Cyclic polyolefin (COP) is an inexpensive hydrophobic material with low auto-fluorescence, high light transmittance and thermal stability, broad chemical resistance and no non-specific protein binding. Here, the hydro...Cyclic polyolefin (COP) is an inexpensive hydrophobic material with low auto-fluorescence, high light transmittance and thermal stability, broad chemical resistance and no non-specific protein binding. Here, the hydrophobic alkane COP was modified to have carbonyl functionalities through oxygen plasma and chemical etching treatments to increase usefulness for chemical and biochemical applications. Then, biotin-hydrazide was used to create biotinylated surfaces that bound streptavidin. A biotinylated target oligonucleotide was subsequently bound to the immobilized biotin-streptavidin and ligation mediated rolling circle amplification-based (L-RCA) SNP detection was demonstrated.展开更多
为微数组上的 DNA 有约束力的蛋白质的质的察觉的一条敏感途径被开发。一个部分双区域从一份 biotin 教材和圆单身者海滨 DNA (ssDNA ) 在被形成的 DNA 建筑群在一个微数组上被看到。endonuclease 识别地点(ERS ) 和 DNA 有约束力的地点...为微数组上的 DNA 有约束力的蛋白质的质的察觉的一条敏感途径被开发。一个部分双区域从一份 biotin 教材和圆单身者海滨 DNA (ssDNA ) 在被形成的 DNA 建筑群在一个微数组上被看到。endonuclease 识别地点(ERS ) 和 DNA 有约束力的地点(DBS ) 在双区域以内并排被安排。察觉系统的工作原则如下被描述：什么时候 DNA 有约束力的蛋白质俘获 DBS， endonuclease 不能在 DNA 建筑群属于 ERS，和使不能调动的教材能被卷圆扩大(RCA ) 沿着圆 ssDNA 扩大。当没有蛋白质保护 DBS 时， ERS 能被 endonuclease 攻击，随后，没有滚动的圆扩大发生。从而，我们能检测顺序特定的 DNA 有约束力的活动与由于 RCA 的信号扩大高敏感。展开更多
Banana (Musa sp.) is a popular and important crop among many communities in East Africa. Banana production is however threatened by the wide-spread banana streak disease (BSD), caused by Banana streak virus (BSV). The...Banana (Musa sp.) is a popular and important crop among many communities in East Africa. Banana production is however threatened by the wide-spread banana streak disease (BSD), caused by Banana streak virus (BSV). The success of BSV management is inherently coupled to the availability of a sensitive indexing method. In this study, the sensitivity of three BSV detection techniques: rolling circle amplification (RCA), immunocapture PCR (with degenerate and Gold finger primers) and standard PCR was compared. A set of 32 BSD-asymptomatic samples were used to compare the techniques. Analysis of variance (ANOVA) for comparison of the four techniques showed that there were significant differences (P Musa tissues for BSV. This study unveils a more reliable BSV detection method, a need that has remained unaddressed for a long while.展开更多
Compared with other types of breast cancer,triple-negative breast cancer(TNBC)has the characteristics of a high degree of malignancy and poor prognosis.Early diagnosis of TNBC through biological markers and timely dev...Compared with other types of breast cancer,triple-negative breast cancer(TNBC)has the characteristics of a high degree of malignancy and poor prognosis.Early diagnosis of TNBC through biological markers and timely development of effective treatment methods can reduce its mortality.Many Research experiments have confirmed that some specific mi RNA expression profiles in TNBC can used as markers for early diagnosis.However,detecting the expression profiles of multiple groups of miRNAs according to traditional detection methods is complicated and consumes many samples.To address this issue,we developed a method for high-throughput,high-sensitivity quantitative detection of multiple sets of miRNAs(including mi R-16,mi R-21,mi R-92,mi R-199,and mi R-342)specifically expressed in TNBC by rolling circle amplification(RCA)on fluorescence-encoded microspheres.Through the optimization of reaction system conditions,the developed method showed an extensive linear dynamic range and high sensitivity for all five miRNAs with the lowest limit of detection of 2 fmol/L.Meanwhile,this high-throughput detection method also appeared reasonable specificity.Only in the presence of a specific target miRNA,the fluorescence signal on the correspondingly encoded microspheres is significantly increased,while the fluorescence signal on other non-correspondingly encoded microspheres is almost negligible.Furthermore,this process exhibited good recovery and reproducibility in serum.The advantages of this method allow us to more conveniently obtain the expression profiles of multiple groups of TNBC-associated mi RNAs,which is beneficial for the early detection of TNBC.展开更多
