE FOR THE PROOF OF CYTOSINE METHYLIERUNGEN

Nucleic acid probe with single fluorophore label bound to internal cytosine for use in loop mediated isothermal amplification

Nucleic acid probe with single fluorophore label bound to internal cytosine for use in loop mediated isothermal amplification Abstract The present invention provides novel probes for use in LAMP detection methods. The probes contain a single fluoro phore label bound to an internal cytosine residue of the probe. The probes are particularly useful in the detection of chlamydia and gonorrhea infections in a patient. ..o z E o ino ...

Nucleic acid sequencing method and system employing enhanced detection of nucleotide-specific ternary complex formation

Provided are methods and systems for detecting formation of nucleotide- specific ternary complexes comprising a DNA polymerase, a nucleic acid, and a nucleotide complementary to the templated base of the primed template nucleic acid. The methods and systems facilitate determination of the next correct nucleotide without requiring chemical incorporation of the nucleotide into the primer. This advantageously improves signal-to-noise ratios and increases the quality of results obtainable in a sequencing-by-binding protocol, and enables extended read lengths. These results can even be achieved in procedures employing unlabeled, native nucleotides.

Methods For Determining Biological States By Assessing Amounts Of Target Nucleic Acids In A Sample

Described herein are compositions for evaluating nucleic acids, standardized mixture for assessing amounts of at least one target nucleic acid in a sample, and kits comprising the same. 1. A method for obtaining a numerical index that indicates a biological state , comprising:providing two samples corresponding to each of a first biological state and a second biological state;assessing an amount of each of two nucleic acids in each of said two samples wherein said assessing can enumerate less than about 1,000 molecules of each of said two nucleic acids;providing said amounts as numerical values wherein said numerical values are directly comparable between a number of samples;mathematically computing said numerical values corresponding to each of said first and said second biological states; anddetermining a mathematical computation that discriminates said first and said second biological states, thereby obtaining said numerical index.2. The method as recited in claim 1 , wherein said two nucleic acids are associated with said first biological state and not with said second biological state.3. The method as recited in claim 2 , wherein one of said two nucleic acids is positively associated with said first biological state and the other of said two nucleic acids is negatively associated with said first biological state.4. The method as recited in claim 3 , wherein said mathematical computation comprises dividing a numerator by a denominator claim 3 , said numerator corresponding to said nucleic acid positively associated with said first biological state and said denominator corresponding to said nucleic acid negatively associated with said first biological state.5. The method as recited in claim 1 , wherein said first biological state is a disease state and said second biological state is a non-disease state.6. The method as recited in claim 1 , wherein the biological state includes: a disease phenotype; a predisposition to a disease state or a non-disease state; a ...

НУКЛЕИНОВО-КИСЛОТНЫЙ ЗОНД

Изобретение относится к биотехнологии. Изобретение включает новые зонды для изотермической амплификации нуклеиновой кислоты и способы обнаружения последовательности-мишени нуклеиновой кислоты с использованием этих зондов. Зонды содержат последовательность олигонуклеотидного зонда, комплементарную области последовательности нуклеиновой кислоты-мишени. Последовательность олигонуклеотидного зонда имеет только одну флуорофорную метку, связанную с внутренним цитозиновым основанием, и не имеет терминатора на 3'-конце. Цитозиновое основание центрально расположено по длине олигонуклеотида, за исключением позиций 1-3 на 3'-конце и позиции 1 на 5'-конце. Данные зонды могут быть использованы для выявления хламидийной и гонорейной инфекций у пациента. 4 н. и 17 з.п. ф-лы, 12 ил., 1 табл., 12 пр.

НАНОЧАСТИЦА С ОДНИМ САЙТОМ ДЛЯ ПРИСОЕДИНЕНИЯ МАТРИЧНОГО ПОЛИНУКЛЕОТИДА

Изобретение относится к области биотехнологии. Описана группа, включающая наночастицу для повышения доступной площади поверхности субстрата для затравки и кластеризации при секвенированиии путем синтеза (SBS) (варианты) и способ повышения плотности затравки и моноклональной кластеризации при SBS (варианты). В одном варианте реализации наночастица содержит каркас, один матричный сайт для связывания матричного полинуклеотида с каркасом и множество вспомогательных сайтов для связывания вспомогательных олигонуклеотидов с каркасом. Изобретение расширяет арсенал средств для повышения доступной площади поверхности субстрата для затравки и кластеризации при секвенированиии путем синтеза (SBS). 6 н. и 40 з.п. ф-лы, 13 ил., 1 табл., 1 пр.

НУКЛЕИНОВО-КИСЛОТНЫЙ ЗОНД

METHOD FOR DETECTING CYTOSINE METHYLATIONS

Disclosed is a method for detecting 5-methylcytosine in genomic DNA samples. First, genomic DNA from a DNA sample is chemically reacted with a reagent, 5- methylcytosine and cytosine reacting differently. The pre-treated DNA is then ampified with primers from a different sequence using a polymerase. In the following step, the amplified genomic DNA is hybridised to an oligonucleotide array and PCR products are obtained which must be provided with an identifying mark. Alternatively, the PCR products can be extended in a Primer Extension Reaction, the extension products also being provided with an identifying mark. The last step involves examining the extended oligonucleotides for the presence of the identifying mark.

NUCLEIC ACID PROBE

The present invention provides novel probes for use in LAMP detection methods. The probes contain a single fluorophore label bound to an internal cytosine residue of the probe. The probes are particularly useful in the detection of chlamydia and gonorrhea infections in a patient.

Method for detecting virus associated with hemorrhagic fever with renal syndrome

USE OF TEMPLATE SWITCHING FOR DNA SYNTHESIS

A method of preparing a DNA copy of a target polynucleotide using template switching is described. The method includes mixing a double stranded template/primer substrate made up of a DNA primer oligonucleotide associated with a complementary oligonucleotide template strand with a target polynucleotide in a reaction medium and adding a suitable amount of a non- retroviral reverse transcriptase to the reaction medium to extend the DNA primer oligonucleotide from its 3' end to provide a DNA copy polynucleotide. The DNA copy polynucleotide includes a complementary target DNA polynucleotide that is synthesized using the target polynucleotide as a template. Methods of adding nucleotides to the double stranded template/primer substrate are also described. The method can be used to facilitate detection, PCR amplification, cloning, and determination of RNA and DNA sequences.

NUCLEIC ACID PROBE WITH SINGLE FLUOROPHORE LABEL BOUND TO INTERNAL CYTOSINE FOR USE IN LOOP MEDIATED ISOTHERMAL AMPLIFICATION

METHOD FOR DETECTING CYTOSINE METHYLATIONS

Disclosed is a method for detecting 5-methylcytosine in genomic DNA samples. First, genomic DNA from a DNA sample is chemically reacted with a reagent, 5-methylcytosine and cytosine reacting differently. The pre-treated DNA is then ampified with primers from a different sequence using a polymerase. In the following step, the amplified genomic DNA is hybridised to an oligonucleotide array and PCR products are obtained which must be provided with an identifying mark. Alternatively, the PCR products can be extended in a Primer Extension Reaction, the extension products also being provided with an identifying mark. The last step involves examining the extended oligonucleotides for the presence of the identifying mark.

Method for detecting cytosine methylations

A method is described for the detection of 5-methylcytosine in genomic DNA samples. First, a genomic DNA from a DNA sample is chemically converted with a reagent, whereby 5-methylcytosine and cytosine react differently. Then the pretreated DNA is amplified with the use of a polymerase with primers of different sequence. In the next step, the amplified genomic DNA is hybridized to an oligonucleotide array and PCR products are obtained, which must be provided with a label. Alternatively, the PCR products can be extended in a primer extension reaction, wherein the extension products are also provided with a label. In the last step, the extended oligonucleotides are investigated for the, presence of the label.

Use of template switching for DNA synthesis

A method of preparing a DNA copy of a target polynucleotide using template switching is described. The method includes mixing a double stranded template/primer substrate made up of a DNA primer oligonucleotide associated with a complementary oligonucleotide template strand with a target polynucleotide in a reaction medium and adding a suitable amount of a non-retroviral reverse transcriptase to the reaction medium to extend the DNA primer oligonucleotide from its 3 end to provide a DNA copy polynucleotide. The DNA copy polynucleotide includes a complementary target DNA polynucleotide that is synthesized using the target polynucleotide as a template. Methods of adding nucleotides to the double stranded template/primer substrate are also described. The method can be used to facilitate detection, PCR amplification, cloning, and determination of RNA and DNA sequences.

Methods of producing nucleic acids using oligonucleotides modified by a stimulus

Provided are methods of producing product nucleic acids involving the use of oligonucleotides that are modified by the application of a stimulus. Aspects of such methods may include producing product nucleic acids using de-activatable oligonucleotides that are deactivated by a de-activating stimulus, as well as methods that may include producing product nucleic acids using activatable oligonucleotides that are activated by an activating stimulus and de-activatable oligonucleotides that are deactivated by a de-activating stimulus. Also provided are kits, compositions and devices that include de-activatable oligonucleotides or activatable and de-activatable oligonucleotides, e.g., for use in performing the methods as described herein.

METHOD FOR DETECTING CYTOSINE METHYLATIONS

Disclosed is a method for detecting 5-methylcytosine in genomic DNA samples. First, genomic DNA from a DNA sample is chemically reacted with a reagent, 5-methylcytosine and cytosine reacting differently. The pre-treated DNA is then ampified with primers from a different sequence using a polymerase. In the following step, the amplified genomic DNA is hybridised to an oligonucleotide array and PCR products are obtained which must be provided with an identifying mark. Alternatively, the PCR products can be extended in a Primer Extension Reaction, the extension products also being provided with an identifying mark. The last step involves examining the extended oligonucleotides for the presence of the identifying mark.

METHOD AND KIT OF DETECTING THE ABSENCE OF MICRO-ORGANISMS

Methods of detecting the absence or presence of a micro-organism in a sample comprising: contacting the sample with a nucleic acid molecule which acts as a substrate for nucleic acid modifying activity of the micro-organism in the sample, incubating the thus contacted sample under conditions suitable for nucleic acid modifying activity; and specifically determining the absence or presence of a modified nucleic acid molecule resulting from the action of the nucleic acid modifying activity on the substrate nucleic acid molecule to indicate the absence or presence of the micro-organism. Corresponding kits are also provided.

MULTIPLEX SNP MARKER COMPOSITION FOR PREDICTING AND DIAGNOSING CANINE HIP DYSPLASIA USING SAME, AND PREDICTION OR DIAGNOSIS METHOD USING SAME

The present invention relates to a composition and a method for predicting or diagnosing canine hip dysplasia and, more specifically, to a composition and a method for predicting or diagnosing canine hip dysplasia using a primer set or a probe set specifically bound to polynucleotide consisting of 10-100 consecutive nucleotides including a single nucleotide polymorphism (SNP) marker which is the 217^th nucleotide of sequence number 1 to sequence number 5, or complementary polynucleotide thereof.

Method of determining the nucleotide sequence of oligonucleotides and DNA molecules

The present invention relates to a novel method for analyzing nucleic acid sequences based on real-time detection of DNA polymerase-catalyzed incorporation of each of the four nucleotide bases, supplied individually and serially in a microfluidic system, to a reaction cell containing a template system comprising a DNA fragment of unknown sequence and an oligonucleotide primer. Incorporation of a nucleotide base into the template system can be detected by any of a variety of methods including but not limited to fluorescence and chemiluminescence detection. Alternatively, microcalorimetic detection of the heat generated by the incorporation of a nucleotide into the extending template system using thermopile, thermistor and refractive index measurements can be used to detect extension reactions.

Verfahren zum Nachweis von Cytosin-Methylierungen

Verfahren zum Nachweis von Cytosin-Methylierungen in DNA, dadurch gekennzeichnet, dass man folgende Arbeitsschritte ausführt: a) eine genomische DNA-Probe wird mit einer Lösung eines Bisulfits im Konzentrationsbereich zwischen 0,1 und 6 mol/l inkubiert, wobei ein denaturierendes Reagenz und/oder Lösemittel sowie mindestens ein Radikalfänger zugegen ist; b) die behandelte DNA-Probe wird mit Wasser oder einer wässrigen Lösung verdünnt; c) die DNA-Probe wird in einer Polymerasereaktion amplifiziert; d) man detektiert, inwieweit sich die Sequenz durch die Behandlung nach Schritt a) gegenüber der genomischen DNA-Probe verändert hat und schliesst auf den Methylierungsstatus zumindest eines Locus in der genomischen DNA-Probe.

Assay ligates random primers to microarray probes to increase efficiency

An assay method suitable for use in microarray hybridization of a target sample includes providing a microarray surface 111 having probes 131, hybridizing the probes 131 to a target sample 140, introducing random primers 150 each having an arbitrary sequence, ligating 210 a free terminal end 131_a of each of the probes to a free terminal end 150_a of the random primers on the probe side, removing the target sample 140 hybridized to each of the probes 131, and measuring the intensity of the remaining random primers. The random primers 150 may be 8 nucleotides in length, and may be ligated 210 directly or via an intermediate polymerase step which may extend the probe or the marker. They may have an integral marker or label 160, or the polymerisation 210 may incorporate fluorescent nucleotides. The probes 131 may be attached using linkers 120 at either the 5' or 3' terminal end.

