公开(公告)号：US20160177372A1

公开(公告)日：2016-06-23

申请号：US14574181

申请日：2014-12-17

Applicant: Roche Molecular Systems, Inc.

Inventor： Stephen G. Will

IPC: C12Q1/68

CPC classification number: C12Q1/6858 ,  C12Q1/6806 ,  C12Q2525/117 ,  C12Q2527/107 ,  C12Q2537/159 ,  C12Q2537/163

Abstract: A method is provided for allele-specific amplification, utilizing a blocking oligonucleotide including at least one nucleotide with a base covalently modified at the exocyclic amino group, the blocking oligonucleotide being perfectly complementary to a wild type (WT) sequence when hybridized forming a first complex having a first melting temperature (Tm), the blocking oligonucleotide being partially non-complementary, at one or more nucleotides, to a target mutant (MT) sequence when hybridized forming a second complex having a second melting temperature (Tm), wherein the first Tm is higher than the second Tm and having at least one nucleotide with a base covalently modified at the exocyclic amino group, wherein the blocking oligonucleotide becomes unhybridized from the target MT sequence during amplification but remains hybridized with the WT sequence inhibiting amplification of the WT sequence utilizing a polymerase lacking 5′-3′ nuclease activity.

Abstract translation: 提供了一种用于等位基因特异性扩增的方法，其利用包含至少一个具有在外界氨基共价修饰的碱基的核苷酸的封闭寡核苷酸，当杂交形成第一个复合物时​​，所述封闭寡核苷酸与野生型（WT）序列完全互补 当杂交形成具有第二熔解温度（Tm）的第二配合物时，具有第一解链温度（Tm），所述封闭寡核苷酸在一个或多个核苷酸处部分不互补，其中所述第一解链温度（Tm） Tm高于第二Tm，并且具有至少一个碱基，其具有在外界氨基处共价修饰的碱基，其中所述阻断寡核苷酸在扩增过程中从目标MT序列变得未杂交，但与WT序列保持杂交，所述WT序列抑制WT序列的扩增 利用缺乏5'-3'核酸酶活性的聚合酶。

公开(公告)号：US20160130641A1

公开(公告)日：2016-05-12

申请号：US14375894

申请日：2013-02-13

Applicant: Janssen Diagnostics, LLC

Inventor： Haiying Wang ,  Yuqiu Jiang ,  Yixin Wang

IPC: C12Q1/68

CPC classification number: C12Q1/6827 ,  C12Q1/6858 ,  C12Q2525/186 ,  C12Q2537/163

Abstract: The disclosed edge-blocker oligonucleotide based AS-NEPB-PCR method amplifies allele specific DNA (or RNA) while dramatically blocking amplification of wild type (WT) DNA (or RNA). The AS-NEPB-PCR design allows ready modification of an existing PCR reaction setup with an edge-blocker oligonucleotide together with an allele specific primer complementary to the mutant sequence to achieve allele specific amplification. The method simplifies assay optimization procedures and achieved sensitivity sufficient to detect a signal present at 0.1% level with close to 100% specificity, which is useful in detecting SNP or genetic mutations. The method was used to detect three different genetic mutations in cancer, in KRAS, BRAF, and EGFR, with three different types of modified edge-blocker oligonucleotides (phosphate, inverted dT and amino-C7). It was possible to detect one copy of mutant DNA in 1000-copy of normal DNA background of a heterogeneous sample, and was far more sensitive than the other blocking method.

