Chemiluminescent detection of DNA hybridization and single-nucleotide polymorphisms on a solid surface using target-primed rolling circle amplification.
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.
Li
Zhengping
Z
Key Laboratory of Medicine Chemistry and Molecular Diagnosis, Ministry of Education, College of Chemistry and Environmental Science, Hebei University, Baoding 071002, Hebei Province, China. lzpbd@hbu.edu.cn
Li
Wei
W
Cheng
Yongqiang
Y
Hao
Longteng
L
D000818:Animals; D004252:DNA Mutational Analysis / Q000379:methods*; D004270:DNA, Circular / Q000235:genetics; D017403:In Situ Hybridization / Q000379:methods; D008163:Luminescent Measurements; D021141:Nucleic Acid Amplification Techniques / Q000379:methods; D015345:Oligonucleotide Probes; D020641:Polymorphism, Single Nucleotide
D004270:DNA, Circular; D015345:Oligonucleotide Probes