Gold nanoparticle enhanced fluorescence anisotropy for the assay of single nucleotide polymorphisms (SNPs) based on toehold-mediated strand-displacement reaction.
We developed a highly differentiating, homogeneous gold nanoparticle (AuNP) enhanced fluorescence anisotropic method for single nucleotide polymorphism (SNP) detection at nanomolar level using toehold-mediated strand-displacement reaction. The template strand, containing a toehold domain with an allele-specific site, was immobilized on the surface of AuNPs, and the solution fluorescence anisotropy was markedly enhanced when the fluorescein-labeled blocking DNA was attached to the AuNP via hybridization. Strand-displacement by the target ssDNA strand resulted in detachment of fluorescein-labeled DNA from AuNPs, and thus decreased fluorescence anisotropy. The drastic kinetic difference in strand-displacement from toehold design was used to distinguish between the perfectly matched and the single-base mismatched strands. Free energy changes were calculated to elucidate the dependence of the differentiation ability on the mutation site in the toehold region. A solid negative signal change can be obtained for single-base mismatched strand in the dynamic range of the calibration curve, and a more than 10-fold signal difference can still be observed in a mixed solution containing 100 times the single-base mismatched strand, indicating the good specificity of the method. This proposed method can be performed with a standard spectrofluorimeter in a homogeneous and cost-effective manner, and has the potential to be extended to the application of fluorescence anisotropy method of SNP detection.
College of Sciences, Shenyang Agricultural University, Shenyang 110161, China.
Xinyi
X
Wang
Mingjian
M
Zou
Hongduan
H
Huang
Yuqian
Y
Ren
Limei
L
Li
Xiaoda
X
Yang
Na
N
Li
D016880:Anisotropy; D001483:Base Sequence; D015374:Biosensing Techniques / Q000295:instrumentation; D004247:DNA / Q000235:genetics*; D004252:DNA Mutational Analysis / Q000295:instrumentation*; D004867:Equipment Design; D019544:Equipment Failure Analysis; D006046:Gold / Q000737:chemistry*; D017404:In Situ Hybridization, Fluorescence / Q000295:instrumentation; D053768:Metal Nanoparticles / Q000737:chemistry* / Q000648:ultrastructure; D008969:Molecular Sequence Data; D036103:Nanotechnology / Q000295:instrumentation; D020641:Polymorphism, Single Nucleotide / Q000235:genetics*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA / Q000295:instrumentation*; D013050:Spectrometry, Fluorescence / Q000295:instrumentation*
D006046:Gold; D004247:DNA