Abstract translation: 所公开的基于边缘阻断剂寡核苷酸的AS-NEPB-PCR方法扩增等位基因特异性DNA（或RNA），同时显着阻断野生型（WT）DNA（或RNA）的扩增。 AS-NEPB-PCR设计允许利用边界阻断剂寡核苷酸与等位基因特异性引物与突变序列互补的等位基因特异性引物即可修改现有PCR反应设置，以实现等位基因特异性扩增。 该方法简化了测定优化程序，并实现了足以检测0.1％水平的信号，具有接近100％的特异性的灵敏度，可用于检测SNP或遗传突变。 该方法用于检测癌症，KRAS，BRAF和EGFR中的三种不同基因突变，其中三种不同类型的修饰的边缘阻断剂寡核苷酸（磷酸盐，反向dT和氨基-C C7）。 可以在异质样品的正常DNA背景的1000拷贝中检测到一个拷贝的突变体DNA，并且比其他阻断方法更敏感。

公开(公告)号：US09187782B2

公开(公告)日：2015-11-17

申请号：US13027926

申请日：2011-02-15

Applicant: Nader Pourmand ,  Muhammad Akram Tariq

Inventor： Nader Pourmand ,  Muhammad Akram Tariq

IPC: C12Q1/68

CPC classification number: C12Q1/6869 ,  C12Q2565/301 ,  C12Q2525/204 ,  C12Q2525/151 ,  C12Q2537/163 ,  C12Q2525/107

Abstract: Disclosed is a method whereby a repetitive nucleic acid sequence, such as a short tandem repeat (STR), may be characterized as to its length. Pyrosequencing is used to sequence an STR repetitive region to measure the length of STRs in a rapid manner. A combinatorial approach is disclosed for the addition of multiple nucleotides (e.g., two mononucleotides) at a time by the polymerase, which reduces the sample analysis time by half. In addition, modified nucleic acids, such as peptide nucleic acids, are used as blocking probe to stop polymerization on the flanking region which makes it possible to use pyrosequencing for DNA length measurement both in the case of homozygous or heterozygous samples for varying repeat patterns of different markers. Further, dideoxynucleotides are added to stop polymerization in the flanking region of the STR.

Abstract translation: 公开了一种重复核酸序列，例如短串联重复（STR）的方法，其特征可在于其长度。 焦磷酸测序用于对STR重复区域进行排序，以快速方式测量STR的长度。 公开了通过聚合酶一次添加多个核苷酸（例如两个单核苷酸）的组合方法，其将样品分析时间减少了一半。 此外，修饰的核酸，例如肽核酸，被用作阻断探针以阻止侧翼区域的聚合，这使得可以在纯合或杂合样品的情况下使用焦磷酸测序进行DNA长度测量，以改变重复模式 不同的标记。 此外，加入双脱氧核苷酸以在STR的侧翼区停止聚合。

公开(公告)号：US09133490B2

公开(公告)日：2015-09-15

申请号：US13834167

申请日：2013-03-15

Applicant: Transgenomic, Inc.

Inventor： Reyes Candau-Chacon

IPC: C12P19/34 ,  C12Q1/68

CPC classification number: C12P19/34 ,  C12Q1/6848 ,  C12Q1/6858 ,  C12Q1/686 ,  C12Q2527/107 ,  C12Q2537/113 ,  C12Q2537/159 ,  C12Q2537/163

Abstract: Methods of using polymerase chain reactions to enrich a target sequence in a sample containing reference sequences and target sequences having high homology and amplifiable by the same primer pair are provided herein. In particular the methods provide a robust means to improve the fold enrichment of the target sequence and minimize reaction-to-reaction, well-to-well and run-to-run variations in the enrichment methods.

Abstract translation: 本文提供了使用聚合酶链反应来富集含有参考序列的样品中的靶序列和具有高同源性并可由相同引物对扩增的靶序列的方法。 特别地，这些方法提供了强化手段，以改善靶序列的倍数富集，并使富集方法中的反应 - 反应，井对井和运行 - 运行变化最小化。

公开(公告)号：US09115392B2

公开(公告)日：2015-08-25

申请号：US13498699

申请日：2010-09-09

Applicant: Martin Schuler ,  Frank Breitenbuecher ,  Sandra Hoffarth ,  Sarah-Luise Stergar

