title: Spectrophotometric and ultrasensitive DNA bioassay by circular-strand displacement polymerization reaction.
abstract: We demonstrated a new spectrophotometric DNA detection approach based on a circular strand-displacement polymerization reaction for the quantitative detection of sequence specific DNA. In this assay, the hybridization of an immobilized hairpin probe on the microtiter plate, to target DNA, results in a conformational change and leads to a stem separation. A short primer thus anneals with the open stem and triggers a polymerization reaction, allowing a cyclic reaction comprising the release of target DNA and hybridization of the target with the remaining immobilized hairpin probe. Through this cyclical process, a large number of duplex DNA complexes are produced. Finally, the biotin modified duplex DNA products can be detected via the HRP catalyzed substrate 3,3',5,5'-tetramethylbenzidine using a spectrophotometer. As a proof of concept, a short DNA sequence (20-nt) related to the South East Asia (SEA) type deletion of α-thalassemia was chosen as the model target. This proposed assay has a very high sensitivity and selectivity with a dynamic response ranging from 0.1 fM to 10 nM and the detection limit was 8 aM. It can be performed within 2 hours, and it can differentiate target SEA DNA from wild-type DNA. By substituting the hairpin probes used in the present work, this assay can be used to detect other subtypes of genetic disorders.
authors.affiliation: Key Laboratory of Regenerative Biology, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China. zeng6@yahoo.com.
authors.forename: Luxin
authors.initials: L
authors.lastname: Yu
authors.forename: Wei
authors.initials: W
authors.lastname: Wu
authors.forename: Junhua
authors.initials: J
authors.lastname: Chen
authors.forename: Zhuo
authors.initials: Z
authors.lastname: Xiao
authors.forename: Chenchen
authors.initials: C
authors.lastname: Ge
authors.forename: Puchang
authors.initials: P
authors.lastname: Lie
authors.forename: Zhiyuan
authors.initials: Z
authors.lastname: Fang
authors.forename: Lingbo
authors.initials: L
authors.lastname: Chen
authors.forename: Ya
authors.initials: Y
authors.lastname: Zhang
authors.forename: Lingwen
authors.initials: L
authors.lastname: Zeng
mesh_terms: D001483:Base Sequence; D055162:Biocatalysis; D001681:Biological Assay / Q000379:methods*; D004247:DNA / Q000032:analysis*; D017931:DNA Primers; D057230:Limit of Detection; D011108:Polymers / Q000737:chemistry*; D015203:Reproducibility of Results
chemical_list: D017931:DNA Primers; D011108:Polymers; D004247:DNA