Ligation-rolling circle amplification combined with γ-cyclodextrin mediated stemless molecular beacon for sensitive and specific genotyping of single-nucleotide polymorphism.
A novel approach for highly sensitive and selective genotyping of single-nucleotide polymorphism (SNP) has been developed based on ligation-rolling circle amplification (L-RCA) and stemless molecular beacon. In this approach, two tailored DNA probes were involved. The stemless molecular beacon, formed through the inclusion interactions of γ-cyclodextrin (γ-CD) and bis-pyrene labeled DNA fragment, was served as signal probe. In the absence of mutant target, the two pyrene molecules were bound in the γ-CD cavity to form an excimer and showed a strong fluorescence at 475 nm. It was here named γ-CD-P-MB. The padlock DNA probe was designed as recognition probe. Upon the recognition of a point mutation DNA targets, the padlock probe was ligated to generate a circular template. An RCA amplification was then initiated using the circular template in the presence of Phi29 polymerase and dNTPs. The L-RCA products, containing repetitive sequence units, subsequently hybridized with the γ-CD-P-MB. This made pyrene molecules away from γ-CD cavity and caused a decrease of excimer fluorescence. As a proof-of-concept, SNP typing of β-thalassemia gene at position -28 was investigated using this approach. The detection limit of mutated target was determined to be 40 fM. In addition, DNA ligase offered high fidelity in distinguishing the mismatched bases at the ligation site, resulting in positive detection of mutant target even when the ratio of the wildtype to the mutant is 999:1. Given these attractive characteristics, the developed approach might provide a great genotyping platform for pathogenic diagnosis and genetic analysis.
State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Changsha 410082, PR China; College of Biology, Hunan University, Changsha 410082, PR China; Key Laboratory for Bio-Nanotechnology and Molecular Engineering of Hunan Province, Changsha 410082, PR China.
Zhen
Z
Zou
State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Changsha 410082, PR China; College of Biology, Hunan University, Changsha 410082, PR China; Key Laboratory for Bio-Nanotechnology and Molecular Engineering of Hunan Province, Changsha 410082, PR China.
Zhihe
Z
Qing
State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Changsha 410082, PR China; College of Biology, Hunan University, Changsha 410082, PR China; Key Laboratory for Bio-Nanotechnology and Molecular Engineering of Hunan Province, Changsha 410082, PR China. Electronic address: xiaoxiaohe@hnu.edu.cn.
Xiaoxiao
X
He
State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Changsha 410082, PR China; College of Biology, Hunan University, Changsha 410082, PR China; Key Laboratory for Bio-Nanotechnology and Molecular Engineering of Hunan Province, Changsha 410082, PR China. Electronic address: kmwang@hnu.edu.cn.
Kemin
K
Wang
State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Changsha 410082, PR China; College of Biology, Hunan University, Changsha 410082, PR China; Key Laboratory for Bio-Nanotechnology and Molecular Engineering of Hunan Province, Changsha 410082, PR China.
Dinggeng
D
He
State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Changsha 410082, PR China; College of Biology, Hunan University, Changsha 410082, PR China; Key Laboratory for Bio-Nanotechnology and Molecular Engineering of Hunan Province, Changsha 410082, PR China.
Hui
H
Shi
State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Changsha 410082, PR China; College of Biology, Hunan University, Changsha 410082, PR China; Key Laboratory for Bio-Nanotechnology and Molecular Engineering of Hunan Province, Changsha 410082, PR China.
Xue
X
Yang
State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Changsha 410082, PR China; College of Biology, Hunan University, Changsha 410082, PR China; Key Laboratory for Bio-Nanotechnology and Molecular Engineering of Hunan Province, Changsha 410082, PR China.
Taiping
T
Qing
State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Changsha 410082, PR China; College of Biology, Hunan University, Changsha 410082, PR China; Key Laboratory for Bio-Nanotechnology and Molecular Engineering of Hunan Province, Changsha 410082, PR China.
Xiaoxiao
X
Yang
D004247:DNA / Q000032:analysis* / Q000737:chemistry / Q000235:genetics; D004270:DNA, Circular / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology; D005456:Fluorescent Dyes / Q000737:chemistry; D005838:Genotype; D006801:Humans; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D009693:Nucleic Acid Hybridization; D009841:Oligonucleotides / Q000032:analysis; D017354:Point Mutation; D020641:Polymorphism, Single Nucleotide; D011721:Pyrenes / Q000737:chemistry; D017086:beta-Thalassemia / Q000235:genetics; D047408:gamma-Cyclodextrins / Q000737:chemistry*
D004270:DNA, Circular; D005456:Fluorescent Dyes; D009841:Oligonucleotides; D011721:Pyrenes; D047408:gamma-Cyclodextrins; D004247:DNA; C030984:pyrene; C023792:gamma-cyclodextrin
Ligation-rolling circle amplification; Single-nucleotide polymorphism; Stemless molecular beacon; γ-Cyclodextrin