Inventor： Martin Schuler ,  Frank Breitenbuecher ,  Sandra Hoffarth ,  Sarah-Luise Stergar

IPC: C12Q1/68 ,  C12P19/34

CPC classification number: C12Q1/6827 ,  C12P19/34 ,  C12Q1/68 ,  C12Q1/6858 ,  C12Q1/6886 ,  C12Q2600/156 ,  C12Q2565/101 ,  C12Q2535/131 ,  C12Q2531/107 ,  C12Q2537/163 ,  C12Q2527/107

Abstract: A method of detecting at least one gene modification such as a mutation in a gene includes carrying out an asymmetric polymerase chain reaction (PCR) with a combined use of at least one detectable mutation-specific hybridization probe (sensor probe) and at least one wild-type specific blocking agent which inhibits a binding of the at least one detectable mutation-specific hybridization probe (sensor probe) to a wild-type gene so as to provide at least one of a selective intensification and an amplification of a detection of a gene segment of a mutation gene having a gene modification.

Abstract translation: 检测基因修饰如基因突变中的至少一种修饰的方法包括使用至少一种可检测突变特异性杂交探针（传感器探针）和至少一种野生型的组合使用不对称聚合酶链式反应（PCR） 型特异性阻断剂，其抑制至少一种可检测突变特异性杂交探针（传感器探针）与野生型基因的结合，从而提供对基因的检测的选择性增强和扩增中的至少一种 具有基因修饰的突变基因片段。

公开(公告)号：US20150031023A1

公开(公告)日：2015-01-29

申请号：US14189762

申请日：2014-02-25

Applicant: Samsung Electronics Co., Ltd.

Inventor： Dong-hyun PARK ,  Yeon-jeong Kim ,  Kyung-yeon Han

IPC: C12Q1/68

CPC classification number: C12Q1/6858 ,  C12Q2537/163

Abstract: Provided are a composition or a kit for detecting a nucleic acid with genetic variation including a first amplification blocking nucleic acid and a second amplification blocking nucleic acid, and a method of detecting a nucleic acid with genetic variation by using the same. Based on the above, a nucleic acid with genetic variation can be detected with high sensitivity and accuracy.

Abstract translation: 提供了用于检测具有遗传变异的核酸的组合物或试剂盒，包括第一扩增阻断核酸和第二扩增阻断核酸，以及通过使用该核酸检测具有遗传变异的核酸的方法。 基于上述，可以以高灵敏度和准确度检测出具有遗传变异的核酸。

公开(公告)号：US20140287410A1

公开(公告)日：2014-09-25

申请号：US14216413

申请日：2014-03-17

Applicant: David A. Shafer

Inventor： David A. Shafer

IPC: C12Q1/68

CPC classification number: C12Q1/6876 ,  C12Q1/6832 ,  C12Q2537/1373 ,  C12Q2537/161 ,  C12Q2537/163

Abstract: Probe systems and methods are provided for detecting nucleic acid targets using labeled polynucleotide probes and antiprobes that interact together and with complementary targets. These interactions result in signaling changes that indicate target frequency and provide error-checking functions that facilitate single base discrimination. These probe:antiprobe compositions enable real-time PCR detection, end-point detection and microarray detection of microbial species, drug resistant mutants, and cancer related variants. The probe:antiprobe may be an internal probe between two primers or may be a primer-probe. The probe also may be modified by introducing a base mismatch to increase thermodynamic discrimination of a correct versus incorrect target differing by a single base. Probe systems also are provided for use in methods of increasing target amplification and detecting specific single base variants.

