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2. 发明公开

EP2772552A1 Amplicon melting analysis with saturation dyes 有权转让
标题翻译：用饱和染料进行扩增子解链分析
公开(公告)号：EP2772552A1
公开(公告)日：2014-09-03
申请号：EP14164889.9
申请日：2003-10-22
申请人： University of Utah Research Foundation , BioFire Defense, LLC
发明人： Wittwer, Carl T , Dujols, Virginie E , Reed, Gudrun , Zhou, Luming
IPC分类号： C12Q1/68 , C12P19/34 , C07D279/16
CPC分类号： C09B23/04 , C07D413/06 , C07D417/06 , C07H21/04 , C12Q1/6827 , C12Q1/6844 , C12Q1/6858 , C12Q1/686 , C12Q2527/125 , C12Q2527/107 , C12Q2565/107 , C12Q2565/101 , C12Q2537/107 , C12Q2537/165 , C12Q2545/113
摘要： The invention provides:
A kit for amplifying and subsequently melting a plurality of target nucleic acids comprising:
(a) amplification reagents;
(b) a dsDNA binding dye having a percent saturation of at least about 90%;
(c) information on amplifying each of the plurality of target nucleic acids in the presence of the dsDNA binding dye using the amplification reagents, to generate a plurality of amplified nucleic acids; and
(d) information on generating a fluorescence melting curve from each of the plurality of amplified nucleic acids and plotting the difference between each of the fluorescence melting curves.
The invention also provides a method of PCR analysis comprising the steps of:
providing replicates of a mixture of a dsDNA binding dye, a target nucleic acid having a specific genotype, and primers configured for amplifying the target nucleic acid,
amplifying each of the replicates of the target nucleic acid in the presence of the dsDNA binding dye,
monitoring fluorescence of the dsDNA binding dye, generating a melting curve for each of the replicates of the target nucleic acid,
repeating the providing, amplifying and generating steps with at least one additional target nucleic acid,
establishing the target nucleic acid melting curve as a standard across temperatures using the replicates of the target nucleic acid having the specific genotype, and
plotting a fluorescence difference between the standard and the melting curve of the at least one additional target nucleic acid.
摘要翻译：本发明提供：用于扩增并随后熔解多种靶核酸的试剂盒，其包含：（a）扩增试剂; （b）具有至少约90％饱和百分比的dsDNA结合染料; （c）使用扩增试剂在dsDNA结合染料存在下扩增多个目标核酸中的每一个以产生多个扩增核酸的信息; （d）关于从多个扩增核酸中的每一个生成荧光解链曲线并绘制每个荧光解链曲线之间的差异的信息。本发明还提供了一种PCR分析方法，该方法包括以下步骤：提供dsDNA结合染料，具有特定基因型的靶核酸和被配置用于扩增靶核酸的引物的混合物的复制品，扩增每个在存在dsDNA结合染料的情况下监测靶核酸，监测dsDNA结合染料的荧光，为靶核酸的每个重复产生解链曲线，用至少一个另外的靶标重复提供步骤，放大步骤和产生步骤使用具有特定基因型的目标核酸的复制品在整个温度范围内建立靶核酸解链曲线作为标准，并绘制标准曲线与至少一个另外的目标核酸的解链曲线之间的荧光差异。

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