Toehold-mediated 链置换(荧光偏振)
合集下载
相关主题
- 1、下载文档前请自行甄别文档内容的完整性，平台不提供额外的编辑、内容补充、找答案等附加服务。
- 2、"仅部分预览"的文档,不可在线预览部分如存在完整性等问题,可反馈申请退款(可完整预览的文档不适用该条件!)。
- 3、如文档侵犯您的权益，请联系客服反馈,我们会尽快为您处理(人工客服工作时间：9:00-18:30)。
& 2012 Elsevier B.V. All rights reserved.
1. Introduction
Single nucleotide polymorphism (SNP) detection is very important in biological and clinical studies because SNP is associated with phenotyping variations, gene functions, anthropometric characteristics, and diseases (Boffa et al., 2008; Marmiroli et al., 2011; Rutters et al., 2011; Simon-Sanchez et al., 2008). Many methodologies have been developed for SNP detection. Representative strategies include allele-specific probe hybridization (Fan et al., 2003; Yang et al., 2006), specific enzymatic cleavage (Chen et al., 2004; Lyamichev et al., 1999), primer extension (Litos et al., 2007; Steemers et al., 2006) as well as ligation (Xu et al., 2008; Wang et al., 2011). There are many methodological approaches to measure the signal output including, to name a few, fluorescence (Duan et al., 2009; Xiao et al., 2009), mass spectroscopy (Sauer et al., 2006; Tost and Gut, 2002),
electrochemistry (Hu et al., 2011; Liu and Lin, 2007), and gel electrophoresis (Cheng et al., 2011; Li et al., 2011). Until now, there has been no consensus for the best SNP detection method in terms of sample throughput, simplicity, robustness, and cost. There is a continuous need for new SNP detection methods to meet specific needs and situations.
Fluorescence anisotropy (r), which is sensitive to changes in the motion of the fluorophore, has substantial advantages in homogeneous and high throughput assays. The variation in fluorescence intensity due to photobleaching and other effects does not significantly affect quantitative fluorescence anisotropy measurements (Fang et al., 2001). Furthermore, fluorescence anisotropy measurement based on recognition reactions, such as aptamer-target reaction, antibody–antigen reaction, and DNA hybridization, do not require separation of reagent blank from the test media. Therefore, fluorescence anisotropy can be used for homogeneous analysis and has the potential for automated highthroughput assays (Jameson and Ross, 2010; Ruta et al., 2009).
article info
Article history: Received 4 September 2012 Accepted 15 September 2012 Available online 24 September 2012
Keywords: Fluorescence anisotropy Gold nanoparticles Single nucleotide polymorphism Strand-displacement
However, the application of fluorescence anisotropy measurement in DNA hybridization has been limited to date, because the
Biosensors and Bioelectronics 41 (2013) 569–575
Contents lists available at SciVerse ScienceDirect
Biosensors and Bioelectronics
journal homepage: www.elsevier.com/locate/bios
abstract
We developed a highly differentiating, homogeneous gold nanoparticle (AuNP) enhanced fluorescence anisotropic method for single nucleotide polymorphism (SNP) detection at nanomolar level using toehold-mediated strand-displacement reaction. The template strand, containing a toehold domain with an allele-specific site, was immobilized on the surface of AuNPs, and the solution fluorescence anisotropy was markedly enhanced when the fluorescein-labeled blocking DNA was attached to the AuNP via hybridization. Strand-displacement by the target ssDNA strand resulted in detachment of fluorescein-labeled DNA from AuNPs, and thus decreased fluorescence anisotropy. The drastic kinetic difference in strand-displacement from toehold design was used to distinguish between the perfectly matched and the single-base mismatched strands. Free energy changes were calculated to elucidate the dependence of the differentiation ability on the mutation site in the toehold region. A solid negative signal change can be obtained for single-base mismatched strand in the dynamic range of the calibration curve, and a more than 10-fold signal difference can still be observed in a mixed solution containing 100 times the single-base mismatched strand, indicating the good specificity of the method. This proposed method can be performed with a standard spectrofluorimeter in a homogeneous and cost-effective manner, and has the potential to be extended to the application of fluorescence anisotropy method of SNP detection.
n Corresponding author. Tel.: þ86 10 62761187; fax: þ86 10 62751708. E-mail address: lina@pku.edu.cn (N. Li).