Nucleic acid probe

A polynucleotide probe suitable for isothermal amplification having a fluorescent label on a cytosine, wherein the cytosine is internal and the oligonucleotide probe does not have a 3 end terminator. The cytosine may be substantially centrally disposed along the oligonucleotides length. The flurophore may comprisie FAM, JOE, TET, HEX,TAMRA, ROX, ALEXA or ATTO. The target nucleic acid may be from a micro-organism, fungi, yeast or virus, in particular Chlamydia trachomatis or Neisseria gonorrhoaeae. Methods of detecting a nucleic acid using said probes and diagnosing Chlamydia and/or Gonorrhea infection as well as kits for isothermal PCR comprising such probes are claimed.

Nucleic acid probe

Primers and assays for linking regions using polymerases

Forward and reverse primers used to link distant regions of a DNA molecule, to remove intermediate regions. A reverse primer R1 can have a first portion complementary to an ending sequence of region A and a second portion having an overlapping sequence. A forward primer F2 can have a first portion complementary to a starting sequence of region B, where the forward primer includes a complementary overlapping sequence that is complementary to the overlapping sequence in R1. Additional Fz and Rz primers that bind to the distant external primers are also included. Additional F3 primer can be included for a region C, and independent claims are included for a primer F4 that also has a complementary overlap sequence with R1. The linked DNA molecules can be used to determine haplotypes of regions. Kits including the particular forward and reverse primers are also described.

SAMPLE PREPARATION METHODS, SYSTEMS AND COMPOSITIONS

The disclosure provides methods, compositions, systems, and kits for the concurrent detection and analysis of different structural and chemical forms of nucleic acids in a sample. Different nucleic acid forms are tagged using DNA-dependent polymerase and reverse transcriptase enzymes with non-templated activity or with ligase enzymes that display a preference for the different nucleic acid forms.

USE OF TEMPLATE SWITCHING FOR DNA SYNTHESIS

A method of preparing a DNA copy of a target polynucleotide using template switching is described. The method includes mixing a double stranded template/primer substrate made up of a DNA primer oligonucleotide associated with a complementary oligonucleotide template strand with a target polynucleotide in a reaction medium and adding a suitable amount of a non- retroviral reverse transcriptase to the reaction medium to extend the DNA primer oligonucleotide from its 3' end to provide a DNA copy polynucleotide. The DNA copy polynucleotide includes a complementary target DNA polynucleotide that is synthesized using the target polynucleotide as a template. Methods of adding nucleotides to the double stranded template/primer substrate are also described. The method can be used to facilitate detection, PCR amplification, cloning, and determination of RNA and DNA sequences.

Methods Of Producing Nucleic Acids Using Oligonucleotides Modified By A Stimulus

Provided are methods of producing product nucleic acids involving the use of oligonucleotides that are modified by the application of a stimulus. Aspects of such methods may include producing product nucleic acids using de-activatable oligonucleotides that are deactivated by a de-activating stimulus, as well as methods that may include producing product nucleic acids using activatable oligonucleotides that are activated by an activating stimulus and de-activatable oligonucleotides that are deactivated by a de-activating stimulus. Also provided are kits, compositions and devices that include de-activatable oligonucleotides or activatable and de-activatable oligonucleotides, e.g., for use in performing the methods as described herein. 1. A method comprising:elongating a de-activatable oligonucleotide via a template nucleic acid-mediated primer extension reaction to produce an extended nucleic acid; anddeactivating the de-activatable oligonucleotide.2. The method according to claim 1 , wherein deactivating the de-activatable oligonucleotide comprises applying a deactivating stimulus selected from the group consisting of: an enzyme claim 1 , a chemical agent and an electromagnetic stimulus.3. The method according to claim 2 , wherein the deactivating stimulus is an enzyme and the enzyme is ribonuclease H (RNase H).4. The method according claim 1 , comprising a template switching reaction comprising a template switching oligonucleotide (TSO) to produce an extended nucleic acid that comprises a sequence complementary to the TSO.5. The method according to claim 4 , wherein the TSO is de-activatable.6. The method according to claim 5 , wherein the de-activatable oligonucleotide and the de-activatable TSO are deactivated by the same stimulus.7. The method according claim 1 , wherein the de-activatable oligonucleotide claim 1 , the TSO claim 1 , or both comprise an adapter sequence claim 1 , a barcode sequence claim 1 , or a combination thereof.8. The method according claim 1 ...

Method of determining the nucleotide sequence of oligonucleotides and DNA molecules

The present invention relates to a novel method for analyzing nucleic acid sequences based on real-time detection of DNA polymerase-catalyzed incorporation of each of the four nucleotide bases, supplied individually and serially in a microfluidic system, to a reaction cell containing a template system comprising a DNA fragment of unknown sequence and an oligonucleotide primer. Incorporation of a nucleotide base into the template system can be detected by any of a variety of methods including but not limited to fluorescence and chemiluminescence detection. Alternatively, microcalorimetic detection of the heat generated by the incorporation of a nucleotide into the extending template system using thermopile, thermistor and refractive index measurements can be used to detect extension reactions.

Nucleic acid probe with single fluorophore label bound to internal cytosine for use in loop mediated isothermal amplification

The present invention provides novel probes for use in LAMP detection methods. The probes contain a single fluorophore label bound to an internal cytosine residue of the probe. The probes are particularly useful in the detection of chlamydia and gonorrhea infections in a patient.

Method for detecting cytosine methylations

Method for determination of DNA methylation

Method of determining the nucleotide sequence of oligonucleotides and DNA molecules

The present invention relates to a novel method for analyzing nucleic acid sequences based on real-time detection of DNA polymerase-catalyzed incorporation of each of the four nucleotide bases, supplied individually and serially in a microfluidic system, to a reaction cell containing a template system comprising a DNA fragment of unknown sequence and an oligonucleotide primer. Incorporation of a nucleotide base into the template system can be detected by any of a variety of methods including but not limited to fluorescence and chemiluminescence detection. Alternatively, microcalorimetic detection of the heat generated by the incorporation of a nucleotide into the extending template system using thermopile, thermistor and refractive index measurements can be used to detect extension reactions.

Exonuclease Enabled Proximity Extension Assays

The present invention relates to a proximity probe based detection assay (“proximity assay”) for an analyte in a sample, specifically a proximity probe extension assay (PEA), an in particular to an improvement in the method to reduce non-specific “background” signals, wherein the improvement comprises the use in such assays of a component comprising 3′ exonuclease activity, said method comprising: (a) contacting said sample with at least one set of at least first and second proximity probes, which probes each comprise an analyte-binding domain and a nucleic acid domain and can simultaneously bind to the analyte; (b) allowing the nucleic acid domains of the proximity probes to interact with each other upon binding of said proximity probes to said analyte, wherein said interaction comprises the formation of a duplex; (c) contacting said sample with a component comprising 3′ exonuclease activity; (d) extending the 3′ end of at least one nucleic acid domain of said duplex to generate an extension product, wherein the step may occur contemporaneously with or after step (c); and (e) amplifying and detecting the extension product.

Nucleic acid sequencing by raman monitoring of uptake of precursors during molecular replication

The methods, compositions and apparatus disclosed herein are of use for nucleic acid sequence determination. The methods involve isolation of one or more nucleic acid template molecules and polymerization of a nascent complementary strand of nucleic acid, using a DNA or RNA polymerase or similar synthetic reagent. As the nascent strand is extended one nucleotide at a time, the disappearance of nucleotide precursors from solution is monitored by Raman spectroscopy or FRET. The nucleic acid sequence of the nascent strand, and the complementary sequence of the template strand, may be determined by tracking the order of incorporation of nucleotide precursors during the polymerization reaction. Certain embodiments concern apparatus comprising a reaction chamber and detection unit, of use in practicing the claimed methods. The methods, compositions and apparatus are of use in sequencing very long nucleic acid templates in a single sequencing reaction.

Primers and assays for linking regions using polymerases

Particular forward and reverse primers may be used to link distant regions of the same large DNA molecule into a smaller DNA molecule. A reverse primer R1 can have a first portion complementary to an ending sequence of region A and can have a second portion having an overlapping sequence. A forward primer F2 can have a first portion complementary to a starting sequence of region B, where the forward primer includes a complementary overlapping sequence (e.g., the same first portion or a second portion) that is complementary to the overlapping sequence. The first portion of F2 may be the entire primer. The smaller DNA molecules can be used to determine haplotypes of regions. Kits including the particular forward and reverse primers are also described.

Reagents and methods for the analysis of circulating microparticles

Use of template switching for DNA synthesis

A method of preparing a DNA copy of a target polynucleotide using template switching is described. The method includes mixing a double stranded template/primer substrate made up of a DNA primer oligonucleotide associated with a complementary oligonucleotide template strand with a target polynucleotide in a reaction medium and adding a suitable amount of a non- retroviral reverse transcriptase to the reaction medium to extend the DNA primer oligonucleotide from its 3' end to provide a DNA copy polynucleotide. The DNA copy polynucleotide includes a complementary target DNA polynucleotide that is synthesized using the target polynucleotide as a template. Methods of adding nucleotides to the double stranded template/primer substrate are also described. The method can be used to facilitate detection, PCR amplification, cloning, and determination of RNA and DNA sequences.

NUCLEIC ACID SEQUENCING METHOD AND SYSTEM EMPLOYING ENHANCED DETECTION OF NUCLEOTIDE-SPECIFIC TERNARY COMPLEX FORMATION

Provided are methods and systems for detecting formation of nucleotide- specific ternary complexes comprising a DNA polymerase, a nucleic acid, and a nucleotide complementary to the templated base of the primed template nucleic acid. The methods and systems facilitate determination of the next correct nucleotide without requiring chemical incorporation of the nucleotide into the primer. This advantageously improves signal-to-noise ratios and increases the quality of results obtainable in a sequencing-by-binding protocol, and enables extended read lengths. These results can even be achieved in procedures employing unlabeled, native nucleotides.

증폭 산물의 농축을 위한 방법 및 조성물

... 일부 측면에서, 본 개시내용은 표적 폴리뉴클레오티드의 적어도 2개 이상의 카피의 콘카테머를 포함하는 앰플리콘 또는 증폭 산물을 농축하는 방법을 제공한다. 일부 실시양태에서, 방법은 표적 폴리뉴클레오티드의 적어도 2개 이상의 카피를 포함하는 앰플리콘을 서열결정하는 단계를 포함한다. 일부 실시양태에서, 표적 폴리뉴클레오티드는 융합 유전자를 비롯하여 점 돌연변이, 단일 뉴클레오티드 다형성, 삽입, 결실 및 전좌를 포함하고 이로 제한되지 않는 염색체 재배열에 의해 생성된 서열을 포함한다. 일부 측면에서, 본 개시내용은 설명되는 방법에 유용한 조성물 및 반응 혼합물을 제공한다.

Detecting the absence of micro-organisms

Method for detecting cytosine methylations

METHODS AND COMPOSITIONS FOR IDENTIFYING AND TREATING LUPUS

A unique set of genetic variations associated with lupus are provided. Also provided are methods for detecting such genetic variations and for assessing risk of developing lupus as well as for diagnosing and treating lupus.

USE OF TEMPLATE SWITCHING FOR DNA SYNTHESIS

A method of preparing a DNA copy of a target polynucleotide using template switching is described. The method includes mixing a double stranded template/primer substrate made up of a DNA primer oligonucleotide associated with a complementary oligonucleotide template strand with a target polynucleotide in a reaction medium and adding a suitable amount of a non- retroviral reverse transcriptase to the reaction medium to extend the DNA primer oligonucleotide from its 3' end to provide a DNA copy polynucleotide. The DNA copy polynucleotide includes a complementary target DNA polynucleotide that is synthesized using the target polynucleotide as a template. Methods of adding nucleotides to the double stranded template/primer substrate are also described. The method can be used to facilitate detection, PCR amplification, cloning, and determination of RNA and DNA sequences.

Method and kit of detecting the absence of micro-oranisms

Methods of detecting the absence or presence of a micro-organism in a sample comprising: contacting the sample with a nucleic acid molecule which acts as a substrate for nucleic acid modifying activity of the micro-organism in the sample, incubating the thus contacted sample under conditions suitable for nucleic acid modifying activity; and specifically determining the absence or presence of a modified nucleic acid molecule resulting from the action of the nucleic acid modifying activity on the substrate nucleic acid molecule to indicate the absence or presence of the micro-organism. Corresponding kits are also provided.

methods and kit diagnosis or prognosis of lupus in a subject.

RECA ASSISTED DETECTION OF MUTATIONS, SINGLE NUCLEOTIDE POLYMORPHISMS AND SPECIFIC SEQUENCES

A method for detecting a specific sequence, a mutation and/or a polymorphisms, including a single nucleotide polymorphism (SNP), is based on the use of RecA-like recombinase protein and primer extension (PE) or oligonucleotide ligation assays (OLA) . RecA coated, specific DNA oligonucleotide probes (RecA filaments) are used for homology searching in duplex DNA. Location of homologous sequences results in the formation of D-loop or double D-loop structures containing a duplex regions comprising the oligonucleotide probe and one strand of the target DNA. In the case of the PE methods, probes are selected to terminate with their 3' end adjacent to the site of mutation or SNP such that a single nucleotide or terminator addition to the primer will be diagnostic of the mutation or SNP. In the case of the OLA methods, sets of oligonucleotide probes (ligation partners) are selected to have adjacent ends terminate at, or adjacent to, the site of mutation or SNP such that ligation is possible only when both ends are correctly base-paired. Successful ligation is diagnostic of the specific sequence, mutation or SNP. Also disclosed are compositions and kits useful for practicing the foregoing methods.

