Integration of rolling circle amplification and cationic conjugated polymer for the homogeneous detection of single nucleotide polymorphisms  

作　　者：TANG ZhiYuan CHENG YongQiang DU Qing ZHANG HongXia LI ZhengPing 

机构地区：[1]Key Laboratory of Medicine Chemistry and Molecular Diagnosis, Ministry of Education, College of Chemistry and Environment Science, HebeiUniversity, Baoding 071002, China

出　　处：《Chinese Science Bulletin》2011年第31期3247-3252,共6页

基　　金：supported by the Specialized Research Fund for the Doctoral Program of Higher Education of China (20070075003, 20091301120003);the Natural Science Foundation of Hebei Province (B2009000170)

摘　　要：A novel, homogeneous and sensitive assay for the detection of single nucleotide polymorphisms (SNPs) by integration of rolling circle amplification (RCA) and cationic conjugated polymer (CCP) has been developed and tested. Mutant DNA serves as the template for specifically circularizing a padlock probe (PLP) with a sequence that is complementary to the mutant DNA. Afterwards, the mutant DNA directly acts as the primer to initiate the RCA reaction in the presence of phi29 DNA polymerase that generates a long, tandem single-strand DNA product. During the RCA reaction, fluorescein-labeled dUTPs are incorporated into the RCA products. When the CCP is introduced, efficient FRET from CCP to fluorescein occurs as a result of the strong electrostatic interactions between the CCP and the DNA produced by RCA. The wild-type DNA contains a single base mismatch with PLP with the result that the PLP is not circularized, RCA is not triggered and inefficient FRET results. By measuring the change of the emission intensities of CCP and fluorescein, it was possible to detect the SNP in a homogeneous manner. The method is sensitive and specific enough to detect 0.1 pmol/L mutant DNA and to determine a mutant allele frequency as low as 2.0%.A novel, homogeneous and sensitive assay for the detection of single nucleotide polymorphisms （SNPs） by integration of rolling circle amplification （RCA） and cationic conjugated polymer （CCP） has been developed and tested. Mutant DNA serves as the template for specifically circularizing a padlock probe （PLP） with a sequence that is complementary to the mutant DNA. After- wards, the mutant DNA directly acts as the primer to initiate the RCA reaction in the presence of phi29 DNA polymerase that generates a long, tandem single-strand DNA product. During the RCA reaction, fluorescein-labeled dUTPs are incorporated into the RCA products. When the CCP is introduced, efficient FRET from CCP to fluorescein occurs as a result of the strong electro- static interactions between the CCP and the DNA produced by RCA. The wild-type DNA contains a single base mismatch with PLP with the result that the PLP is not circularized, RCA is not triggered and inefficient FRET results. By measuring the change of the emission intensities of CCP and fluorescein, it was possible to detect the SNP in a homogeneous manner. The method is sensitive and specific enough to detect 0.1 pmol/L mutant DNA and to determine a mutant allele frequency as low as 2.0%.

关 键 词：单核苷酸多态性 多态性检测 共轭聚合物 阳离子 同质化 滚环 一体化 DNA序列 

分 类 号：Q503[生物学—生物化学]