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。

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。