Combination of DNA Ligase Reaction and Gold Nanoparticle-Quenched Fluorescent Oligonucleotides: A Simple and Efficient Approach for Fluorescent Assaying of Single-Nucleotide Polymorphisms

Abstract

A new fluorescent sensing approach for detection of single-nucleotide polymorphisms (SNPs) is proposed based on the ligase reaction and gold nanoparticle (AuNPs)-quenched fluorescent oligonucleotides. The design exploits the strong fluorescence quenching of AuNPs for organic dyes and the difference in noncovalent interactions of the nanoparticles with single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA), where ssDNA can be adsorbed onto the surface of AuNPs while dsDNA cannot be. In the assay, two half primer DNA probes, one being labeled with a dye and the other being phosphorylated, were first incubated with a target DNA template. In the presence of DNA ligase, the two captured ssDNAs are linked for the perfectly matched DNA target to form a stable duplex, but the duplex could not be formed by the single-base mismatched DNA template. After addition of AuNPs, the fluorescence of dye-tagged DNA probe will be efficiently quenched unless the perfectly matched DNA target is present. To demonstrate the feasibility of this design, the performance of SNP detection using two different DNA ligases, T4 DNA ligase and Escherichia coli DNA ligase, were investigated. In the case of T4 DNA ligase, the signal enhancement of the dye-tagged DNA for perfectly matched DNA target is 4.6-fold higher than that for the single-base mismatched DNA. While in the presence of E. coli DNA ligase, the value raises to be 30.2, suggesting excellent capability for SNP discrimination.

Ultrasensitive Detection of Single Nucleotide Polymorphism in Human Mitochondrial DNA Utilizing Ion-Mediated Cascade Surface-Enhanced Raman Spectroscopy Amplification

“…Then the P1 was added and the ligation reaction was conducted according to our previous work. 27 To differentially denature the duplex formed by T1 or T2, the solutions were heated to 43°C for 20 min, which was identified by thermally melting. Then the solutions were centrifuged at 1000 rpm for 3 min and the precipitate was collected.…”

DNA template-synthesized silver nanoparticles: A new platform for high-performance fluorescent biosensing of biothiols

“…Through fluorescence resonance energy transfer (FRET) process or photo-induced electron transfer (PET) process, most fluorescent dyes could be quenched by metal nanoparticles [6][7][8][9][10][11][12][13]. When gold nanoparticles (GNPs) were employed as metal nanoquenchers to establish fluorescent platforms for biosensing and received great success during last decades, however, little attention was paid to such applications of AgNPs.…”

“…In recent years, to develop sensitive, rapid and cost-effective biosensors, metal nanoparticles were employed as super nanoquenchers instead of traditional organic dyes in design of fluorescent sensors [6][7][8][9][10][11][12][13]. For the much higher quenching efficiency, metal nanoparticle-based fluorescent sensors were successfully developed with advantages of lower background and better sensitivity.…”

“…An alternative way is to non-cross link them by weak interactions as electrostatic force and π-π stacking interaction (Scheme 1(b)). Sensors for label-free fluorescent detections are developed under this strategy with advantages of simplicity and fast responding [8][9][10][11][12][13]. However, the relatively weak interactions make the non-cross linked combinations unstable in complex conditions, which increase the background and limit their applications in real samples.…”

Gold nanoparticle fluorescent molecular beacon for low-resolution DQ2 gene HLA typing

“…Au surfaces (16)(17)(18) and even more gold nanoparticles (AuNPs) (19)(20)(21)(22)(23)(24)(25) have found application as highly efficient and universal quenchers. Moreover the use of Au based quenching has been shown to be characterised by minimal background fluorescence and subsequently improved signal-tobackground ratio (9,23,26).…”