title: Controllable Mismatched Ligation for Bioluminescence Screening of Known and Unknown Mutations.
abstract: Single-nucleotide polymorphisms (SNPs) are closely related to human diseases and individual drug responses, and the accurate detection of SNPs is crucial to both clinical diagnosis and development of personalized medicine. Among various SNPs detection methods, ligase detection reaction (LDR) has shown great potential due to its low detection limit and excellent specificity. However, frequent involvement of expensive labels increases the experimental cost and compromises the assay efficiency, and the requirement of careful predesigned probes limits it to only known SNPs assays. In this research, we develop a controllable mismatched ligation for bioluminescence screening of both known and unknown mutations. Especially, the ligation specificity of E. coli ligase is tunable under different experimental conditions. The mismatches locating on the 3'-side of the nick cannot be ligated efficiently by E. coli ligase, whereas all mismatches locating on the 5'-side of the nick can be ligated efficiently by E. coli ligase. We design a 3'-discriminating probe (3'-probe) for the discrimination of known mutation and introduce a T7 Endo I for the detection of unknown mutation. With the integration of bioluminescence monitoring of ligation byproduct adenosine 5'-monophosphate (AMP), both known and unknown SNPs can be easily detected without the involvement of any expensive labels and labor-intensive separation. This method is simple, homogeneous, label-free, and cost-effective and may provide a valuable complement to current sequencing technologies for disease diagnostics, personalized medicine, and biomedical research.
authors.affiliation: Single-Molecule Detection and Imaging Laboratory, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences , Shenzhen, Guangdong 518055, China.
authors.forename: Qinfeng
authors.initials: Q
authors.lastname: Xu
authors.affiliation: Single-Molecule Detection and Imaging Laboratory, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences , Shenzhen, Guangdong 518055, China.
authors.forename: Si-qiang
authors.initials: SQ
authors.lastname: Huang
authors.affiliation: College of Chemistry, Chemical Engineering and Materials Science, Collaborative Innovation Center of Functionalized Probes for Chemical Imaging in Universities of Shandong, Key Laboratory of Molecular and Nano Probes, Ministry of Education, Shandong Provincial Key Laboratory of Clean Production of Fine Chemicals, Shandong Normal University , Jinan, Shandong 250014, China.
authors.forename: Fei
authors.initials: F
authors.lastname: Ma
authors.affiliation: College of Chemistry, Chemical Engineering and Materials Science, Collaborative Innovation Center of Functionalized Probes for Chemical Imaging in Universities of Shandong, Key Laboratory of Molecular and Nano Probes, Ministry of Education, Shandong Provincial Key Laboratory of Clean Production of Fine Chemicals, Shandong Normal University , Jinan, Shandong 250014, China.
authors.forename: Bo
authors.initials: B
authors.lastname: Tang
authors.affiliation: College of Chemistry, Chemical Engineering and Materials Science, Collaborative Innovation Center of Functionalized Probes for Chemical Imaging in Universities of Shandong, Key Laboratory of Molecular and Nano Probes, Ministry of Education, Shandong Provincial Key Laboratory of Clean Production of Fine Chemicals, Shandong Normal University , Jinan, Shandong 250014, China.|Single-Molecule Detection and Imaging Laboratory, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences , Shenzhen, Guangdong 518055, China.
authors.forename: Chun-yang
authors.initials: CY
authors.lastname: Zhang
mesh_terms: D004247:DNA / Q000235:genetics*; D011088:DNA Ligases / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology; D006367:HeLa Cells; D006801:Humans; D008163:Luminescent Measurements; D009154:Mutation; D020641:Polymorphism, Single Nucleotide / Q000235:genetics
chemical_list: D004247:DNA; D011088:DNA Ligases