BARCODE-FREE SINGLE VESICLE MULTIPLEXED PROTEIN AND RNA ANALYSIS

According to various embodiments, a system and method for characterizing protein and nucleic acid content of a plurality of individual particles. The method includes encapsulating individual particles into compartments also containing analyte specific binding complements with oligonucleotide tags comprising a unique molecular identifier sequence, a sequence to identify the analyte specific binding complement, and a homology domain sequence. Allowing the oligonucleotide tags to hybridize on homology domain to form initial tag pairs, amplifying the tag pairs, using an enzyme to cut at the homology domain, allowing tags to re-hybridize, pooling the compartments, and sequencing. Finally, predicting co-encapsulated analytes by computational identification of clusters based on more frequently found oligonucleotide tag pairs. 1. A method for characterizing protein and nucleic acid content of individual particles , the method comprising:encapsulating a plurality of particles into compartments, the compartments also containing analyte specific binding complements with oligonucleotide tags, the tags including a sequence to identify the analyte specific binding complement and two unique molecular identifier (UMI) sequences separated by a restriction enzyme cleavage site;amplifying the tags;using an enzyme to cut at the restriction site;allowing cut tags to re-hybridize;pooling the compartments;sequencing the nucleic acid sequences; andpredicting co-encapsulated analytes by computational identification of clusters based on more frequently found UMI pairs.2. The method of claim 1 , wherein the particles are lipid vesicles such as a cells claim 1 , extracellular vesicles claim 1 , exosomes claim 1 , lipid nanoparticles claim 1 , enveloped viruses claim 1 , or any other biological entities of similar size range.3. The method of claim 1 , wherein the oligonucleotide tags include a nucleic acid binding end claim 1 , rather or in addition to the analyte specific binding complement ...

Compositions, systems, and methods for detecting the presence of polymer subunits using chemiluminescence

Under one aspect, a composition includes a substrate; a first polynucleotide coupled to the substrate; a second polynucleotide hybridized to the first polynucleotide; and a catalyst coupled to a first nucleotide of the second polynucleotide, the catalyst being operable to cause a chemiluminogenic molecule to emit a photon. Under another aspect, a method includes providing a catalyst operable to cause a first chemiluminogenic molecule to emit a photon; providing a substrate; providing a first polynucleotide coupled to the substrate; hybridizing a second polynucleotide to the first polynucleotide; coupling a first quencher to a first nucleotide of the second polynucleotide; and inhibiting, by the first quencher, photon emission by the first chemiluminogenic molecule.

Microarray hybridization assay methods

Methods for the analysis of circulating microparticles

Reagents and methods for the analysis of cell free biomolecules (e.g. cell free nucleic acid molecules and cell free polypeptides) of circulating microparticles (i.e. microparticles originating from blood) are provided. The methods comprise analysing a sample that comprises a circulating microparticle or a sample derived from a circulating microparticle. The methods include methods of measuring at least two linked signals, each signal corresponding to the presence, absence and/or level of a biomolecule of a circulating microparticle. The methods also include methods of determining the presence, absence and/or level of a biomolecule of a ciruclatling microparticle using a barcoded affinity probe. In certain methods both nucleic acid biomolecules and non- nucleic acid biomolecules of a circulatling microparticle are analysed together. Reagents for use in the methods are also provided.

METHODS AND COMPOSITIONS FOR IDENTIFYING LIGANDS ON ARRAYS USING INDEXES AND BARCODES

Some embodiments provided herein include methods and compositions for the detection of target ligands on an array. In some embodiments, a capture probe specifically binds to a target ligand from a sample, the location of a bead comprising the capture probe in an array is determined, and the bead is decoded to identify the capture probe and the sample. In some embodiments, a barcode is indicative of a capture probe attached to a bead; and an index is indicative of a subpopulation of beads. In some embodiments, the barcode and the index are determined by sequencing.

NO DETAILS

METHODS FOR DETECTION OF GENETIC DISORDERS

Nucleic acid sequencing by raman monitoring of uptake of precursors during molecular replication

RAPID ANALYSIS OF VARIATIONS IN A GENOME

METHOD OF DETERMINING THE NUCLEOTIDE SEQUENCE OF OLIGONUCLEOTIDES AND DNA MOLECULES

The present invention relates to a novel method for analyzing nucleic acid sequences based on real-time detection of DNA polymerase-catalyzed incorporation of each of the four nucleotide bases, supplied individually and serially in a microfluidic system, to a reaction cell containing a template system comprising a DNA fragment of unknown sequence and an oligonucleotide primer. Incorporation of a nucleotide base into the template system can be detected by any of a variety of methods including but not limited to fluorescence and chemiluminescence detection. Alternatively, microcalorimetic detection of the heat generated by the incorporation of a nucleotide into the extending template system using thermopile, thermistor and refractive index measurements can be used to detect extension reactions.

Methods and compositions for identifying and treating lupus

Nucleic acid sequencing method and system employing enhanced detection of nucleotide-specific ternary complex formation

Provided are methods and systems for detecting formation of nucleotide- specific ternary complexes comprising a DNA polymerase, a nucleic acid, and a nucleotide complementary to the templated base of the primed template nucleic acid. The methods and systems facilitate determination of the next correct nucleotide without requiring chemical incorporation of the nucleotide into the primer. This advantageously improves signal-to-noise ratios and increases the quality of results obtainable in a sequencing-by-binding protocol, and enables extended read lengths. These results can even be achieved in procedures employing unlabeled, native nucleotides.

METHOD OF DETERMINING THE NUCLEOTIDE SEQUENCE OF OLIGONUCLEOTIDES AND DNA MOLECULES

The present invention relates to a novel method for analyzing nucleic acid sequences based on real-time detection of DNA polymerase-catalyzed incorporation of each of the four nucleotide bases, supplied individually and serially in a microfluidic system, to a reaction cell containing a template system comprising a DNA fragment of unknown sequence and an oligonucleotide primer. Incorporation of a nucleotide base into the template system can be detected by any of a variety of methods including but not limited to fluorescence and chemiluminescence detection. Alternatively, microcalorimetic detection of the heat generated by the incorporation of a nucleotide into the extending template system using thermopile, thermistor and refractive index measurements can be used to detect extension reactions.

USE OF TEMPLATE SWITCHING FOR DNA SYNTHESIS

A method of preparing a DNA copy of a target polynucleotide using template switching is described. The method includes mixing a double stranded template/primer substrate made up of a DNA primer oligonucleotide associated with a complementary oligonucleotide template strand with a target polynucleotide in a reaction medium and adding a suitable amount of a non-retroviral reverse transcriptase to the reaction medium to extend the DNA primer oligonucleotide from its 3' end to provide a DNA copy polynucleotide. The DNA copy polynucleotide includes a complementary target DNA polynucleotide that is synthesized using the target polynucleotide as a template. Methods of adding nucleotides to the double stranded template/primer substrate are also described. The method can be used to facilitate detection, PCR amplification, cloning, and determination of RNA and DNA sequences.

Methods and compositions for identifying ligands on arrays using indexes and barcodes

Some embodiments provided herein include methods and compositions for the detection of target ligands on an array. In some embodiments, a capture probe specifically binds to a target ligand from a sample, the location of a bead comprising the capture probe in an array is determined, and the bead is decoded to identify the capture probe and the sample. In some embodiments, a barcode is indicative of a capture probe attached to a bead; and an index is indicative of a subpopulation of beads. In some embodiments, the barcode and the index are determined by sequencing.

Use of template switching for DNA synthesis

A method of preparing a DNA copy of a target polynucleotide using template switching is described. The method includes mixing a double stranded template/primer substrate made up of a DNA primer oligonucleotide associated with a complementary oligonucleotide template strand with a target polynucleotide in a reaction medium and adding a suitable amount of a non- retroviral reverse transcriptase to the reaction medium to extend the DNA primer oligonucleotide from its 3' end to provide a DNA copy polynucleotide. The DNA copy polynucleotide includes a complementary target DNA polynucleotide that is synthesized using the target polynucleotide as a template. Methods of adding nucleotides to the double stranded template/primer substrate are also described. The method can be used to facilitate detection, PCR amplification, cloning, and determination of RNA and DNA sequences.

Method of determining the nucleotide sequence of oligonucleotides and DNA molecules

The present invention relates to a novel method for analyzing nucleic acid sequences based on real-time detection of DNA polymerase-catalyzed incorporation of each of the four nucleotide bases, supplied individually and serially in a microfluidic system, to a reaction cell containing a template system comprising a DNA fragment of unknown sequence and an oligonucleotide primer. Incorporation of a nucleotide base into the template system can be detected by any of a variety of methods including but not limited to fluorescence and chemiluminescence detection. Alternatively, microcalorimetic detection of the heat generated by the incorporation of a nucleotide into the extending template system using thermopile, thermistor and refractive index measurements can be used to detect extension reactions.

métodos para a detecção de distúrbios genéticos

Method of detecting target nucleotide sequence, detection target structure to be used in embodying the method, process for producing the same and assay kit for detecting target nucleotide sequence

It is intended to provide a process for producing a detection target structure which is useful in detecting a target nucleotide sequence. For example, a process for producing a detection target structure from a target nucleic acid (1). The target nucleic acid (1) has a target nucleotide sequence 2 as a part thereof (Fig. 1A). According to the above process, the target nucleic acid is extended by using primers so that the part other than the target nucleotide sequence 2 is made double-stranded, thereby giving a detection target structure 4 (Fig. 1B). The detection target structure thus produced, a method of detecting a target nucleotide sequence using the same, a kit for producing a detection target structure and an assay kit for detecting a target nucleotide sequence are also disclosed.

EFFICIENT, EXPANSIVE, USER-DEFINED DNA MUTAGENESIS

The presently disclosed subject matter provides methods for the creation of one or more user-defined mutations that can be located anywhere in a target sequence, such as in a gene. These mutations can comprise single mutations, multiple mutations, or a comprehensive codon mutagenesis library, in which all possible single codon substitutions in a gene are created. These methods can be used on a single-stranded or double-stranded template.

Self-adjusting single contact voltage sensor

Disclosed is a voltage sensing apparatus comprising a signal generator coupled to a first conducting layer and a conductive element having a first voltage, the signal generator configured to superimpose a second voltage to the first voltage. The voltage sensing apparatus also comprises a meter disposed between the first conducting layer and a second conducting layer or between the signal generator and the second conducting layer. An output parameter of the meter is a function of one or more of the group consisting of: the first voltage and the second voltage. The output parameter and the second voltage can be used to adjust a determination of the first voltage.

Nucleic acid sequencing by Raman monitoring of uptake of nucleotides during molecular replication

The methods and apparatus disclosed herein are useful for detecting nucleotides, nucleosides, and bases and for nucleic acid sequence determination. The methods involve detection of a nucleotide, nucleoside, or base using surface enhanced Raman spectroscopy (SERS) or surface enhanced coherent anti-Stokes Raman spectroscopy (SECARS). The detection can be part of a nucleic acid sequencing reaction to detect uptake of a deoxynucleotide triphosphate during a nucleic acid polymerization reaction, such as a nucleic acid sequencing reaction. The nucleic acid sequence of a synthesized nascent strand, and the complementary sequence of the template strand, can be determined by tracking the order of incorporation of nucleotides during the polymerization reaction. Methods for enhancing the SERS signal of a nucleotide or nucleoside by cleaving the base from a sugar moiety are provided. Furthermore, methods for detecting single base repeats are provided.

Mutant Taq Polymerase for Faster Amplification

The invention includes a mutant Taq polymerase, which can significantly extend and amplify a target sequence where the extension conditions are time limited to as little as one second. The mutant Taq polymerase, or a biologically active fragment thereof, has one or more substitutions differing from the wild type as shown in Table I.

method for detecting cytosine methylations

Beschrieben wird ein Verfahren zum Nachweis von 5-Methylcytosin in genomischen DNA-Proben. Zuerst wird eine genomische DNA aus einer DNA-Probe mit einem Reagenz chemisch umgesetzt, wobei 5-Methylcytosin und Cytosin unterschiedlich reagieren. Anschließend wird die vorbehandelte DNA unter Verwendung einer Polymerase mit Primern unterschiedlicher Sequenz amplifiziert. Im nächsten Schritt wird die amplifizierte genomische DNA auf einen Oligonukleotid Array hybridisiert und PCR-Produkte erhalten, die mit einer Markierung versehen sein müssen. Alternativ können die PCR-Produkte in einer Primer Extention Reaktion verlängert werden, wobei auch die Verlängerungsprodukte mit einer Markierung versehen sind. Im letzten Schritt werden die verlängerten Oligonukleotide auf das Vorhandensein der Markierung untersucht.

