滚环扩增阳离子共轭聚合物均相检测microRNA.pdfVIP

摘 要 摘 要 microRNA（miRNA）是一种长度一般在20-24个核苷酸左右的非编码小分子RNA， 它在基因调控和表达中起着重要作用。由于它在生物个体发育分化等生命活动中有着重 要调节作用，因此对miRNA的检测在研究生物功能和临床诊断等方面具有重要意义。 本论文应用滚环扩增技术结合阳离子共轭聚合物均相检测，提出了一种在均相溶液中进 行microRNA检测的新方法。设计的锁式探针（PLP）与目标分子miRNA-221两侧序列 完全互补，然后在T4RNA连接酶2的作用下，PLP探针首尾相接被连接成环。随后目 标分子miRNA-221可直接作为引物，以环形PLP探针为模板在Phi29DNA聚合酶作用 下引发滚环扩增反应，生成上万个碱基的DNA单链产物。随后在扩增产物中加入与其 互补的荧光标记探针，并使其充分杂交。未杂交的荧光标记探针应用核酸外切酶I降解 为单核苷酸分子。当加入阳离子共轭聚合物时，由于阳离子共轭聚合物和DNA链之间 很强的静电吸引作用，在二者之间会产生高效率的荧光共振能量转移（FRET）。通过测 量共轭聚合物荧光发射强度的变化，在均相溶液中实现了microRNA检测。该方法对于 miRNA-221检测的线性范围为0.5-20pmol/L，检测限为0.2pmol/L。 关键词： miRNA 滚环扩增技术 阳离子共轭聚合物 荧光共振能量转移 I Abstract Abstract Abstract AAbbssttrraacctt MicroRNAs (miRNAs) are a class of non-coding small RNAgenes, and their sequences are22-24ntthatplay important roles inthe regulation of translation and expression.As its role in gene expression and control in the ontogeny, it is important for us to detect miRNAs to study its biological function and clinical diagnosis. In the test, we develop a novel and homogeneous assay for the detection of miRNAs by integration of rolling circle amplification (RCA) and cationic conjugated polymer (CCP). MiRNA-221 serves as the template for specifically circularizing the padlock probe (PLP) with a sequence that is complementary to the miRNA-221, and T4 RNA ligase2 connect the both ends of the padlock probe to form a ring. Afterwards, miRNA-221 directly acts as the primer to initiate the RCA reaction in the presence of phi29 DNApolymerase that generates a long, tandem single-strand DNAproduct. When products fully hybridied with labeled-DNA strands, we introduced the CCP, efficient FRET from CCP to fluorescein occurs as a result of t