基于NUPACK预测设计的Toehold诱导链置换反应及其在
以阿尔兹海默症相关的基因片段rs242557为研究对象, 通过NUPACK软件预测Toehold(黏性末端)长度对链置换反应的影响, 设计具有高特异性的双链核苷酸探针; 富G的核苷酸序列首先被掩蔽在Watson-Crick双链结构中, 当体系加入目标基因后, 通过引发链置换反应解开该双链结构, 形成的DNA酶可催化氧化鲁米诺化学发光反应, 最终采用微流控化学发光法实现单核苷酸多态性(SNP)分析. 该方法不仅具有设计简单、 免标记及检测通量高等优点, 而且在仅消耗2 μL试样的条件下实现了单碱基突变的rs242557目标基因的SNP分析(区分因子达56), 正常目标基因的检出限为1.7 nmol/L.
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邬期望, 沈宏. 基于NUPACK预测设计的Toehold诱导链置换反应及其在DNA酶催化微流控化学发光单核苷酸多态性分析中的应用. 高等学校化学学报, 2015, 36(12): 2386-2393.
WU Qiwang, SHEN Hong. NUPACK Prediction Assisted of Toehold Induced Strand Displacement Reaction and Its Application in SNPs Genotyping by DNAzyme-catalyzed Microfluidic Chemiluminescence Detection†. Chem. J. Chinese Universities, 2015, 36(12): 2386-2393.
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Table 1 Sequences of the oligonucleotides used in this work*
Table 1 Sequences of the oligonucleotides used in this work*
Scheme 1 Schematic diagram of sequential injection chemiluminescence system
Scheme 1 Schematic diagram of sequential injection chemiluminescence system
Scheme 2 Schematic diagram of SNP typing utilizing toehold-mediated strand displacement reaction and DNAzyme-catalyzed chemiluminescence detection
Scheme 2 Schematic diagram of SNP typing utilizing toehold-mediated strand displacement reaction and DNAzyme-catalyzed chemiluminescence detection
Fig.1 Impact of toehold length on SNP typing (A) Predicted by NUPACK simulations; (B) CL analysis; (C) DF calculation results. The concentrations of P strands, B strands and rs242557 are 150, 300 and 80 nmol/L, respectively.
Fig.1 Impact of toehold length on SNP typing (A) Predicted by NUPACK simulations; (B) CL analysis; (C) DF calculation results. The concentrations of P strands, B strands and rs242557 are 150, 300 and 80 nmol/L, respectively.
Fig.2 Native PAGE results(A) and UV-Vis absorption of hemin-oligonucleotide interaction(B) (A) Lane 1: P strand, lane 2: P strand annealed with B strand, lane 3: P/B reacted with rs242557-G, lane 4: P strand annealed with rs242557-G, lane 5: rs242557-G, lane 6: rs242557-A, lane 7: P/B reacted with rs242557-A; (B) hemin: 1.5 μmol/L, P strand: 300 nmol/L, B strand: 600 nmol/L, rs242557 strand: 200 nmol/L. a. Hemin; b. a+P/B; c. b+rs242557-A; d. b+rs242557-G.
Fig.2 Native PAGE results(A) and UV-Vis absorption of hemin-oligonucleotide interaction(B) (A) Lane 1: P strand, lane 2: P strand annealed with B strand, lane 3: P/B reacted with rs242557-G, lane 4: P strand annealed with rs242557-G, lane 5: rs242557-G, lane 6: rs242557-A, lane 7: P/B reacted with rs242557-A; (B) hemin: 1.5 μmol/L, P strand: 300 nmol/L, B strand: 600 nmol/L, rs242557 strand: 200 nmol/L. a. Hemin; b. a+P/B; c. b+rs242557-A; d. b+rs242557-G.
Fig.3 Influence of reaction time on DNA detection Control: blank sample solution, sample rs242557-G solution: 80 nmol/L.
Fig.3 Influence of reaction time on DNA detection Control: blank sample solution, sample rs242557-G solution: 80 nmol/L.
Fig.4 Influence of H2O2(A) and luminol(B) concentration on DNA detection Sample: 80 nmol/L rs242557-G solution.
Fig.4 Influence of H2O2(A) and luminol(B) concentration on DNA detection Sample: 80 nmol/L rs242557-G solution.
Fig.5 Influence of flow rate on DNA detection Sample: 80 nmol/L rs242557-G solution.
Fig.5 Influence of flow rate on DNA detection Sample: 80 nmol/L rs242557-G solution.
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