Single-molecule targeted sequencing method, device and system and application

本发明提供了一种单分子靶向测序(SMTS)方法、装置、系统及应用，所述方法包括：(i)将靶向引物连接到基底表面；(ii)将靶向引物与末端修饰有光学检测标记的模板核酸进行杂交形成靶向引物/模板核酸复合物；(iii)对复合物进行成像；(iv)添加带光学检测标记的核苷酸，使之延伸至探针引物链的3’端；(v)对延伸中的靶向引物链进行成像以鉴定步骤(iv)引入的核苷酸种类；(vi)除去延伸产物上带光学检测标记的核苷酸的可断裂的基团：(vii)重复步骤(iii)至(vi)一次或多次。本发明提供的单分子靶向测序方案通过靶向引物捕获模板核酸，实现单分子靶向测序。

Compositions of standardized mixtures for determining an amount of a nucleic acid

The present invention is directed to methods and compositions for evaluating nucleic acids, methods of preparing such compositions, and applications and business methods employing such compositions and methods. In particular, the present invention provides business methods for operating a gene expression measurement service.

핵산 물질의 팩킹방법

본 발명은 주형 핵산, 상기 주형에 적어도 부분적으로 상보적인 서열을 갖는 프라이머, 폴리머라제 및 변형된-NTP(nucleoside triphosphate)를 포함하는 반응 혼합물을 인큐베이션 시켜 상기 프라이머를 연장시키는 단계를 포함하는, 변형된-NTP를 적어도 부분적으로 포함하는 핵산 물질을 증폭하는 방법으로서, 상기 변형된-NTP는 ATP, TTP, CTP, GTP 및 UTP로 이루어진 군으로부터 선택된 하나 이상이 변형된 것을 포함하는 것인 방법 및 상기 방법에 의해 생산된 핵산 물질을 포함하는 조성물에 관한 것이다. 또한 본 발명은 상기 방법에 이용될 NTP를 포함하고, 상기 NTP가 변형된-NTP를 포함하는 적어도 부분적으로 핵산 패킹 키트를 제공한다. The present invention relates to a method for producing a modified (or modified) nucleic acid comprising the steps of incubating a reaction mixture comprising a template nucleic acid, a primer having a sequence at least partially complementary to said template, a polymerase and a modified nucleoside triphosphate Wherein the modified NTP comprises a modification of at least one selected from the group consisting of ATP, TTP, CTP, GTP and UTP, and a method of amplifying the nucleic acid material at least partially comprising the method To a composition comprising a nucleic acid material produced by the process. The present invention also provides an at least partially nucleic acid packing kit comprising an NTP to be used in the method, wherein the NTP comprises modified NTP.

GERÄT DAS DIE FREIGABE VON NUKLEOTIDEN WÄHREND EINER REAKTION STEUERT

NUCLEIC ACID DETECTION METHOD

The present invention relates to methods for the detection of nucleic acids of defined sequence and kits for use in said methods. The methods employ nicking agent(s), polymerase and oligonucleotide probes to produce probe fragments in the presence of a target nucleic acid. 2. A method according to wherein the second oligonucleotide probe (P2) is attached to a solid material or to a moiety that permits its attachment to a solid material.3. (canceled)4. A method according to wherein the probe (P1) derived strand of the double-stranded nucleic acid amplifier comprises one or more modifications that render it resistant to cleavage by the first and/or second nicking agent(s) claim 1 , such as a phosphorothioate linkage.5. (canceled)6. A method according to wherein the double-stranded nucleic acid amplifier contains two or more nicking agent cleavage sites.7. A method according to wherein the sample is also contacted with a target nucleic acid primer which optionally contains the cleavage site for the first nicking agent.8. A method according to wherein the first oligonucleotide probe (P1) comprises a complementarity region at its 3′ end that is capable of sequence specific hybridisation to the target nucleic acid; whereby claim 1 , following hybridisation of probe (P1) to the target nucleic acid claim 1 , extension of probe (P1) by the polymerase forms a double-stranded nucleic acid that contains the cleavage site for the first nicking agent within the target nucleic acid strand; and whereby the first nicking agent specifically recognises said double-stranded nucleic acid and cleaves said target nucleic acid strand to produce a primer that remains hybridised to the probe (P1) derived strand; and the polymerase extends the primer to produce the double-stranded nucleic acid amplifier which is cleaved in step a) (A).9. A method according to wherein probe (P1) contains the recognition sequence for a probe (P1) nicking agent and whereby the double-stranded nucleic acid ...

METHOD FOR DETECTING GENETIC MUTATION

A method for detecting a genetic mutation is provided. The method comprising: carrying out PCR using a template DNA, a forward primer, a reverse primer and a wild-type oligonucleotide; and detecting an amplified DNA. In the method, the wild-type oligonucleotide comprises LNA or BNA, and on a DNA strand of the template, with which the wild-type oligonucleotide is hybridized, a region with which the wild-type oligonucleotide is a hybridized and a region with which the forward primer or the reverse primer is hybridized partially overlap each other, or the regions are separated from each other by 1 to 18 bases. 1. A method for detecting a genetic mutation , the method comprising:carrying out PCR using a template DNA comprising a target genetic mutation site, a forward primer and a reverse primer each configured to amplify a region containing the target genetic mutation site, and a wild-type oligonucleotide comprising a base sequence complementary to wild type template DNA; anddetecting an amplified DNA comprising a mutant-type genetic mutation site on the basis of the result of PCR amplification,wherein the wild-type oligonucleotide comprises LNA or BNA, andwherein on a DNA strand of the template, with which the wild-type oligonucleotide is hybridized, a region with which the wild-type oligonucleotide is a hybridized and a region with which the forward primer or the reverse primer is hybridized partially overlap each other, or the regions are separated from each other by 1 to 18 bases.2. The method according to claim 1 , wherein the wild-type oligonucleotide comprises BNA.3. The method according to claim 1 , whereinin the PCR amplification, a nucleic acid chain-extending reaction may be carried out using a DNA polymerase at a temperature at which the wild-type oligonucleotide is hybridized with a wild type template DNA comprising a wild-type genetic mutation site, and the wild-type oligonucleotide is substantially not hybridized with a mutant type template DNA ...

CHEMICAL SENSING DEVICE

An apparatus with a transducer having a first output signal and arranged to receive an electrical input. The transducer switches the first output signal between an ON and OFF state. The apparatus has a chemical sensing surface coupled to the transducer arranged to receive a chemical input. A signal generator oscillates one or more of said inputs to vary the switching point of the transducer. The oscillating input may be the chemical input and/or the electrical input. The output signal may be a pulse whose period ON or OFF is determined by the oscillating input modulated by the chemical input. 1. A method of measuring ion concentration in a buffered fluid , the method comprising:(i) monitoring an electrical output signal from an ISFET exposed to the fluid;(ii) releasing or adsorbing a chemical from a titration electrode to the fluid to change said ion concentration until the output signal reaches a predetermined threshold; and(iii) when that predetermined threshold is reached, determining the quantity of chemical released or adsorbed.2. A method according to claim 1 , further comprising (iv) determining the initial ion concentration from knowledge of the buffer capacity and amount of chemical released or adsorbed.3. A method according to claim 1 , further comprising repeating parts (ii) and (iii) at two different times or with different fluids and then:(iv) determining the difference in initial ion concentration from knowledge of the difference in amount of chemical released or adsorbed in each part (ii), wherein the buffer capacity before each part (ii) is substantially the same.4. A method according to claim 3 , further comprising (v) undoing the effects of part (ii) by adsorbing to or releasing from the titration electrode a substantially equal quantity of said chemical from or to the fluid.5. A method according to claim 1 , wherein the threshold is selected from the group consisting of: a predetermined change in the output signal; a predetermined rate of change ...

OPTIMIZED REAL-TIME NUCLEIC ACID DETECTION PROCESSES

This invention provides for compositions for use in real time nucleic acid detection processes. Such real time nucleic acid detection processes are carried out with energy transfer elements attached to nucleic acid primers, nucleotides, nucleic acid probes or nucleic acid binding agents. Real time nucleic acid detection allows for the qualitative or quantitative detection or determination of single-stranded or double-stranded nucleic acids of interest in a sample. Other processes are provided by this invention including processes for removing a portion of a homopolymeric sequence, e.g., poly A sequence or tail, from an analyte or library of analytes. Compositions useful in carrying out such removal processes are also described and provided. Paneling and multiplex analyses of more than one nucleic acid analyte using one sample are also provided. 1. A composition of matter comprising: wherein one or both of the two adjacent oligonucleotides of the first set comprises a first energy transfer element of an energy transfer element pair, and', 'wherein in the presence of the target nucleic strand and a ligase, the two oligonucleotides of the first set are ligatable to each other to form a first ligation product complementary to the target nucleic acid strand; and, 'a first set of two adjacent oligonucleotides complementary to a target nucleic acid strand,'} wherein one or both of the two adjacent oligonucleotides of the second set comprises a second energy transfer element of the energy transfer element pair, and', 'wherein in the presence of the first ligation product and a ligase, the two adjacent oligonucleotides of the second set are ligatable to each other to form a second ligation product complementary to the first ligation product,, 'a second set of two adjacent oligonucleotides complementary to the first ligation product,'}wherein either (i) the first energy transfer element is an energy donor and the second energy transfer element is an energy acceptor, or (ii) ...

LINEARLY-AMPLIFIED INTERNAL CONTROL FOR NUCLEIC ACID AMPLIFICATION REACTION

The present disclosure provides, among other things, systems, methods, and kits include internal amplification controls. Provided internal amplification controls are or include non-target sequences that are amplified during nucleic acid amplification. Provided internal amplification controls are linearly amplified. Provided internal amplification controls are useful for systems, methods and kits for nucleic acid amplification. 1. A method of performing a nucleic acid amplification reaction , comprising steps of:providing an internal amplification control, comprising a single oligonucleotide primer and a nucleic acid template;contacting the internal amplification control with a nucleic acid amplification reagent in a reaction vessel; andperforming a nucleic acid amplification reaction,wherein the nucleic acid amplification template is linearly amplified, andwherein when a target nucleic acid sample is also present in the reaction vessel, the target nucleic acid sample is exponentially or linearly amplified.2. The method of any of the preceding claims , wherein a sequence of the nucleic acid template is or comprises the single oligonucleotide primer or a sequence complementary to the single oligonucleotide primer.3. The method of any of the preceding claims , wherein when the single oligonucleotide primer binds with a nucleic acid template , the single oligonucleotide primer is extended by a polymerase.4. The method of any of the preceding claims , wherein when the single oligonucleotide primer is extended by the polymerase , the probe activates.5. The method of any of the preceding claims , wherein the nucleic acid amplification reagent comprises target specific proteins.6. The method of any of the preceding claims , wherein the target nucleic acid sample is an environmental sample.7. The method of any of the preceding claims , wherein the nucleic acid amplification reagent comprises a DNA polymerase at a concentration of at least about 8.0 U/reaction and a target ...

COMPOSITIONS AND METHODS FOR NUCLEIC ACID SEQUENCING

Provided herein are methods and compositions for improved sequencing techniques using, for example, polymeric particles and/or three-dimensional structures. 114.-. (canceled)15. A composition comprising a plurality of cores surrounded by a shell polymer , wherein:(a) each core of the plurality of cores is surrounded by the shell polymer;(b) the core is formed by polymerized units of core monomers forming a core polymer;(c) a core polynucleotide primer is attached to the core polymer within the core;(d) a target nucleic acid is hybridized to the core primer;(e) the shell polymer is formed by polymerized units of shell monomers; and(f) the shell polymer is not attached to a polynucleotide primer.1628.-. (canceled)29. The composition of claim 15 , wherein the core polymer claim 15 , the shell polymer claim 15 , or both are a hydrogel.30. The composition of claim 15 , wherein each core has a core diameter claim 15 , the shell polymer surrounding each core has a thickness defining an outer shell diameter claim 15 , and the core diameter is (i) about 20% to about 80% of the outer shell diameter claim 15 , or (ii) about 50% of the shell diameter.31. The composition of claim 15 , wherein each core has a core diameter claim 15 , wherein the shell polymer surrounding each core has a thickness defining an outer shell diameter claim 15 , and further wherein: (i) the core diameter is about 200-1200 nm claim 15 , and/or (ii) the shell diameter is about 0.25-5 μm.32. The composition of claim 15 , wherein the core polynucleotide primer is covalently attached to the core polymer.33. (canceled)34. The composition of claim 15 , wherein the core polymer and shell polymer are permeable to a polymerase.3537.-. (canceled)38. The composition of claim 15 , wherein each core further comprises a silica claim 15 , magnetic claim 15 , or paramagnetic bead.39112.-. (canceled)113. The composition of claim 15 , wherein each core further comprises one or more reagents for amplifying the target ...

Methods of enriching and determining target nucleotide sequences

The present invention provides methods, compositions and kits for enriching and determining nucleotide sequences of a plurality of target loci from a sample comprising nucleic acids. The methods comprise one or more cycles of primer extension followed by PCR amplification of target sequences using nested target-specific primers.

