Li Zhengping, Li Wei, Cheng Yongqiang, Hao Longteng
Key Laboratory of Medicine Chemistry and Molecular Diagnosis, Ministry of Education, College of Chemistry and Environmental Science, Hebei University, Baoding 071002, Hebei Province, China.
Analyst. 2008 Sep;133(9):1164-8. doi: 10.1039/b807368f. Epub 2008 Jul 25.
A new chemiluminescent (CL) method has been developed for the sensitive detection of DNA hybridization and single-nucleotide polymorphisms (SNPs) with target-primed rolling circle amplification (RCA). The capture oligonucleotide probe is firstly immobilized on a polystyrene well plate and then hybridized with the wild DNA target. A designed padlock probe is circularized after perfect hybridization to the DNA target. Then the RCA reaction can be initiated from the DNA target that acts as a primer and generates a long tandem single-strand of DNA with repeat sequences. In contrast, the mutant DNA target, which contains a mismatched base with the padlock probe, cannot initiate the RCA reaction and primes only a limited extension with the unligated padlock probe. Afterwards, a biotinylated oligonucleotide is used to hybridize with the RCA product in each repeat sequence and streptavidin-alkaline phosphatase (STV-AP) is employed to combine the anchored biotin. The DNA target is detected with the CL reaction of STV-AP and 3-(2'-spiroadamantane)-4-methoxy-4-(3''-phosphoryloxy)phenyl-1,2-dioxetane (AMPPD). With the RCA-based method, the sensitivity of DNA detection can be increased by about two orders of magnitude compared with that of direct DNA hybridization. A DNA target as low as 3.6 pM can be detected. Wild-type DNA and the one-base mutant DNA can be differentiated with high selectivity through this RCA reaction.
一种新的化学发光（CL）方法已被开发出来，用于通过靶标引发的滚环扩增（RCA）灵敏检测DNA杂交和单核苷酸多态性（SNP）。捕获寡核苷酸探针首先固定在聚苯乙烯微孔板上，然后与野生型DNA靶标杂交。设计的锁式探针与DNA靶标完美杂交后环化。然后RCA反应可以从充当引物的DNA靶标开始，生成具有重复序列的长串联单链DNA。相比之下，与锁式探针含有错配碱基的突变型DNA靶标不能引发RCA反应，只能用未连接的锁式探针引发有限的延伸。之后，用生物素化的寡核苷酸与每个重复序列中的RCA产物杂交，并使用链霉亲和素-碱性磷酸酶（STV-AP）结合锚定的生物素。通过STV-AP与3-(2'-螺金刚烷)-4-甲氧基-4-(3''-磷酰氧基)苯基-1,2-二氧杂环丁烷（AMPPD）的CL反应检测DNA靶标。与直接DNA杂交相比，基于RCA的方法可将DNA检测的灵敏度提高约两个数量级。可检测低至3.6 pM的DNA靶标。通过这种RCA反应，可以高选择性地区分野生型DNA和单碱基突变型DNA。
