公开(公告)号：WO2014077822A1

公开(公告)日：2014-05-22

申请号：PCT/US2012/065376

申请日：2012-11-15

Applicant: FLUIDIGM CORPORATION

Inventor： LIVAK, Kenneth ,  MEYERS, Stacy ,  WANG, Xiaohui ,  WANG, Jun

IPC: C12Q1/68

CPC classification number: C12Q1/6827 ,  C12Q1/686 ,  C12Q2521/101 ,  C12Q2525/185 ,  C12Q2535/125 ,  C12Q2563/107 ,  C12Q2565/101 ,  C12Q2565/629

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 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，并检测扩增产物，在PCR扩增反应中扩增标记的靶核酸序列，调节序列退火， 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：WO2012054933A2

公开(公告)日：2012-04-26

申请号：PCT/US2011/057536

申请日：2011-10-24

Applicant: FLUIDIGM CORPORATION ,  LIVAK, Kenneth ,  WEST, Jason A., A. ,  JONES, Robert, C.

Inventor： LIVAK, Kenneth ,  WEST, Jason A., A. ,  JONES, Robert, C.

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, said 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，与靶序列5'的第一探针结合序列和第二探针结合序列3的扩增产物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片段和与第二探针结合序列杂交的第二片段，从而产生标记的扩增产物