ENZYMATIC NUCLEIC ACID SYNTHESIS: COMPOSITIONS AND METHODS FOR INHIBITING PYROPHOSPHOROLYSIS

Nucleotide triphosphate probes containing a molecular and/or atomic tag on a γ and/or β phosphate group and/or a base moiety having a detectable property are disclosed, and kits and method for using the tagged nucleotides in sequencing reactions and various assay. Also, phosphate and polyphosphate molecular fidelity altering agents are disclosed. 1. A method for sequencing nucleic acids , comprising:a) flowing labeled deoxynucleotide triphosphates onto a plurality of template nucleic acids which are hybridized to primer nucleic acids, wherein the plurality of template nucleic acids or the primers are attached to a support in an addressable array format, wherein the labeled nucleotide triphosphates include a molecular tag attached to their base moiety and the molecular tag emits a detectable signal, and wherein the labeled nucleotide triphosphates lack a quencher moiety that absorbs the detectable signal emitted by the molecular tag;b) incorporating with a polymerase the labeled nucleotide triphosphates into the primers; andc) detecting incorporation of the labeled nucleotide triphosphates.2. The method of claim 1 , wherein the molecular tag which is attached to the base moiety comprises a fluorophore moiety.3. The method of claim 1 , wherein the labeled deoxynucleotide triphosphates further comprise a molecular tag attached to their sugar moiety.4. The method of claim 3 , wherein the molecular tag is attached to the 3′ position of the sugar moiety.5. The method of claim 4 , wherein the molecular tag is removable from the 3′ position of the sugar moiety.6. The method of claim 3 , wherein the molecular tag comprises an alkyl group claim 3 , aryl group claim 3 , alkaryl group claim 3 , aralkyl group.7. The method of claim 1 , wherein the labeled deoxynucleotide triphosphates further comprise a molecular tag attached to their beta phosphate or gamma phosphate.8. The method of claim 7 , wherein the molecular tag comprises an alkyl group claim 7 , aryl group claim 7 , ...

SINGLE-MOLECULE NANOFET SEQUENCING SYSTEMS AND METHODS

Real time electronic sequencing devices, chips, and systems are described. Arrays of nanoFET devices are used to provide sequence information about a template nucleic acid in a polymerase-template complex bound to the nanoFET. The nanoFET devices typically have a source, a drain and a gate comprising a nanowire. A single polymerase enzyme complex comprising a polymerase enzyme complexed with the template nucleic acid is bound to the gate. The polymerase is bound to the gate non-covalently through a polymeric binding agent that has two strands, each strand interacting with the nanowire such that the polymerase is in a central location between the strands with the polymeric binding agent extending away from the polymerase complex along the nanowire in both directions. 1. A nanoFET device for sequencing a single nucleic acid template molecule comprising:a source, a drain and a gate, the gate comprising a nanowire;a single polymerase enzyme complex bound to the gate, the polymerase enzyme complex comprising a polymerase enzyme complexed with the template nucleic acid;wherein the polymerase is bound to the gate non-covalently through a polymeric binding agent that has two strands, each strand interacting with the nanowire such that the polymerase is in a central location between the strands with the polymeric binding agent extending away from the polymerase complex along the nanowire in both directions.2. The nanoFET device of wherein the nanowire comprises a carbon nanotube.3. The nanoFET device of wherein the nanowire comprises a silicon nanowire.4. The nanoFET device of wherein the polymeric binding agent comprises a protein or polypeptide.5. The nanoFET device of wherein the polymerase comprises a phi29 type polymerase.6. The nanoFET device of wherein the polymerase comprises a modified phi29 polymerase.7. The nanoFET device of wherein the polymerase is covalently bound to the polymeric binding agent.8. The nanoFET device of wherein the nanowire further comprises ...

DETECTION OF TARGET NUCLEIC ACID SEQUENCE BY PTO CLEAVAGE AND EXTENSION-DEPENDENT SIGNALING OLIGONUCLEOTIDE HYBRIDIZATION ASSAY

The present invention relates to the detection of a target nucleic acid sequence by a PCE-SH (PTO Cleavage and Extension-Dependent Signaling Oligonucleotide Hybridization) assay. The present invention does not use probes to be hybridized with target nucleic acid sequences for providing target signals. Interestingly, the present invention uses probes (signaling oligonucleotides) to be hybridized with the extended strand formed in a target-dependent manner in which the extended strand is synthesized using the CTO artificially selected as templates. 1. A kit for detecting a target nucleic acid sequence from a DNA or a mixture of nucleic acids by a PCE-SH (PTO Cleavage and Extension-Dependent Signaling Oligonucleotide Hybridization) assay , comprising:(a) a probing and targeting oligonucleotide (PTO); wherein the PTO comprises (i) a 3′-targeting portion comprising a hybridizing nucleotide sequence complementary to the target nucleic acid sequence and (ii) a 5′-tagging portion comprising a nucleotide sequence non-complementary to the target nucleic acid sequence; the 5′-tagging portion, the 3′-targeting portion or a junction site between the 5′-tagging portion and the 3′-targeting portion has a cleavage site for an enzyme having a 5′ nuclease activity;(b) an upstream oligonucleotide comprising a hybridizing nucleotide sequence complementary to the target nucleic acid sequence; wherein the upstream oligonucleotide is located upstream of the PTO;(c) a capturing and templating oligonucleotide (CTO); wherein the CTO comprises in a 3′ to 5′ direction (i) a capturing portion comprising a nucleotide sequence complementary to the 5′-tagging portion or a part of the 5′-tagging portion of the PTO and (ii) a templating portion comprising a nucleotide sequence non-complementary to the 5′-tagging portion and the 3′-targeting portion of the PTO; wherein the CTO is not labeled;(d) a signaling oligonucleotide (SO) having at least one label; wherein the SO comprises a complementary ...

BARCODE-FREE SINGLE VESICLE MULTIPLEXED PROTEIN AND RNA ANALYSIS

According to various embodiments, a system and method for characterizing protein and nucleic acid content of a plurality of individual particles. The method includes encapsulating individual particles into compartments also containing analyte specific binding complements with oligonucleotide tags comprising a unique molecular identifier sequence, a sequence to identify the analyte specific binding complement, and a homology domain sequence. Allowing the oligonucleotide tags to hybridize on homology domain to form initial tag pairs, amplifying the tag pairs, using an enzyme to cut at the homology domain, allowing tags to re-hybridize, pooling the compartments, and sequencing. Finally, predicting co-encapsulated analytes by computational identification of clusters based on more frequently found oligonucleotide tag pairs. 1. A method for characterizing protein and nucleic acid content of individual particles , the method comprising:encapsulating a plurality of particles into compartments, the plurality of particles including nucleic acid sequences, the compartments also containing analyte specific binding complements with oligonucleotide tags, the tags including a sequence to identify the analyte specific binding complement and two unique molecular identifier (UMI) sequences separated by a restriction enzyme cleavage site;amplifying the tags;using an enzyme to cut at the restriction site;allowing cut tags to re-hybridize;pooling the compartments;sequencing the nucleic acid sequences of the particles; andpredicting co-encapsulated analytes by computational identification of clusters based on more frequently found UMI pairs.2. The method of claim 1 , wherein the particles are lipid vesicles.3. The method of claim 1 , wherein the oligonucleotide tags include a nucleic acid binding end to bind to nucleic acids associated with the encapsulated individual particle.4. The method of claim 3 , wherein the nucleic acid binding end is a poly-A tail.5. The method of claim 1 , ...

Linearly-amplified internal control for nucleic acid amplification reaction

The present disclosure provides, among other things, systems, methods, and kits include internal amplification controls. Provided internal amplification controls are or include non-target sequences that are amplified during nucleic acid amplification. Provided internal amplification controls are linearly amplified. Provided internal amplification controls are useful for systems, methods and kits for nucleic acid amplification.

SEQUENCING METHOD, APPARATUS, SYSTEM AND APPLICATION THEREOF

A sequencing method comprising: (i) combining a template nucleic acid having a first optical detection label at the end with a primer to obtain a first complex; (ii) imaging the first complex to obtain a first image; (iii) mixing the first complex, polymerase, and one or more of nucleotides with a optical detection label to obtain an extension product by polymerization reaction; (iv) imaging the extension product to obtain a second image; (v) removing the cleavable group of the nucleotides with the optical detection label from the extension product to obtain a second complex; (vi) repeating the above steps (ii) to (v) once or more times to determine the template nucleic acid sequence. The sequencing method can achieve single molecule sequencing by capturing template nucleic acids through primers. 1. A single molecule sequencing method comprising:(i) combining a template nucleic acid having a first optical detection label at the end with a primer to obtain a first complex, wherein the primer being attached to a surface of a substrate;(ii) imaging the first complex to obtain a first image;(iii) mixing the first complex, polymerase, and one or more of nucleotides with a second optical detection label and thus adding the one or more of nucleotides with the second optical detection label to the first complex by polymerization reaction, thereby obtaining an extension product, wherein the nucleotides with the second optical detection label comprises a second optical detection label, a cleavable group and a nucleotide that are sequentially connected;(iv) imaging the extension product to obtain a second image;(v) removing the cleavable group from the extension product to obtain a second complex; and(vi) replacing the first complex with the second complex; and repeating the above steps (ii) to (v) once or more times to determine the template nucleic acid sequence.2. A method according to claim 1 , wherein the first optical detection label is a non-light breakable label.3. A ...

METHODS AND COMPOSITIONS TO GENERATE UNIQUE SEQUENCE DNA PROBES, LABELING OF DNA PROBES AND THE USE OF THESE PROBES

The invention relates generally to the field of the identification of DNA sequences, genes or chromosomes. Methods and composition to obtain Unique Sequence DNA probes are provided. Compositions comprised of and double stranded DNA containing Unique Sequences from which the repetitive sequences are eliminated according to the method described in this invention. The invention also relates to the preservation of cells that have been identified after immunomagnetic selection and fluorescent labeling in order to further interrogate the cells of interest. Furthermore the invention relates to genetic analysis of cells that have been identified after immunomagnetic selection and fluorescent labeling. 1a. a syringe pump for transfer of fluids in cartridge, wherein said fluids are buffer, fixative, or hybridization buffer;b. a sample engagement system for reagent transfer;c. an aspiration probe for drying; andd. a heater block wherein said block operates through a feedback thermocouple, control thermocouple, and temperature controller.. A device for preparing a population of suspect cancer cells for fluorescent in situ hybridization comprising: This application is a divisional application of U.S. application Ser. No. 12/067,532 filed on Oct. 10, 2008, which is a national stage application of PCT/US06/36656 filed on Sep. 20, 2006, which claims priority to U.S. Provisional Applications 60/713,676, filed Sep. 20, 2005; 60/729,536, filed Oct. 24, 2005; and 60/786,117, filed Mar. 27, 2006, all of which are hereby incorporated by reference in their entireties.Field of the InventionThe invention relates generally to the field of identification of DNA sequences, genes or chromosomes.Human genomic DNA is a mixture of unique sequences and repetitive sequences that are present in multiple copies throughout the genome. In some applications, nucleic acid hybridization probes to detect repetitive sequences are desirable. These probes have shown utility in the fields of fetal cell ...

Compositions and methods for single-molecule construction of dna

The present invention provides for compositions and methods for single-molecule construction of DNA. The present invention provides for a method comprising: (a) providing a reaction chamber comprising a solid support bound to a single starter double-stranded (ds) DNA molecule comprising a free end, (b) introducing one or more extension molecules and one or more enzymes capable of joining a payload region of an extension molecule to the free end of starter dsDNA molecule to the reaction chamber wherein the extension molecule comprises an cleavable linker.

Probe Density Considerations and Elongation of Self-Complementary Looped Probes Where Probes Are Attached to a Solid Phase

In a multiplexed assay method carried out in solution, wherein the solution contains nucleic acid targets and, wherein several different types of oligonucleotide probes, each type having a different sequence in a region designated as a target binding domain, are used to detect the nucleic acid targets, said assay method including a method for increasing the effective concentration of the nucleic acid targets at the surface of a bead to which the oligonucleotide probes are bound, by one or more of the following steps:

DNA SEQUENCING BY SYNTHESIS USING RAMA AND IFRARED SPECTROSCOPY DETECTION

This invention provides a process of labeling a polynucleotide analogue to be detected by Raman and/or infrared spectroscopy detection. 25-. (canceled)6. The nucleoside triphosphate analogue of having a Raman spectroscopy peak with wavenumber from 2100 cmto 2260 cm.919-. (canceled)2225-. (canceled)2728-. (canceled)29E. Coli. The process of claim 26 , wherein the DNA polymerase is 9° N polymerase or a variant thereof claim 26 , DNA polymerase I claim 26 , Bacteriophage T4 DNA polymerase claim 26 , Sequenase claim 26 , Taq DNA polymerase or 9° N polymerase (exo-)A485L/Y409V.3176-. (canceled) This application claims priority of U.S. Provisional Application No. 61/489,191, filed May 23, 2011, the content of which is hereby incorporated by reference in its entirety.This invention was made with government support under grant no. R01 HG003582 from the National Institutes of Health. The U.S. Government has certain rights in this invention.Throughout this application, certain publications are referenced in parentheses. Full citations for these publications may be found immediately preceding the claims. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to describe more fully the state of the art to which this invention relates.Sequencing by Synthesis (SBS) has driven much of the “next generation” sequencing technology, allowing the field to approach the $100,000 Genome (1-4). With further improvements in nucleotide incorporation detection methods, SBS could be an engine that drives third-generation platforms leading to the reality of the “$1,000 Genome”. At the same time, since non-fluorescent detection approaches are likely to decrease the cost of obtaining data by avoiding expensive cameras and imaging tools, SBS also offers the possibility of high sensitivity, leading to both longer reads and permitting single molecule sequencing, thereby removing one of the most time-consuming and biased steps, ...

