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

doi:10.7503/cjcu20150427

高等学校化学学报 ›› 2015, Vol. 36 ›› Issue (12): 2386.doi: 10.7503/cjcu20150427

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

邬期望, 沈宏()

浙江大学化学系, 微分析系统研究所, 杭州 310058

收稿日期:2015-05-26 出版日期:2015-12-10 发布日期:2015-11-17
作者简介:联系人简介: 沈宏, 男, 博士, 副教授, 主要从事分析化学研究. E-mail:shzju@zju.edu.cn
基金资助:
国家自然科学基金(批准号: 21075110)、浙江省自然科学基金(批准号: LY16B050004)和浙大青年教师交叉研究种子基金(批准号: JCZZ-2013010)资助

NUPACK Prediction Assisted of Toehold Induced Strand Displacement Reaction and Its Application in SNPs Genotyping by DNAzyme-catalyzed Microfluidic Chemiluminescence Detection^†

WU Qiwang, SHEN Hong^*()

Institute of Microanalytical Systems, Department of Chemistry, Zhejiang University, Hangzhou 310058, China

Received:2015-05-26 Online:2015-12-10 Published:2015-11-17
Contact: SHEN Hong E-mail:shzju@zju.edu.cn
Supported by:
† Supported by the National Natural Science Foundation of China(No.21075110), the Natural Science Foundation of Zhejiang Province, China(No.LY16B050004) and the Interdisciplinary Seed Research Fund of Zhejiang University, China(No.JCZZ-2013010)

摘要/Abstract

摘要：

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.

关键词: NUPACK软件, 链置换反应, 单核苷酸多态性, DNA酶, 微流控

Abstract:

Highly sensitive and selective method for single nucleotide polymorphisms(SNPs) genotyping is of great importance for early diagnosis and treatment of various diseases. By taking rs242557, an Alzheimer’s disease related gene fragment, as a target model, the impact of toehold length on strand displacement reaction was predicted by NUPACK software simulation in order to rationalize the design of duplex DNA probe for high genotyping efficiency. The G-rich DNA sequence was initially blocked in the duplex strand structure. With the addition of target gene fragment to trigger the blocked-domain dehybridization and the toehold-mediated strand displacement reaction, the DNAzyme was formed. Combined with microfluidic chemiluminescence detection, a novel SNP genotyping method had been developed, which was not only label-free, easy to design and exhibited high throughput ability, but also displayed a remarkable specificity to target rs242557 against single base mutation, achieving a discrimination factor of 56 and a detection limit of 1.7 nmol/L with just 2 μL of sample solution consumption.

Key words: NUPACK, Strand displacement reaction, Single nucleotide polymorphisms(SNP), DNAzyme, Microfluidic

中图分类号:

O657.3

TrendMD:

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.

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.

图/表 11

Table 1 Sequences of the oligonucleotides used in this work*

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

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.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.5 Influence of flow rate on DNA detection Sample: 80 nmol/L rs242557-G solution.

Fig.6 CL response to the different concentrations of sample rs242557-G solution(A) and corresponding calibration plot between net CL response and sample rs242557-G solution from 5 to 400 nmol/L(B) Insert of (B): magnification of the plots in the range of 5—200 nmol/L.

Fig.7 CL responses on control, wild type and mutant type sample solutions Target sample solutions: 80 nmol/L. a. Control; b. rs242557-A; c. rs242557-G; d. rs242557-A34; e. rs242557-G34.

Table 2 DNA detection in biological samples

Sample	Added/(nmol·L^-1)	Found/(nmol·L^-1)	Recovery(%)	RSD(%, n=5)
1	40	36.8	92.0	4.02
2	60	63.4	105.6	2.79
3	80	74.7	93.4	4.51

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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

NUPACK Prediction Assisted of Toehold Induced Strand Displacement Reaction and Its Application in SNPs Genotyping by DNAzyme-catalyzed Microfluidic Chemiluminescence Detection^†

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