0956-5663/$ - see front matter & 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.bios.2012.09.023
Gold nanoparticle enhanced fluorescence anisotropy for the assay of single nucleotide polymorphisms (SNPs) based on toehold-mediated strand-displacement reaction
Xinyi Wang a,b,c, Mingjian Zou来自百度文库b, Hongduan Huang b, Yuqian Ren b, Limei Li a, Xiaoda Yang c, Na Li b,n
a College of Sciences, Shenyang Agricultural University, Shenyang 110161, China b Beijing National Laboratory for Molecular Sciences (BNLMS), The Key Laboratory of Bioorganic Chemistry and Molecular Engineering, Ministry of Education, College of Chemistry and Molecular Engineering, Yihueyuan Road no 5, Peking University, Haidian District, Beijing 100871, China c Department of Chemical Biology, School of Pharmaceutical Sciences, Peking University, Beijing 100083, China
1. Introduction
Single nucleotide polymorphism (SNP) detection is very important in biological and clinical studies because SNP is associated with phenotyping variations, gene functions, anthropometric characteristics, and diseases (Boffa et al., 2008; Marmiroli et al., 2011; Rutters et al., 2011; Simon-Sanchez et al., 2008). Many methodologies have been developed for SNP detection. Representative strategies include allele-specific probe hybridization (Fan et al., 2003; Yang et al., 2006), specific enzymatic cleavage (Chen et al., 2004; Lyamichev et al., 1999), primer extension (Litos et al., 2007; Steemers et al., 2006) as well as ligation (Xu et al., 2008; Wang et al., 2011). There are many methodological approaches to measure the signal output including, to name a few, fluorescence (Duan et al., 2009; Xiao et al., 2009), mass spectroscopy (Sauer et al., 2006; Tost and Gut, 2002),
electrochemistry (Hu et al., 2011; Liu and Lin, 2007), and gel electrophoresis (Cheng et al., 2011; Li et al., 2011). Until now, there has been no consensus for the best SNP detection method in terms of sample throughput, simplicity, robustness, and cost. There is a continuous need for new SNP detection methods to meet specific needs and situations.
Fluorescence anisotropy (r), which is sensitive to changes in the motion of the fluorophore, has substantial advantages in homogeneous and high throughput assays. The variation in fluorescence intensity due to photobleaching and other effects does not significantly affect quantitative fluorescence anisotropy measurements (Fang et al., 2001). Furthermore, fluorescence anisotropy measurement based on recognition reactions, such as aptamer-target reaction, antibody–antigen reaction, and DNA hybridization, do not require separation of reagent blank from the test media. Therefore, fluorescence anisotropy can be used for homogeneous analysis and has the potential for automated highthroughput assays (Jameson and Ross, 2010; Ruta et al., 2009).
article info
Article history: Received 4 September 2012 Accepted 15 September 2012 Available online 24 September 2012
Keywords: Fluorescence anisotropy Gold nanoparticles Single nucleotide polymorphism Strand-displacement
However, the application of fluorescence anisotropy measurement in DNA hybridization has been limited to date, because the
Biosensors and Bioelectronics 41 (2013) 569–575
Contents lists available at SciVerse ScienceDirect
Biosensors and Bioelectronics
journal homepage: www.elsevier.com/locate/bios
abstract
We developed a highly differentiating, homogeneous gold nanoparticle (AuNP) enhanced fluorescence anisotropic method for single nucleotide polymorphism (SNP) detection at nanomolar level using toehold-mediated strand-displacement reaction. The template strand, containing a toehold domain with an allele-specific site, was immobilized on the surface of AuNPs, and the solution fluorescence anisotropy was markedly enhanced when the fluorescein-labeled blocking DNA was attached to the AuNP via hybridization. Strand-displacement by the target ssDNA strand resulted in detachment of fluorescein-labeled DNA from AuNPs, and thus decreased fluorescence anisotropy. The drastic kinetic difference in strand-displacement from toehold design was used to distinguish between the perfectly matched and the single-base mismatched strands. Free energy changes were calculated to elucidate the dependence of the differentiation ability on the mutation site in the toehold region. A solid negative signal change can be obtained for single-base mismatched strand in the dynamic range of the calibration curve, and a more than 10-fold signal difference can still be observed in a mixed solution containing 100 times the single-base mismatched strand, indicating the good specificity of the method. This proposed method can be performed with a standard spectrofluorimeter in a homogeneous and cost-effective manner, and has the potential to be extended to the application of fluorescence anisotropy method of SNP detection.
n Corresponding author. Tel.: þ86 10 62761187; fax: þ86 10 62751708. E-mail address: lina@pku.edu.cn (N. Li).
0956-5663/$ - see front matter & 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.bios.2012.09.023
Gold nanoparticle enhanced fluorescence anisotropy for the assay of single nucleotide polymorphisms (SNPs) based on toehold-mediated strand-displacement reaction
Xinyi Wang a,b,c, Mingjian Zou来自百度文库b, Hongduan Huang b, Yuqian Ren b, Limei Li a, Xiaoda Yang c, Na Li b,n
a College of Sciences, Shenyang Agricultural University, Shenyang 110161, China b Beijing National Laboratory for Molecular Sciences (BNLMS), The Key Laboratory of Bioorganic Chemistry and Molecular Engineering, Ministry of Education, College of Chemistry and Molecular Engineering, Yihueyuan Road no 5, Peking University, Haidian District, Beijing 100871, China c Department of Chemical Biology, School of Pharmaceutical Sciences, Peking University, Beijing 100083, China