Method for Detecting multiple DNA Mutations and Copy Number Variations

Disclosed are methods for detecting DNA mutations of target genes in a DNA sample by combining single-molecule clonal amplification and mutant primer specific extension detection. In the method, thousands and millions of DNA molecules are locally amplified to form immobilized DNA clusters of identical sequences. Mutation specific primers are used to anneal to the mutant sequences in the DNA clusters and are extended by DNA polymerase to make labeled DNA strands. The labeled DNA clusters are detected to identify the DNA clusters of mutant sequences. This method enables detection of single mutation molecule and direct enumeration of mutation molecules in the sample. Once generated from a DNA sample, the immobilized DNA clusters can be reused many times for detection of different mutations or sequences of interest. Methods for determining differential gene expression and chromosome copy number variation are also disclosed. 1. A method for detecting a DNA mutation of a target gene in a DNA sample , comprising the steps of:a) performing a single-molecule clonal amplification on the DNA sample to obtain a large number of immobilized DNA clusters of identical DNA sequences, wherein each DNA cluster is spatially separated from one another and has a distinguishable physical location;b) adding a first mutation specific primer to the DNA clusters, and annealing the first mutation specific primer to a first mutant sequence, if present, within the DNA clusters;c) adding a DNA polymerase and a dNTP mix containing a first labeled nucleotide to the DNA cluster, and extending the annealed first mutation specific primer to make a first mutation specific strand incorporated with labeled nucleotides; andd) detecting the firstly labeled DNA clusters, thereby determining the number of first mutation molecules in the DNA sample.2. The method of claim 1 , further comprisinge) adding a second mutation specific primer to the DNA clusters, and annealing the second mutation specific primer to ...

Mutant Taq Polymerase for Faster Amplification

The invention includes a mutant Taq polymerase, which can significantly extend and amplify a target sequence where the extension conditions are time limited to as little as one second. The mutant Taq polymerase, or a biologically active fragment thereof, has one or more substitutions differing from the wild type as shown in Table I.

METHOD OF DETERMINING THE NUCLEOTIDE SEQUENCE OF OLIGONUCLEOTIDES AND DNA MOLECULES

The present invention relates to a novel method for analyzing nucleic acid sequences based on real-time detection of DNA polymerase-catalyzed incorporation of each of the four nucleotide bases, supplied individually and serially in a microfluidic system, to a reaction cell containing a template system comprising a DNA fragment of unknown sequence and an oligonucleotide primer. Incorporation of a nucleotide base into the template system can be detected by any of a variety of methods including but not limited to fluorescence and chemiluminescence detection. Alternatively, microcalorimetic detection of the heat generated by the incorporation of a nucleotide into the extending template system using thermopile, thermistor and refractive index measurements can be used to detect extension reactions 1. A method for sequencing DNA , comprising:a) providing a primer/template system comprising a template sequence hybridized to a primer oligonucleotide and a DNA polymerase;b) contacting the primer/template system with a single type of deoxyribonucleotide under conditions that produce a detectable signal when the DNA polymerase incorporates a deoxyribonucleotide onto the 3′ end of the primer oligonucleotide, wherein the single type of deoxyribonucleotide is an unlabeled and unblocked deoxyribonucleotide, and wherein the contacting occurs in a reaction chamber;c) converting, with a device, the detectable signal into an electrical signal based on an electrical potential generated across the device by the detectable signal, wherein the converting occurs in the reaction chamber;d) detecting the electrical signal generated by the detectable signal produced in the reaction chamber; ande) identifying the deoxyribonucleotide that is incorporated onto the 3′ end of the primer oligonucleotide.2. The method of claim 1 , further comprising flushing the reaction chamber with a buffer that is free of deoxyribonucleotides.3. The method of claim 1 , wherein step (b) further comprises: ...

COMPOSITIONS, SYSTEMS, AND METHODS FOR DETECTING THE PRESENCE OF POLYMER SUBUNITS USING CHEMILUMINESCENCE

Under one aspect, a composition includes a substrate; a first polynucleotide coupled to the substrate; a second polynucleotide hybridized to the first polynucleotide; and a catalyst coupled to a first nucleotide of the second polynucleotide, the catalyst being operable to cause a chemiluminogenic molecule to emit a photon. Under another aspect, a method includes providing a catalyst operable to cause a first chemiluminogenic molecule to emit a photon; providing a substrate; providing a first polynucleotide coupled to the substrate; hybridizing a second polynucleotide to the first polynucleotide; coupling a first quencher to a first nucleotide of the second polynucleotide; and inhibiting, by the first quencher, photon emission by the first chemiluminogenic molecule. 1. A composition comprising:a catalyst operable to cause a chemiluminogenic molecule to emit a photon;a substrate;a first polynucleotide coupled to the substrate;a second polynucleotide hybridized to the first polynucleotide; anda quencher coupled to a first nucleotide of the second polynucleotide, the quencher operable to inhibit photon emission by the chemiluminogenic molecule.2. (canceled)3. (canceled)4. (canceled)5. (canceled)6. The composition of claim 1 , wherein the catalyst is coupled to the substrate or coupled to the first polynucleotide.7. (canceled)8. The composition of claim 1 , wherein the quencher is cleavable from the first nucleotide.9. The composition of claim 1 , wherein the quencher is coupled to the first nucleotide via a first claim 1 , and a second moiety is coupled to the first moiety and to the quencher.10. (canceled)11. (canceled)12. The composition of claim 1 , wherein the catalyst is selected from the group consisting of an enzyme claim 1 , a metallic catalyst claim 1 , and a metalorganic catalyst claim 1 , and wherein the enzyme is selected from the group consisting of: a luciferase claim 1 , a 1 claim 1 ,2-dioxetane cleaver and a peroxide generator.13. The composition of ...

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 hydrogen ion concentration (pH), changes in other analyte concentration, and/or binding events associated with chemical processes relating to DNA synthesis. 1. (canceled)2. A chemical detection device , comprising:a substrate of a first semiconductor type; and a chemically-sensitive field effect transistor formed in the substrate, the chemically-sensitive field effect transistor having a floating gate structure and a body terminal coupled to a body biasing voltage source line;', 'a first switch consisting of a first field effect transistor formed in the substrate;', 'a second switch consisting of a second field effect transistor formed in the substrate, wherein a channel of the chemically-sensitive field effect transistor, a channel of the first field effect transistor and the second field effect transistor are each a same semiconductor type; and', 'a well of a second semiconductor type that is formed in the substrate, wherein the chemically-sensitive transistor, the first field effect transistor and the second field effect transistor are each formed in the well., 'a chemical detection pixel including3. The chemical detection device of claim 2 , wherein at least one of the first field effect transistor and the second field effect transistor has a body coupled ...

COMPOSITIONS, SYSTEMS, AND METHODS FOR DETECTING THE PRESENCE OF POLYMER SUBUNITS USING CHEMILUMINESCENCE

Under one aspect, a composition includes a substrate; a first polynucleotide coupled to the substrate; a second polynucleotide hybridized to the first polynucleotide; and a catalyst coupled to a first nucleotide of the second polynucleotide, the catalyst being operable to cause a chemiluminogenic molecule to emit a photon. Under another aspect, a method includes providing a catalyst operable to cause a first chemiluminogenic molecule to emit a photon; providing a substrate; providing a first polynucleotide coupled to the substrate; hybridizing a second polynucleotide to the first polynucleotide; coupling a first quencher to a first nucleotide of the second polynucleotide; and inhibiting, by the first quencher, photon emission by the first chemiluminogenic molecule. 1. A composition including:a substrate;a first polynucleotide coupled to the substrate;a second polynucleotide hybridized to the first polynucleotide; anda catalyst coupled to a first nucleotide of the second polynucleotide, the catalyst being operable to cause a chemiluminogenic molecule to emit a photon.2. The composition of claim 1 , further including a plurality of the chemiluminogenic molecules.3. The composition of claim 2 , the catalyst causing each of the chemiluminogenic molecules to emit a corresponding photon.4. The composition of claim 3 , further including a plurality of reagent molecules claim 3 , the catalyst causing each of the chemiluminogenic molecules to emit a corresponding photon by oxidizing that chemiluminogenic molecule using a reagent molecule.5. The composition of claim 4 , the oxidized chemiluminogenic molecule having an excited state that decays by emitting the corresponding photon.6. The composition of claim 1 , wherein the catalyst is cleavable from the first nucleotide.7. The composition of claim 1 , the catalyst being coupled to the first nucleotide via a first moiety coupled to the nucleotide claim 1 , and a second moiety coupled to the first moiety and to the catalyst.8. ...

METHODS FOR RNA QUANTIFICATION

The disclosure provides for methods, compositions, systems, devices, and kits for quantification of RNA, and determination of efficiency of re verse transcription of RNA to cDNA. 1. A method for determining the functional integrity of an RNA sample , comprising:hybridizing a plurality of oligonucleotides comprising a target specific region that specifically binds to a target in an mRNA molecule of one or more reference genes in the RNA sample, wherein at least 10% of the plurality of oligonucleotides comprises different molecular labels;extending the plurality of oligonucleotides to generate a plurality of nucleic acid molecules of the one or more reference genes;counting the number of molecular labels associated with the plurality of nucleic acid molecules; anddetermining the functional integrity of the RNA sample based on the number of molecular labels associated with the one or more reference genes.2. The method of claim 1 , wherein at least one of the one or more reference genes is a housekeeping gene.3. (canceled)4. (canceled)5. (canceled)6. The method of claim 1 , wherein the RNA sample comprises total RNA.7. The method of claim 1 , wherein the RNA sample comprises mRNA.8. The method of claim 1 , wherein the RNA sample comprises partially degraded RNA.9. The method of claim 1 , wherein the extending step comprises reverse transcription of the mRNA molecule.10. The method of claim 1 , wherein each of the plurality of oligonucleotides comprises a binding site for a primer claim 1 , and comprising amplifying the plurality of nucleic acid molecules using a primer that binds to the binding site.11. (canceled)12. The method of claim 10 , wherein each of the plurality of oligonucleotides or the primer comprises an optically detectable label.13. The method of claim 12 , wherein the optically detectable label is a fluorescent label.14. The method of claim 12 , wherein the plurality of oligonucleotides comprises different fluorescent labels for each of the one or more ...

Sample preparation methods, systems and compositions

The disclosure provides methods, compositions, systems, and kits for the concurrent detection and analysis of different structural and chemical forms of nucleic acids in a sample.

SAMPLE PREPARATION METHODS, SYSTEMS AND COMPOSITIONS

The disclosure provides methods, compositions, systems, and kits for the concurrent detection and analysis of different structural and chemical forms of nucleic acids in a sample. 1114-. (canceled)115. A method of performing an amplification reaction on a first RNA and a first DNA , comprising:a) providing a sample comprising a mixture of said first DNA and said first RNA, wherein said first DNA does not comprise a sequence complementary to said first RNA;b) tagging said first DNA with a first tag without using a transposase;c) tagging said first RNA with a second tag;d) performing an amplification or primer extension reaction on said first DNA with a polymerase that is selective for DNA templates; ande) synthesizing a complementary cDNA strand from said first RNA with a reverse transcriptase.116. The method of claim 115 , wherein said first DNA is selected from the group comprising single-stranded DNA claim 115 , double-stranded DNA claim 115 , triple-stranded DNA claim 115 , or a Holliday junction.117. The method of claim 115 , wherein said first RNA is selected from the group comprising single-stranded RNA claim 115 , double-stranded RNA claim 115 , or a ribozyme.118. The method of claim 115 , wherein said first DNA is cell-free DNA.119. The method of claim 115 , wherein said sample is selected from the group consisting of blood claim 115 , plasma claim 115 , serum claim 115 , cerebrospinal fluid claim 115 , synovial fluid claim 115 , bronchio-alveolar lavage claim 115 , urine claim 115 , stool claim 115 , saliva claim 115 , nasal swab claim 115 , and any combination thereof.120. The method of claim 115 , comprising performing said amplification to generate amplified products.121. The method of claim 120 , further comprising sequencing said amplified products.122. A method of sequencing nucleic acids comprising:a) providing a sample comprising double-stranded nucleic acids and single-stranded nucleic acids; andb) attaching a first adapter to an end of said double ...

Methods and composition to generate unique sequence DNA probes, labeling of DNA probes and the use of these probes

The invention relates generally to the field of the identification of DNA sequences, genes or chromosomes. Methods and composition to obtain Unique Sequence DNA probes are provided. Compositions comprises of and double stranded DNA containing Unique Sequences from which the repetitive sequences are eliminated according to the method described in this invention. The invention also relates to the preservation of cells that have been identified after immunomagnetic selection and fluorescent labeling in order to further interrogate the cells of interest. Furthermore the invention relates to genetic analysis of cells that have been identified after immunomagnetic selection and fluorescent labeling.

