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    • 24. 发明申请
    • USE OF PRODUCTS OF PCR AMPLIFICATION CARRYING ELEMENTS OF SECONDARY STRUCTURE TO IMPROVE PCR-BASED NUCLEIC ACID DETECTION
    • 利用PCR扩增产物实现二级结构元件改进基于PCR的核酸检测
    • WO2007127999A3
    • 2008-12-11
    • PCT/US2007067836
    • 2007-04-30
    • KUTYAVIN IGOR
    • KUTYAVIN IGOR
    • C12Q1/68C07H21/04C12P19/34
    • C12Q1/6853C12Q1/686C12Q2561/109C12Q2537/143C12Q2525/301C12Q2565/1015
    • Particular aspects comprise amplifying target nucleic acid using PCR and an oligonucleotide primer pair wherein at least one of the primers is designed to incorporate a 5'-specialty sequence to provide for an amplification product that intramolecularly folds into a secondary structure; and detecting the amplification product by a method comprising: providing an oligonucleotide cleavage component, hybridizing said oligonucleotide cleavage component with the amplification product to form a three-strand cleavage structure wherein two strands of the three-strand cleavage structure are provided by the secondary structure of the amplification product, cleaving 3'- or 5'-strands of the three-strand cleavage structure using a duplex-specific nuclease activity resulting in a cleavage product, and detecting the cleavage product indicative of the presence of the target nucleic acid. In certain aspects both primers incorporate a 5'-specialty sequence and detecting comprises cleaving 3'- or 5'-strands of a three-strand cleavage structure using duplex-specific nuclease to provide a cleavage product.
    • 具体方面包括使用PCR扩增靶核酸和寡核苷酸引物对,其中至少一个引物被设计为掺入5'-特异性序列以提供分子内折叠成二级结构的扩增产物; 并通过包括以下方法检测扩增产物:提供寡核苷酸切割组分,将所述寡核苷酸切割组分与扩增产物杂交以形成三链切割结构,其中三股裂解结构的两条链由 扩增产物,使用产生切割产物的双链体特异性核酸酶活性切割三链切割结构的3'-或5'链,并检测指示靶核酸存在的切割产物。 在某些方面,两个引物都包含5'特异性序列,检测包括使用双链体特异性核酸酶切割三链切割结构的3'或5'链以提供切割产物。