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    • 7. 发明公开
    • ENGINEERED TEMPLATES AND THEIR USE IN SINGLE PRIMER AMPLIFICATION
    • 工程模板及其在单一主放大中的使用
    • EP1711615A2
    • 2006-10-18
    • EP04814163.4
    • 2004-12-15
    • ALEXION PHARMACEUTICALS, INC.
    • MARUYAMA, ToshiakiFREDERICKSON, ShanaBOWDISH, Katherine, S.RENSHAW, MarkLIN, Ying-Chi
    • C12Q1/00
    • C07K16/4291C07K16/005C07K16/082C07K2317/55C07K2317/56C07K2317/565C12N15/1093C12Q1/6844C12Q1/6853C12Q1/6855C12Q1/686Y10S530/861C12Q2525/161C12Q2525/155C12Q2537/157C12Q2533/101C12Q2525/301C12Q2525/131C12Q2525/186
    • Methods of amplifying nucleic acid have now been discovered which include the steps of: a) annealing a primer to a template nucleic acid sequence, the primer having a first portion which anneals to the template and a second portion of predetermined sequence; b) synthesizing a polynucleotide that anneals to and is complementary to the portion of the template between the location at which the first portion of the primer anneals to the template and the end of the template, the polynucleotide having a first end and a second end, wherein the first end incorporates the primer; c) separating the polynucleotide synthesized in step (b) from the template; d) annealing a nested oligonucleotide to the second end of the polynucleotide synthesized in step (b), the nested oligonucleotide having a first portion that anneals to the second end of the polynucleotide and a second portion having the same predetermined sequence as the second portion of the primer; e) extending the polynucleotide synthesized in step (b) to provide a terminal portion thereof that is complementary to the predetermined sequence; and f) amplifying the extended polynucleotide using a single primer having the predetermined sequence. In particularly useful embodiments, the methods are used to amplify a repertoire of IgA antibodies.
    • 现在已经发现了扩增核酸的方法,其包括以下步骤:a)将引物与模板核酸序列退火,所述引物具有与模板退火的第一部分和预定序列的第二部分; b)合成与所述模板的所述部分退火并与所述模板的所述部分互补的多核苷酸,所述位置在所述引物的第一部分与所述模板退火的位置与所述模板的末端之间退火,所述多核苷酸具有第一末端和第二末端, 其中第一末端包含引物; c)将步骤(b)中合成的多核苷酸与模板分离; d)将巢式寡核苷酸退火至在步骤(b)中合成的多核苷酸的第二末端,所述巢式寡核苷酸具有与多核苷酸的第二末端退火的第一部分和与第二部分具有相同预定序列的第二部分 底漆; e)延伸步骤(b)中合成的多核苷酸以提供与预定序列互补的其末端部分; 和f)使用具有预定序列的单一引物扩增延伸的多核苷酸。 在特别有用的实施方案中,所述方法用于扩增IgA抗体库。