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    • 4. 发明申请
    • METHODS AND ARRAYS FOR PRODUCING AND SEQUENCING MONOCLONAL CLUSTERS OF NUCLEIC ACID
    • 用于生产和测序核酸单核苷酸的方法和阵列
    • WO2016075204A1
    • 2016-05-19
    • PCT/EP2015/076353
    • 2015-11-11
    • ILLUMINA, INC.ILLUMINA CAMBRIDGE LTD.
    • GUNDERSON, Kevin L.BAI, JingweiKELLINGER, Matthew WilliamBEIERLE, John M.BOUTELL, Jonathan MarkRIGATTI, RobertoROGERT BACIGALUPO, Maria CandelariaBOYANOV, BoyanMAISINGER, Klaus
    • C12Q1/68B01J19/00C40B40/06C40B50/14
    • C12Q1/6874B01J19/0046B01J2219/00317B01J2219/00585B01J2219/00596B01J2219/00608B01J2219/00637B01J2219/00722C12Q1/6806C12Q1/6837C12Q1/6853C40B40/06C40B50/14C12Q2565/513C12Q2565/543
    • Methods for capturing and amplifying target polynucleotides on a solid surface, in particular in a well in a microarray, wherein the microarray may comprise a) a substrate comprising at least one well, a surface surrounding the well and an inner well surface; b) a first layer covering the inner well surface and comprising at least one first capture primer pair; and c) a second layer covering the first layer and the surface surrounding the well. Alternatively, the microarray may comprise a) a substrate comprising at least one well, a surface surrounding the well and an inner well surface; and b) a layer covering the inner well surface and comprising at least one first capture primer pair and at least one second capture primer pair. In particular kinetic exclusion amplification is used in creating monoclonal populations of the nucleic acids in the wells. The application also discloses a method for modifying an immobilized capture primer comprising: a) contacting a substrate comprising a plurality of immobilized capture primers with a plurality of template nucleic acids to produce one or more immobilized template nucleic acids,wherein the plurality of immobilized capture primers comprises a first plurality of primers comprising a 3'-terminal universal capture region Y, e.g. primer P5, and a second plurality of primers comprising a 3'-terminal universal capture region Z, e.g. primer P7; and wherein each template nucleic acid is flanked by a 5'-terminal and a 3'-terminal universal capture region Y or Z and comprises one or more, e.g. SapI, restriction sites and a target-specific capture region between the one or more restriction sites and the 3'-terminal universal capture region; and b) extending one or more immobilized capture primer. Finally, the application discloses a method for modifying an immobilized capture primer comprising: a) contacting a substrate comprising a plurality of immobilized capture primers with a plurality of different seed nucleic acids to produce a plurality of different immobilized seed nucleic acids; b) extending two or more of the immobilized capture primers to produce a plurality of different immobilized extension products complementary to two or more of the plurality of different immobilized seed nucleic acids; and c) activating one immobilized extension product of the plurality of different immobilized extension products, to form an activated capture primer.
    • 用于在固体表面上特别是在微阵列中的阱中捕获和扩增靶多核苷酸的方法,其中所述微阵列可以包括a)包含至少一个阱的衬底,围绕所述阱的表面和内部阱表面; b)覆盖所述内部孔表面并包含至少一个第一捕获引物对的第一层; 以及c)覆盖所述第一层和围绕所述孔的表面的第二层。 或者,微阵列可以包括:a)包含至少一个阱的衬底,围绕阱的表面和内部阱表面; 以及b)覆盖所述内部孔表面并包含至少一个第一捕获引物对和至少一个第二捕获引物对的层。 特别地,动力学排除扩增用于产生孔中核酸的单克隆群体。 本申请还公开了一种修饰固定化捕获引物的方法,其包括:a)使包含多个固定的捕获引物的底物与多个模板核酸接触以产生一种或多种固定的模板核酸,其中所述多个固定的捕获引物 包括包含3'末端通用捕获区Y的第一多个引物,例如 引物P5和包含3'末端通用捕获区Z的第二多个引物。 引物P7; 并且其中每个模板核酸侧接有5'-末端和3'末端通用捕获区Y或Z,并且包含一个或多个,例如, SapI,限制性位点和一个或多个限制性位点与3'-末端通用捕获区之间的靶标特异性捕获区域; 和b)延伸一个或多个固定的捕获引物。 最后,本申请公开了一种用于修饰固定化捕获引物的方法,其包括:a)使包含多个固定的捕获引物的底物与多种不同的种子核酸接触以产生多种不同的固定化种子核酸; b)延伸两个或更多个固定的捕获引物以产生与所述多个不同固定化种子核酸中的两种或更多种互补的多种不同的固定化延伸产物; 和c)激活所述多个不同的固定化延伸产物的一个固定化的延伸产物,以形成活化的捕获引物。