960化工网
Highly specific DNA detection from massive background nucleic acids based on rolling circle amplification of target dsDNA†
Xingyu Wang,Xin Yu,Xiaoliang Wang,Masatomo Suzuki,Hiroyuki Asanuma,Ping Dong,Wei Wu,Xingguo Liang
RSC Advances Pub Date : 08/26/2014 00:00:00 , DOI:10.1039/C4RA05642F
Abstract

An advanced rolling circle amplification (RCA) strategy based on the target-circularization of the targeted double-stranded DNA (dsDNA) was established. Different from the traditional padlock-RCA, in which single-stranded probe DNA was circularized and amplified as signal amplification, our new approach could amplify a double-stranded DNA target. This special circularization of the target was realized by the ligation of target DNA with a biotin-labelled duplex adaptor containing 9 nt sticky ends by complementary base pairing. High specificity was obtained using two primers targeting the target sequence but not the probe itself in traditional padlock-RCA. With the help of streptavidin magnetic beads that immobilized the ligated dsDNA amplicon, the background nucleic acids contributing the most to non-specific amplification were eliminated. Under optimized conditions, less than 60 copies of the target sequence could be detected in the presence of massive background nucleic acids (>1012 copies of unrelated sequences). The sensitivity and specificity can rival canonical PCR. Without thermal cycles, the reduced handling and simpler equipment requirements render this assay a simple and rapid alternative to conventional methods. Based on these advantages, this method is a promising candidate in practical applications such as detecting contaminated food-borne pathogens in comprehensive food samples.

Graphical abstract: Highly specific DNA detection from massive background nucleic acids based on rolling circle amplification of target dsDNA
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