公开(公告)号：US20160348149A1

公开(公告)日：2016-12-01

申请号：US15144529

申请日：2016-05-02

Applicant: Fluidigm Corporation

Inventor： Kenneth J. Livak ,  Jason A. A. West ,  Robert C. Jones

IPC: C12Q1/68

CPC classification number: C12Q1/682 ,  C12Q1/6876 ,  C12Q2600/178 ,  C12Q2525/155 ,  C12Q2525/307 ,  C12Q2537/161

Abstract: Reagents and methods are provided for detecting the presence of a target polynucleotide in a sample are disclosed. In one aspect, a method for producing a labeled amplification product by amplifying a target nucleic acid sequence to produce an amplification product comprising the target sequence, a first probe-binding sequence 5′ to the target sequence, and a second probe-binding sequence 3′ to the target sequence, thereby producing an amplification product; and hybridizing a first detection probe to the amplification product, the first detection probe comprising a first segment that hybridizes to the first probe-binding sequence and a second segment that hybridizes to the second probe-binding sequence, thereby producing a labeled amplification product is disclosed.

Abstract translation: 提供了用于检测样品中靶多核苷酸的存在的试剂和方法。 一方面，通过扩增靶核酸序列以产生包含靶序列的扩增产物，与靶序列的第一探针结合序列5'和第二探针结合序列3来产生标记的扩增产物的方法 '到靶序列，从而产生扩增产物; 并且将第一检测探针与扩增产物杂交，所述第一检测探针包含与第一探针结合序列杂交的第一区段和与第二探针结合序列杂交的第二区段，从而产生标记的扩增产物 。

公开(公告)号：US09644231B2

公开(公告)日：2017-05-09

申请号：US14713887

申请日：2015-05-15

Applicant: Fluidigm Corporation

Inventor： Kenneth J. Livak ,  Stacey N. Meyers ,  Xiaohui Wang ,  Jun Wang

IPC: G01N21/64 ,  C40B30/04 ,  C12Q1/68

CPC classification number: C12Q1/6818 ,  C12Q2525/151

Abstract: The invention provides a method for detecting a target nucleotide sequence by tagging the nucleotide sequence with a nucleotide tag, providing a probe oligonucleotide with a melting temperature Tm1, comprising a regulatory sequence and a nucleotide tag recognition sequence; incorporating the probe oligonucleotide into the tagged polynucleotide in a polynucleotide amplification reaction, providing a regulatory oligonucleotide with a melting temperature Tm2, comprising a sequence segment that complementary to the regulatory sequence and a tail segment that does not hybridize to the probe nucleotide when the sequence segment and the regulatory sequence are annealed, amplifying the tagged target nucleic acid sequence in a PCR amplification reaction using the probe oligonucleotide as a primer, and using a DNA polymerase with high strand displacement activity and low 5′-nuclease activity, and detecting the amplification product; wherein Tm1 and Tm2 are higher than the annealing temperature associated with the polynucleotide amplification reaction.

公开(公告)号：US09587272B2

公开(公告)日：2017-03-07

申请号：US14340446

申请日：2014-07-24

Applicant: Fluidigm Corporation

Inventor： Kenneth J. Livak ,  Stacey N. Meyers ,  Jun Wang ,  Xiaohui Wang

IPC: C07H21/04 ,  C12Q1/68

CPC classification number: C12Q1/6818 ,  C12Q2525/151

Abstract: The invention provides a method for detecting a target nucleotide sequence by tagging the nucleotide sequence with a nucleotide tag, providing a probe oligonucleotide with a melting temperature Tm1, comprising a regulatory sequence and a nucleotide tag recognition sequence; incorporating the probe oligonucleotide into the tagged polynucleotide in a polynucleotide amplification reaction, providing a regulatory oligonucleotide with a melting temperature Tm2, comprising a sequence segment that is at least partially complementary to the regulatory sequence, amplifying the tagged target nucleic acid sequence in a PCR amplification reaction using the probe oligonucleotide as a primer, and detecting the amplification product; wherein Tm1 and Tm2 are higher than the annealing temperature associated with the polynucleotide amplification reaction.

Abstract translation: 本发明提供了通过用核苷酸标签标记核苷酸序列来检测靶核苷酸序列的方法，提供了包含调节序列和核苷酸标签识别序列的具有解链温度Tm1的探针寡核苷酸; 在多核苷酸扩增反应中将探针寡核苷酸掺入标记的多核苷酸中，提供具有解链温度Tm2的调节性寡核苷酸，其包含与调节序列至少部分互补的序列片段，在PCR扩增中扩增标记的靶核酸序列 使用探针寡核苷酸作为引物的反应，检测扩增产物; 其中Tm1和Tm2高于与多核苷酸扩增反应相关的退火温度。

公开(公告)号：US20150147755A1

公开(公告)日：2015-05-28

申请号：US14340446

申请日：2014-07-24

Applicant: Fluidigm Corporation

Inventor： Kenneth J. Livak ,  Stacey N. Myers ,  Jun Wang ,  Xiaohui Wang

IPC: C12Q1/68

CPC classification number: C12Q1/6818 ,  C12Q2525/151

Abstract: The invention provides a method for detecting a target nucleotide sequence by tagging the nucleotide sequence with a nucleotide tag, providing a probe oligonucleotide with a melting temperature Tm1, comprising a regulatory sequence and a nucleotide tag recognition sequence; incorporating the probe oligonucleotide into the tagged polynucleotide in a polynucleotide amplification reaction, providing a regulatory oligonucleotide with a melting temperature Tm2, comprising a sequence segment that is at least partially complementary to the regulatory sequence, amplifying the tagged target nucleic acid sequence in a PCR amplification reaction using the probe oligonucleotide as a primer, and detecting the amplification product; wherein Tm1 and Tm2 are higher than the annealing temperature associated with the polynucleotide amplification reaction.