Methods and compositions to generate unique sequence DNA probes, labeling of DNA probes and the use of these probes

The invention relates generally to the field of the identification of DNA sequences, genes or chromosomes. Methods and composition to obtain Unique Sequence DNA probes are provided. Compositions comprised of and double stranded DNA containing Unique Sequences from which the repetitive sequences are eliminated according to the method described in this invention. The invention also relates to the preservation of cells that have been identified after immunomagnetic selection and fluorescent labeling in order to further interrogate the cells of interest. Furthermore the invention relates to genetic analysis of cells that have been identified after immunomagnetic selection and fluorescent labeling.

System for the detection and enumeration of suspect target cells in a mixed cell population

The invention relates generally to the field of identification of DNA sequences, genes or chromosomes. Methods and composition to obtain Unique Sequence DNA probes are provided. Composition comprises of any double stranded DNA containing Unique Sequences from which the repetitive sequences are eliminated according to the method described in this invention. The invention also relates to the preservation of cells that have been identified after immunomagnetic selection and fluorescent labeling in order to further interrogate the cells of interest. Furthermore the invention relates to genetic analysis of cells that have been identified after immunomagnetic selection and fluorescent labeling.

Optimization of gene expression analysis using immobilized capture probes

Disclosed are methods of multiplexed analysis of oligonucleotides in a sample, including a method of preventing a significant reduction in duplexes detectable in a hybridization assay involving (i) selecting probe lengths for sets of oligonucleotide probes, wherein probes include different subsequences such that at least one subsequence is complementary to a subsequence in a cognate target; wherein probes for longer cognate targets are longer in length than probes for shorter cognate targets, (ii) selecting, for each set of probes, a density of oligonucleotides probes attached per unit area on a solid phase carrier which is below a limit at which the significant reduction in detectable duplexes is predicated to take place, (iii) producing the probes and affixing them to different solid phase carriers at the selected density, and (iv) annealing targets to the probes, wherein signal intensities of probes and targets of different lengths are about the same.

Methods for sequencing a polynucleotide template

The invention relates to methods for pairwise sequencing of a polynucleotide template which result in the sequential determination of nucleotide sequence in two distinct and separate regions of the polynucleotide template.

Methods for sequencing a polynucleotide template

The invention relates to methods for pairwise sequencing of a polynucleotide template which result in the sequential determination of nucleotide sequence in two distinct and separate regions of the polynucleotide template.

Methods and compositions for identifying and treating lupus

본 발명은 루푸스와 연관된 특유한 세트의 유전자 변이를 제공한다. 그러한 유전자 변이를 검출하고, 루푸스가 발병할 위험을 평가하고, 또한 루푸스를 진단 및 치료하기 위한 방법을 또한 제공한다.

self-assembled ribonucleoprotein nanoparticles

본 발명은 Single guide RNA(sgRNA) 영역과 small interfering RNA(siRNA) 영역을 갖는 복수 개의 제1 반복단위를 포함하는 폴리리보뉴클레오티드; 및 하나 이상의 상기 sgRNA 영역에 결합된 뉴클레아제 단백질을 포함하는, 스스로 조립되는 폴리리보뉴클레오티드-단백질 복합체 및 그 용도에 관한 것이다. The present invention relates to a polyribonucleotide comprising a plurality of first repeating units having a single guide RNA (sgRNA) region and a small interfering RNA (siRNA) region; And a nuclease protein bound to at least one of said sgRNA regions, and to uses thereof.

Methods for detecting analytes of varying abundance

본 발명은, 샘플에서 다수의 분석물을 검출하는 방법을 제공하되, 상기 분석물은 상기 샘플에서 다양한 수준의 풍부도(abundance)를 가지며, 상기 방법은, (i) 상기 샘플로부터 다수의 분취물들(aliquots)을 제공하는 단계; 및 (ii) 각각의 분취물에서, 각각의 분취물에 대해 별개의 멀티플렉스 어세이(multiplex assay)를 수행함으로써 상기 분석물의 상이한 서브세트(subset)를 검출하는 단계, 각각의 서브세트에서 상기 분석물은 상기 샘플 중의 그들의 예측된 풍부도에 기초하여 선택됨;을 포함한다.

Methods of sequencing polynucleotides

본 발명은 고처리율 핵산 서열분석 방법의 개선, 특히 페어와이즈(pairwise) 서열분석 동안 연장 반응을 수행하는 방법에 대한 개선에 관한 것이다. 본 발명은 페어와이즈 서열분석 동안 가닥 재합성 연장 반응을 수행하는 방법에 관한 것으로, 상기 가닥 재합성 연장 반응은 제1 서열분석 판독과 제2 서열분석 판독 사이에서 수행되고, 상기 가닥 재합성 연장 반응은 하나 이상의 고정된 프라이머를 연장하여 제1 주형 가닥을 복사하여 제2 고정된 주형 가닥을 생성하고; 그러한 가닥 재합성 연장 반응은 55℃ 미만의 온도에서, 바람직하게는 38℃에서 비-열안정성 가닥 치환 폴리머라제를 사용하여 수행되는 것을 특징으로 한다.

Compositions, systems, and methods for detecting the presence of polymer subunits using chemiluminescence

Under one aspect, a composition includes a substrate; a first polynucleotide coupled to the substrate; a second polynucleotide hybridized to the first polynucleotide; and a catalyst coupled to a first nucleotide of the second polynucleotide, the catalyst being operable to cause a chemiluminogenic molecule to emit a photon. Under another aspect, a method includes providing a catalyst operable to cause a first chemiluminogenic molecule to emit a photon; providing a substrate; providing a first polynucleotide coupled to the substrate; hybridizing a second polynucleotide to the first polynucleotide; coupling a first quencher to a first nucleotide of the second polynucleotide; and inhibiting, by the first quencher, photon emission by the first chemiluminogenic molecule.

Methods for RNA quantification

Provided herein are methods, compositions, systems, devices, and kits for quantification of RNA, and determination of efficiency of reverse transcription of RNA to cDNA.

Methods and compositions for identifying and treating lupus

A unique set of genetic variations associated with lupus are provided. Also provided are methods for detecting such genetic variations and for assessing risk of developing lupus as well as for diagnosing and treating lupus.

Methods for selectively suppressing non-target sequences

The invention generally relates to negative selection of nucleic acids. The invention provides methods and systems that remove unwanted segments of nucleic acid in a sample so that a target gene or region of interest may be analyzed without interference from the unwanted segments. A sample is obtained that includes single-stranded nucleic acid with one or more unwanted segments. Complementary nucleic acid is added to the single-stranded nucleic acid to create a double-stranded region that includes the unwanted segment. The double-stranded region is then digested, leaving single-stranded nucleic acid that includes the target gene or region of interest. This allows paralogs, pseudogenes, repetitive elements, and other segments of the genome that may be similar to the target gene or region of interest to be removed from the sample.

Array and method for detecting spatial information of nucleic acids

Provided are a method for detecting spatial information of nucleic acids in a sample, as well as a nucleic acid array used in the method and a method for producing the nucleic acid array.

Method for retaining even coverage of short insert libraries

The invention relates to a method of preparing a library of template polynucleotides with uniform sequence representation and to use of a library of templates prepared using this method for solid-phase nucleic acid amplification. In particular, the invention relates to a method of preparing a library of template polynucleotides which have common sequences at their 5′ ends and at their 3′ ends, which contains even representation of all the fragments present in a starting sample of nucleic acid before fragmentation. The invention is especially applicable to the preparation of short insert libraries, where the sample fragments are less than 150 base pairs in length.

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 hydrogen ion concentration (pH), changes in other analyte concentration, and/or binding events associated with chemical processes relating to DNA synthesis.

Chemically-sensitive sensor array device

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 hydrogen ion concentration (pH), changes in other analyte concentration, and/or binding events associated with chemical processes relating to DNA synthesis.

Method for measuring analytes using large scale chemfet 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 hydrogen ion concentration (pH), changes in other analyte concentration, and/or binding events associated with chemical processes relating to DNA synthesis.

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 hydrogen ion concentration (pH), changes in other analyte concentration, and/or binding events associated with chemical processes relating to DNA synthesis.

Chemically-sensitive sensor array calibration circuitry

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 hydrogen ion concentration (pH), changes in other analyte concentration, and/or binding events associated with chemical processes relating to DNA synthesis.

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 hydrogen ion concentration (pH), changes in other analyte concentration, and/or binding events associated with chemical processes relating to DNA synthesis.

Sensor arrays and nucleic acid sequencing applications

Embodiments of the present invention provide devices methods for sequencing DNA using arrays of reaction regions containing electronic sensors to monitor changes in solutions contained in the reaction regions. Test and fill reaction schemes are disclosed that allow DNA to be sequenced. By sequencing DNA using parallel reactions contained in large arrays, DNA can be rapidly sequenced.

Method of determining the nucleotide sequence of oligonucleotides and DNA molecules

The present invention relates to a novel method for analyzing nucleic acid sequences based on real-time detection of DNA polymerase-catalyzed incorporation of each of the four nucleotide bases, supplied individually and serially in a microfluidic system, to a reaction cell containing a template system comprising a DNA fragment of unknown sequence and an oligonucleotide primer. Incorporation of a nucleotide base into the template system can be detected by any of a variety of methods including but not limited to fluorescence and chemiluminescence detection. Alternatively, microcalorimetic detection of the heat generated by the incorporation of a nucleotide into the extending template system using thermopile, thermistor and refractive index measurements can be used to detect extension reactions.

Methods and compositions for identifying ligands on an array using indices and barcodes

本文提供的一些实施方案包括用于在阵列上检测靶配体的方法和组合物。在一些实施方案中，捕获探针特异性结合到来自样本的靶配体，确定阵列中包含该捕获探针的珠的位置，并且对该珠进行解码以识别该捕获探针和该样本。在一些实施方案中，条形码指示附接于珠的捕获探针；并且索引指示珠亚群。一些实施方案涉及在珠阵列上对几个不同核酸样本的靶多核苷酸进行测序。

Method for determining snp genotype

본 발명은 단일 뉴클레오타이드 변이에 대한 프로빙 앤드 태깅 올리고뉴클레오타이드(Probing and Tagging Oligonucleotide for Single Nucleotide Variation; PTO-SNV)를 이용하여 단일 뉴클레오타이드 다형성(single nucleotide polymorphism; SNP) 유전자형을 결정하는 신규한 방법에 관한 것이다. 본 발명은 SNP 유전자형 분석 반응에서 오직 하나의 대립 유전자-특이적 올리고뉴클레오타이드만으로, 분석되는 타겟 핵산 서열이 관심 있는 SNP 대립 유전자에 대해 동형접합인지 또는 이형접합인지, 또는 관심 있는 SNP 대립 유전자가 존재하지 않는지를 결정할 수 있는 SNP 유전자형 분석을 위한 신규한 프로토콜을 제공한다. The present invention relates to a novel method for determining single nucleotide polymorphism (SNP) genotypes using Probing and Tagging Oligonucleotide for Single Nucleotide Variation (PTO-SNV) . The present invention is based on the finding that only one allele-specific oligonucleotide in the SNP genotyping reaction is capable of detecting whether the target nucleic acid sequence being analyzed is homozygous or heterozygous for the SNP allele of interest or the SNP allele of interest SNP genotypic analysis that can be used to determine whether or not genotypic SNPs are present.

Nucleotide compositions and uses thereof

The present invention relates to preparation of nucleotide compositions and uses thereof for conducting nucleic acid analyzes. The compositions and methods embodied in the present invention are particularly useful for nucleic acid analyzes that require high-resolution detection of labeled nucleotides or labeled nucleic acid targets.

Detection of Nephrotic Hemorrhagic Fever Virus

본 발명은 서열 목록에서 SEQ ID Nos. 1 내지 5로 표시되는 핵산 및 그것의 상보 서열들로 이루어지는 군으로부터 선택된 최소한 하나의 핵산의 일부분을 역전사 및 2단계의 중합효소 연쇄 반응(PCR)에 의해 증폭시키는 것으로 이루어지는, 신증후성 출혈열 바이러스(이하 HFRSV라 언급한다)의 검출 방법, 이 방법에 의해 DNA가 증폭되는 시험될 샘플중의 HFRSV를 검출하기 위한 방법, 및 특정의 핵산 서열 또는 그것의 변이 서열을 가지는, HFRSV 검출에 사용하기 위한 프라이머에 관한 것이다. The present invention relates to SEQ ID Nos. Nephrotic hemorrhagic fever virus (hereinafter referred to as amplification of a portion of at least one nucleic acid selected from the group consisting of nucleic acids represented by 1 to 5 and its complementary sequences by reverse transcription and two-step polymerase chain reaction (PCR)) A method for detecting HFRSV in a sample to be tested in which DNA is amplified by the method, and a primer for use in detecting HFRSV having a specific nucleic acid sequence or a variant sequence thereof. It is about.

Simultaneous spatio-temporal measurement of gene expression and cellular activity

Provided herein are methods for simultaneous spatio-temporal measurement of gene expression and cellular activity.

LASH method for single molecule sequencing and target nucleic acid detection

발광 반응을 위한 성분을 이용하여 단일 핵산 분자를 서열분석 또는 검출하기 위한 방법 및 시스템이 본 명세서에 제공된다.