Abstract translation: 提供了探针系统和方法，用于使用标记的多核苷酸探针和与辅助靶相互作用的对映体来检测核酸靶。 这些相互作用导致了指示目标频率的信号变化，并提供了有助于单基地辨别的错误检查功能。 这些探针：反钩组合物可实现微生物物种，耐药性突变体和癌症相关变异体的实时PCR检测，终点检测和微阵列检测。 探针：反接头可以是两个引物之间的内部探针，也可以是引物探针。 探针也可以通过引入碱基不匹配来增加对单个碱基不同的正确对不正确目标的热力学鉴别。 提供探针系统用于增加靶扩增和检测特异性单碱基变体的方法。

公开(公告)号：US20140017685A1

公开(公告)日：2014-01-16

申请号：US13979480

申请日：2012-01-16

Applicant: Guoliang Fu

Inventor： Guoliang Fu

IPC: C12Q1/68

CPC classification number: C12Q1/6883 ,  C12Q1/6827 ,  C12Q1/6853 ,  C12Q2527/107 ,  C12Q2537/163 ,  C12Q2565/1015

Abstract: The present invention provides methods, compositions and kits for detecting the presence, absence or amount of a target nucleic acid or at least one variant nucleotide in one or more nucleic acids contained in a sample.

Abstract translation: 本发明提供用于检测样品中所含的一个或多个核酸中靶核酸或至少一个变体核苷酸的存在，不存在或量的方法，组合物和试剂盒。

公开(公告)号：US08623603B2

公开(公告)日：2014-01-07

申请号：US13042549

申请日：2011-03-08

Applicant: Gerassimos Makrigiorgos

Inventor： Gerassimos Makrigiorgos

IPC: C12Q1/68 ,  C12P19/34

CPC classification number: C12Q1/6848 ,  C12Q1/6858 ,  C12Q2527/107 ,  C12Q2537/159 ,  C12Q2537/163

Abstract: The present invention is directed to methods, compositions and software for enriching low abundance alleles in a sample. It is directed in particular to the use of an excess amount of reference blocking sequence in an amplification reaction mixture in order to improve the enrichment efficiency, and reduce cycle time, of full COLD-PCR.

Abstract translation: 本发明涉及用于丰富样品中低丰度等位基因的方法，组合物和软件。 特别涉及在扩增反应混合物中使用过量的参考封闭序列，以提高完全COLD-PCR的富集效率，缩短循环时间。

公开(公告)号：US20120108440A1

公开(公告)日：2012-05-03

申请号：US12952563

申请日：2010-11-23

Applicant: Patrick Lau ,  Danny Lee

Inventor： Patrick Lau ,  Danny Lee

IPC: C40B20/00 ,  C40B50/12 ,  C40B50/06 ,  C07H21/00

CPC classification number: C12N15/1068 ,  C12Q1/6855 ,  C40B50/06 ,  C12Q2537/163 ,  C12Q2533/107 ,  C12Q2525/301 ,  C12Q2525/197

Abstract: Provided herein is a method of reducing adapter dimer formation comprising contacting a sample comprising target nucleic acid sequences with 5′ and 3′ adapters in the presence of one or more hairpin oligonucleotides. Also provided is a method of preparing a library of nucleic acid sequences comprising contacting first adapter oligonucleotides with a sample comprising target nucleic acid sequences under conditions to form first ligation products, contacting the sample with one or more hairpin oligonucleotides that binds to the first adapter oligonucleotides, and contacting the sample with second adapter oligonucleotides under conditions to bind to the first ligation products and form second ligation products, wherein the second ligation products form the library of nucleic acid sequences.

Abstract translation: 本文提供了减少适配体二聚体形成的方法，包括在一种或多种发夹寡核苷酸存在下使包含靶核酸序列的样品与5'和3'衔接子接触。 还提供了制备核酸序列文库的方法，包括在条件下使第一衔接子寡核苷酸与包含靶核酸序列的样品接触以形成第一连接产物，使样品与一种或多种结合第一衔接子寡核苷酸的发夹寡核苷酸接触 并且在与第一连接产物结合的条件下使样品与第二衔接子寡核苷酸接触并形成第二连接产物，其中第二连接产物形成核酸序列文库。