Abstract translation: 本发明提供了通过用核苷酸标签标记核苷酸序列来检测靶核苷酸序列的方法，提供了具有调节序列和核苷酸标签识别序列的融解温度Tm1的探针寡核苷酸; 在多核苷酸扩增反应中将探针寡核苷酸掺入标记的多核苷酸中，提供具有解链温度Tm2的调节性寡核苷酸，其包含与调节序列至少部分互补的序列片段，在PCR扩增中扩增标记的靶核酸序列 使用探针寡核苷酸作为引物的反应，检测扩增产物; 其中Tm1和Tm2高于与多核苷酸扩增反应相关的退火温度。

公开(公告)号：US20160153026A1

公开(公告)日：2016-06-02

申请号：US14880112

申请日：2015-10-09

Applicant: FLUIDIGM CORPORATION

Inventor： Kenneth J. Livak ,  Marc Unger

IPC: C12Q1/68

CPC classification number: C12Q1/686 ,  C12Q1/6834 ,  C12Q2565/629

Abstract: High throughput methods are used that combine the features of using a matrix-type microfluidic device, labeled nucleic acid probes, and homogenous assays to detect and/or quantify nucleic acid analytes. The high throughput methods are capable of detecting nucleic acid analyses with high PCR and probe specificity, producing a low fluorescence background and therefore, a high signal to noise ratio. Additionally, the high throughput methods are capable of detecting low copy number nucleic acid analyte per cell.

Abstract translation: 使用高通量方法，其结合使用基质型微流体装置，标记的核酸探针和均质测定的特征来检测和/或定量核酸分析物。 高通量方法能够以高PCR和探针特异性检测核酸分析，产生低荧光背景，因此具有高的信噪比。 此外，高通量方法能够检测每个细胞的低拷贝数核酸分析物。

公开(公告)号：US20140315197A1

公开(公告)日：2014-10-23

申请号：US14184600

申请日：2014-02-19

Applicant: Fluidigm Corporation

Inventor： Kenneth J. Livak

IPC: C12Q1/68

CPC classification number: C12Q1/6876 ,  C12Q1/6818 ,  C12Q1/6823 ,  C12Q2525/161 ,  C12Q2525/197 ,  C12Q2565/1015

Abstract: Methods and reagents for detection and analysis of nucleic acids are provided. Certain methods involves an encoding amplification in which a target sequence is associated with probe-binding sequences and optionally with indexing sequences, (2) an optional distribution step in which the product of the encoding amplification is split into multiple aliquots, and (3) a decoding and detection step in which the presence, absence, quantity, or relative amount of the target sequence in the aliquots is determined. The detection step makes use of a multifunctional “self-digesting” molecular probe comprising a primer polynucleotide and a probe oligonucleotide, linked in a 5′-5′ orientation.

Abstract translation: 提供了用于检测和分析核酸的方法和试剂。 某些方法涉及编码扩增，其中靶序列与探针结合序列和任选的索引序列相关，（2）任选的分布步骤，其中编码扩增的产物被分成多个等分试样，和（3）a 解码和检测步骤，其中确定等分试样中靶序列的存在，不存在，数量或相对量。 检测步骤使用包含以5'-5'取向连接的引物多核苷酸和探针寡核苷酸的多功能“自消化”分子探针。

公开(公告)号：US20150361486A1

公开(公告)日：2015-12-17

申请号：US14713887

申请日：2015-05-15

Applicant: FLUIDIGM CORPORATION

Inventor： Kenneth J. Livak ,  Stacey N. Meyers ,  Xiaohui Wang ,  Jun Wang

IPC: C12Q1/68

CPC classification number: C12Q1/6818 ,  C12Q2525/151

Abstract: The invention provides a method for detecting a target nucleotide sequence by tagging the nucleotide sequence with a nucleotide tag, providing a probe oligonucleotide with a melting temperature Tm1, comprising a regulatory sequence and a nucleotide tag recognition sequence; incorporating the probe oligonucleotide into the tagged polynucleotide in a polynucleotide amplification reaction, providing a regulatory oligonucleotide with a melting temperature Tm2, comprising a sequence segment that complementary to the regulatory sequence and a tail segment that does not hybridize to the probe nucleotide when the sequence segment and the regulatory sequence are annealed, amplifying the tagged target nucleic acid sequence in a PCR amplification reaction using the probe oligonucleotide as a primer, and using a DNA polymerase with high strand displacement activity and low 5′-nuclease activity, and detecting the amplification product; wherein Tm1 and Tm2 are higher than the annealing temperature associated with the polynucleotide amplification reaction.

Abstract translation: 本发明提供了通过用核苷酸标签标记核苷酸序列来检测靶核苷酸序列的方法，提供了具有调节序列和核苷酸标签识别序列的融解温度Tm1的探针寡核苷酸; 在多核苷酸扩增反应中将探针寡核苷酸掺入标记的多核苷酸，提供具有解链温度Tm2的调节性寡核苷酸，其包含与调节序列互补的序列区段和当序列片段不与探针核苷酸杂交时的尾段 并使用探针寡核苷酸作为引物，并使用具有高链置换活性和低5'-核酸酶活性的DNA聚合酶，在PCR扩增反应中扩增标记的靶核酸序列，并检测扩增产物 ; 其中Tm1和Tm2高于与多核苷酸扩增反应相关的退火温度。