Nucleic acid sequencing by Raman monitoring of uptake of precursors during molecular replication

The methods, compositions and apparatus disclosed herein are of use for nucleic acid sequence determination. The methods involve isolation of one or more nucleic acid template molecules and polymerization of a nascent complementary strand of nucleic acid, using a DNA or RNA polymerase or similar synthetic reagent. As the nascent strand is extended one nucleotide at a time, the disappearance of nucleotide precursors from solution is monitored by Raman spectroscopy or FRET. The nucleic acid sequence of the nascent strand, and the complementary sequence of the template strand, may be determined by tracking the order of incorporation of nucleotide precursors during the polymerization reaction. Certain embodiments concern apparatus comprising a reaction chamber and detection unit, of use in practicing the claimed methods. The methods, compositions and apparatus are of use in sequencing very long nucleic acid templates in a single sequencing reaction.

Reagents, kits and methods for molecular barcoding

Multimeric barcoding reagents for labelling a target nucleic acid comprise: first and second barcode molecules linked together, wherein each of the barcode molecules comprises a nucleic acid sequence comprising a barcode region; and first and second barcoded oligonucleotides. The multimeric barcoding reagents enable spatial sequencing. A single multimeric barcoding reagent can be used to label sub-sequences of an intact nucleic acid molecule or co-localised fragments of a nucleic acid molecule. The labelled sub-sequences can be sequenced and the sequencing data processed to determine the sequence of sub-sequences from a single intact nucleic acid molecule or from co-localised fragments of a nucleic acid molecule. Corresponding libraries, kits, methods and uses are provided.

Methods of enriching and determining target nucleotide sequences

The present invention provides methods, compositions and kits for enriching and determining nucleotide sequences of a plurality of target loci from a sample comprising nucleic acids. The methods comprise one or more cycles of primer extension followedby PCR amplification of target sequences using nested target-specific primers.

Intermittent detection during analytical reactions

Methods, devices, and systems for performing intermittent detection during analytical reactions are provided. Such methods facilitate collection of reaction data from disparate reaction times. Further, such methods are useful for reducing photo-induced damage of one or more reactants in an illuminated analytical reaction at a given reaction time. In preferred embodiments, the reaction mixture is subjected to at least one illuminated and non-illuminated period and allowed to proceed such that the time in which the reaction mixture is illuminated is less than a photo-induced damage threshold period.

Ligation assays in liquid phase

Ligation assays in liquid phase for detecting nucleic acid sequences.

LASH method for single molecule sequencing and target nucleic acid detection

本明細書において、発光反応のための成分を利用して単一の核酸分子を配列決定または検出するための方法およびシステムが提供される。【選択図】図１

Method for detecting analytes of varying abundance

The present invention provides a method of detecting multiple analytes in a sample, wherein said analytes have varying levels of abundance in the sample, said method comprising: (i) providing multiple aliquots from the sample; and (ii) in each aliquot, detecting a different subset of the analytes by performing a separate multiplex assay for each aliquot, wherein the analytes in each subset are selected based on their predicted abundance in the sample.

Single molecule sequencing method, device, system, and application

A sequencing method comprising: (i) combining a template nucleic acid having a first optical detection label at the end with a primer to obtain a first complex; (ii) imaging the first complex to obtain a first image; (iii) mixing the first complex, polymerase, and one or more of nucleotides with a optical detection label to obtain an extension product by polymerization reaction; (iv) imaging the extended first complex to obtain a second image; (v) removing the cleavable group of the nucleotides with the optical detection label from the extension product to obtain a second complex; (vi) repeating the above steps (ii) to (v) once or more times to determine the template nucleic acid sequence. The sequencing method can achieve single molecule sequencing by capturing template nucleic acids through primers.

DNA sequencing by synthesis using Raman and infrared spectroscopy detection

This invention provides a process of labeling a polynucleotide analogue to be detected by Raman and/or infrared spectroscopy detection.

Array and method for detecting spatial information of nucleic acids

Provided are a method for detecting spatial information of nucleic acids in a sample, as well as a nucleic acid array used in the method and a method for producing the nucleic acid array.

Array and method for detecting spatial information of nucleic acids

Provided are a method for detecting spatial information of nucleic acids in a sample, as well as a nucleic acid array used in the method and a method for producing the nucleic acid array.

Nucleic acid probe with single fluorophore label bound to internal cytosine for use in loop mediated isothermal amplification

The present invention provides novel probes for use in LAMP detection methods. The probes contain a single fluorophore label bound to an internal cytosine residue of the probe. The probes are particularly useful in the detection of chlamydia and gonorrhea infections in a patient.

Nucleic acid sequencing by Raman monitoring of uptake of nucleotides during molecular replication

The methods and apparatus disclosed herein are useful for detecting nucleotides, nucleosides, and bases and for nucleic acid sequence determination. The methods involve detection of a nucleotide, nucleoside, or base using surface enhanced Raman spectroscopy (SERS) or surface enhanced coherent anti-Stokes Raman spectroscopy (SECARS). The detection can be part of a nucleic acid sequencing reaction to detect uptake of a deoxynucleotide triphosphate during a nucleic acid polymerization reaction, such as a nucleic acid sequencing reaction. The nucleic acid sequence of a synthesized nascent strand, and the complementary sequence of the template strand, can be determined by tracking the order of incorporation of nucleotides during the polymerization reaction. Methods for enhancing the SERS signal of a nucleotide or nucleoside by cleaving the base from a sugar moiety are provided. Furthermore, methods for detecting single base repeats are provided.

Template switch is used for the purposes of DNA synthesis

描述了使用模板转换制备靶多核苷酸的DNA拷贝的方法。所述方法包括：将双链模板/引物底物（其由与互补寡核苷酸模板链结合的DNA引物寡核苷酸组成）与靶多核苷酸一起在反应介质中混合，并将合适量的非逆转录病毒逆转录酶加入反应介质中，以使DNA引物寡核苷酸从它的3’末端延伸从而提供DNA拷贝多核苷酸。所述DNA拷贝多核苷酸包括使用靶多核苷酸作为模板合成的互补的靶DNA多核苷酸。还描述了向双链模板/引物底物添加核苷酸的方法。所述方法可以用于便利RNA和DNA序列的检测、PCR扩增、克隆和测定。

Compositions and methods for nucleic acid sequencing

Provided herein are methods and compositions for improved sequencing techniques using, for example, polymeric particles and/or three-dimensional structures.

Processes for detection of nucleic acids

This invention provides for compositions for use in real time nucleic acid detection processes. Such real time nucleic acid detection processes are carried out with energy transfer elements attached to nucleic acid primers, nucleotides, nucleic acid probes or nucleic acid binding agents. Real time nucleic acid detection allows for the qualitative or quantitative detection or determination of single-stranded or double-stranded nucleic acids of interest in a sample. Other processes are provided by this invention including processes for removing a portion of a homopolymeric sequence, e.g., poly A sequence or tail, from an analyte or library of analytes. Compositions useful in carrying out such removal processes are also described and provided.

A method for detecting multiple dna mutations and copy number variations

Disclosed are methods for detecting DNA mutations of target genes in a DNA sample by combining single-molecule clonal amplification and mutant primer specific extension detection. In the method, thousands and millions of DNA molecules are locally amplified to form immobilized DNA clusters of identical sequences. Mutation specific primers are used to anneal to the mutant sequences in the DNA clusters and are extended by DNA polymerase to make labeled DNA strands. The labeled DNA clusters are detected to identify the DNA clusters of mutant sequences. This method enables detection of single mutation molecule and direct enumeration of mutation molecules in the sample. Once generated from a DNA sample, the immobilized DNA clusters can be reused many times for detection of different mutations or sequences of interest. Methods for determining differential gene expression and chromosome copy number variation are also disclosed.

Methods and reagents for molecular barcoding

Methods and reagents for preparing nucleic acid samples for sequencing are provided. The samples include formalin-fixed paraffin-embedded (FFPE) samples. The methods comprise contacting a nucleic acid sample with a multimeric barcoding reagent comprising barcode regions linked together and appending barcode sequences to nucleic acid sequences of a target nucleic acid molecule. Methods are also provided that additionally use in-vitro transposition, coupling sequences and/or primer-extension to append barcode sequences to nucleic acid sequences of a target nucleic acid molecule.

Bisulfite conversion of DNA

The present invention provides an improved method for the bisulfite conversion of DNA, and facilitates the analysis of cytosine methylation of genomic DNA. Novel combinations of denaturing solvents, new reaction conditions and new purification methods provide surprisingly efficacious methods for bisulfite conversion of DNA relative to prior art methods. The converted DNA may subsequently be analyzed by many different methods.

Methods and compositions for assessing nucleic acids

The present invention is directed to methods and compositions for evaluating nucleic acids, methods of preparing such compositions, and applications and business methods employing such compositions and methods. In particular, the present invention provides business methods for operating a gene expression measurement service.

Methods and compositions for identifying and treating lupus.

La presente invención se refiere a un método para diagnosticar lupus o valorar el riesgo de desarrollar lupus en un sujeto, el método comprendiendo: a) detectar en una muestra biológica del sujeto, la presencia de una firma genética indicativa de lupus o riesgo de desarrollar lupus, en donde dicha firma genética comprende la ausencia de un alelo menor de polimorfismo de nucleótido sencillo (SNP) rs4963128 y la presencia de un alelo menor de SNP rs2269368; y b) terminar que el sujeto tiene lupus o está en riesgo de desarrollar lupus cuando la firma genética se encuentra presente.

Synthetic Sequencing Method Using Continuous Labeling Method

본 발명은 표적 폴리뉴클레오티드 분자를 시퀀싱하는 방법을 제공한다. 일부 실시형태에서, 본 발명은 합성에 의한 시퀀싱 방법을 제공하며, 이때 뉴클레오티드-접합체 복합체들의 서로 다른 서브세트들은 복수의 신생 핵산 복제 가닥들의 각 반복적 연장 동안 순차적으로 형성되고 검출되며, 이때 각각의 신생 핵산 복제 가닥은 복수의 표적 폴리뉴클레오티드 분자들 중 하나에 상보적이다. 일부 실시형태에서, 이러한 복수의 표적 폴리뉴클레오티드 분자들은 고체 지지체 상에 배열된다.

Systems and methods for detecting multiple analytes

A method for detecting different analytes includes mixing different analytes with sensing probes, wherein at least some of the sensing probes are specific to respective ones of the analytes. The analytes respectively are captured by the sensing probes that are specific to those analytes. Fluorophores respectively are coupled to sensing probes that captured respective analytes. The sensing probes are mixed with beads, wherein the beads are specific to respective ones of the sensing probes, and wherein the beads include different codes identifying the analytes to which those sensing probes are specific. The sensing probes respectively are coupled to beads that are specific to those sensing probes. The beads are identified that are coupled to the sensing probes that captured analytes using at least fluorescence from the fluorophores coupled to those sensing probes. The analytes that are captured are identified.

Age predicting method using dna methylation

The present invention relates to an age predicting method using DNA methylation. More specifically, the present invention relates to a method for predicting the age of a test subject by measuring methylation levels (ratio of DNA methylation to non-methylation) for CpG markers of target genes in a sample derived from the test subject.

Methods and compositions for identifying ligands on arrays using indexes and barcodes

Some embodiments provided herein include methods and compositions for the detection of target ligands on an array. In some embodiments, a capture probe specifically binds to a target ligand from a sample, the location of a bead comprising the capture probe in an array is determined, and the bead is decoded to identify the capture probe and the sample. In some embodiments, a barcode is indicative of a capture probe attached to a bead; and an index is indicative of a subpopulation of beads. Some embodiments relate to sequencing target polynucleotides from several different nucleic acids samples on a bead array.

Method for detecting cytosine methylations

Disclosed is a method for detecting 5-methylcytosine in genomic DNA samples. First, genomic DNA from a DNA sample is chemically reacted with a reagent, 5-methylcytosine and cytosine reacting differently. The pre-treated DNA is then ampified with primers from a different sequence using a polymerase. In the following step, the amplified genomic DNA is hybridised to an oligonucleotide array and PCR products are obtained which must be provided with an identifying mark. Alternatively, the PCR products can be extended in a Primer Extension Reaction, the extension products also being provided with an identifying mark. The last step involves examining the extended oligonucleotides for the presence of the identifying mark.

Real-time nucleic acid detection processes and compositions

This invention provides compositions for use in real time nucleic acid detection processes. Such real time nucleic acid detection processes are carried out with energy transfer elements attached to nucleic acid primers, nucleotides, nucleic acid probes or nucleic acid binding agents. Real time nucleic acid detection allows for the qualitative or quantitative detection or determination of single-stranded or double-stranded nucleic acids of interest in a sample. Other processes are provided by this invention including processes for removing a portion of a homopolymeric sequence, e.g., poly A sequence or tail, from an analyte or library of analytes. Compositions useful in carrying out such removal processes are also described and provided.

Business methods for assessing nucleic acids

The present invention is directed to methods and compositions for evaluating nucleic acids, methods of preparing such compositions, and applications and business methods employing such compositions and methods. In particular, the present invention provides business methods for operating a gene expression measurement service.

Use of template switching for dna synthesis