title: Detection of single-nucleotide polymorphisms with novel leaky surface acoustic wave biosensors, DNA ligation and enzymatic signal amplification.
abstract: This manuscript describes a new technique for detecting single-nucleotide polymorphisms (SNPs) by integrating a leaky surface acoustic wave (LSAW) biosensor, enzymatic DNA ligation and enzymatic signal amplification. In this technique, the DNA target is hybridized with a capture probe immobilized on the surface of a LSAW biosensor. Then, the hybridized sequence is ligated to biotinylated allele-specific detection probe using Taq DNA ligase. The ligation does not take place if there is a single-nucleotide mismatch between the target and the capture probe. The ligated detection probe is transformed into a streptavidin-horseradish peroxidase (SA-HRP) terminal group via a biotin-streptavidin complex. Then, the SA-HRP group catalyzes the polymerization of 3,3-diaminobenzidine (DAB) to form a surface precipitate, thus effectively increasing the sensitivity of detecting surface mass changes and allowing detection of SNPs. Optimal detection conditions were found to be: 0.3 mol/L sodium ion concentration in PBS, pH 7.6, capture probe concentration 0.5 μmol/L and target sequence concentration 1.0 μmol/L. The detection limit was found to be 1 × 10(-12)mol/L. Using this technique, we were able to detect a single-point mutation at nucleotide A2293G in Japanese encephalitis virus.
authors.affiliation: Department of Clinical Laboratory Medicine, Institute of Surgery Research, Daping Hospital, The Third Military Medical University, Chongqing 400042, China.
authors.forename: Qinghua
authors.initials: Q
authors.lastname: Xu
authors.forename: Kai
authors.initials: K
authors.lastname: Chang
authors.forename: Weiping
authors.initials: W
authors.lastname: Lu
authors.forename: Wei
authors.initials: W
authors.lastname: Chen
authors.forename: Yi
authors.initials: Y
authors.lastname: Ding
authors.forename: Shuangrong
authors.initials: S
authors.lastname: Jia
authors.forename: Kejun
authors.initials: K
authors.lastname: Zhang
authors.forename: Fake
authors.initials: F
authors.lastname: Li
authors.forename: Jianfeng
authors.initials: J
authors.lastname: Shi
authors.forename: Liang
authors.initials: L
authors.lastname: Cao
authors.forename: Shaoli
authors.initials: S
authors.lastname: Deng
authors.forename: Ming
authors.initials: M
authors.lastname: Chen
mesh_terms: D015100:3,3'-Diaminobenzidine / Q000737:chemistry; D001426:Bacterial Proteins / Q000737:chemistry; D015374:Biosensing Techniques / Q000379:methods*; D001710:Biotin / Q000031:analogs & derivatives / Q000737:chemistry; D004247:DNA / Q000737:chemistry*; D006735:Horseradish Peroxidase / Q000737:chemistry; D006801:Humans; D006863:Hydrogen-Ion Concentration; D020641:Polymorphism, Single Nucleotide; D012680:Sensitivity and Specificity; D013016:Sound
chemical_list: D001426:Bacterial Proteins; C066114:biotin-streptavidin complex; D015100:3,3'-Diaminobenzidine; D001710:Biotin; D004247:DNA; D006735:Horseradish Peroxidase