Exosomes secreted by tumor cells carry abundant molecular biomarkers that reflect the status of their originating cells.These tumor-derived exosomes(TDEs)have emerged as attractive diagnostic targets.However,the ident...Exosomes secreted by tumor cells carry abundant molecular biomarkers that reflect the status of their originating cells.These tumor-derived exosomes(TDEs)have emerged as attractive diagnostic targets.However,the identification and characterization of highly heterogeneous TDEs remain practically challenging.Here,we report a dual rolling circle amplification(DRCA)-assisted approach for the selective encapsulation of single TDEs for fluorescence microscopic and flow cytometric analysis.TDEs have been targeted by aptamers that recognized their surface tumor marker and exosomal marker CD63,following DRCA that produced entangling polymeric DNA chains,resulting in facile particle enlargement that allows single-particle fluorescence profiling of exosome heterogeneity.We have demonstrated the use of a dual-marker positive ratio for exosome differentiation and applied division and multiplication operations for normalized andmagnified marker heterogeneity analysis.We further applied this assay to distinguish lung adenocarcinoma and pulmonary nodule patients and found an accuracy of 90%.We anticipate promising transformations of this straightforward assay into clinically implantable diagnostic methods.展开更多
During the development of structural DNA nanotechnology,the emerging of scaffolded DNA origami is marvelous.It utilizes DNA double helix inherent specificity of Watson-Crick base pairing and structural features to cre...During the development of structural DNA nanotechnology,the emerging of scaffolded DNA origami is marvelous.It utilizes DNA double helix inherent specificity of Watson-Crick base pairing and structural features to create self-assembling structures at the nanometer scale exhibiting the addressable character.However,the assembly of DNA origami is disorderly and unpredictable.Herein,we present a novel strategy to assemble the DNA origami using rolling circle amplification based DNA nanoribbons as the linkers.Firstly,long single-stranded DNA from Rolling Circle Amplification is annealed with several staples to form kinds of DNA nanoribbons with overhangs.Subsequently,the rectangle origami is formed with overhanged staple strands at any edge that would hybridize with the DNA nanoribbons.By mixing them up,we illustrate the one-dimensional even two-dimensional assembly of DNA origami with good orientation.展开更多
A novel, homogeneous and sensitive assay for the detection of single nucleotide polymorphisms (SNPs) by integration of rolling circle amplification (RCA) and cationic conjugated polymer (CCP) has been developed and te...A novel, homogeneous and sensitive assay for the detection of single nucleotide polymorphisms (SNPs) by integration of rolling circle amplification (RCA) and cationic conjugated polymer (CCP) has been developed and tested. Mutant DNA serves as the template for specifically circularizing a padlock probe (PLP) with a sequence that is complementary to the mutant DNA. Afterwards, the mutant DNA directly acts as the primer to initiate the RCA reaction in the presence of phi29 DNA polymerase that generates a long, tandem single-strand DNA product. During the RCA reaction, fluorescein-labeled dUTPs are incorporated into the RCA products. When the CCP is introduced, efficient FRET from CCP to fluorescein occurs as a result of the strong electrostatic interactions between the CCP and the DNA produced by RCA. The wild-type DNA contains a single base mismatch with PLP with the result that the PLP is not circularized, RCA is not triggered and inefficient FRET results. By measuring the change of the emission intensities of CCP and fluorescein, it was possible to detect the SNP in a homogeneous manner. The method is sensitive and specific enough to detect 0.1 pmol/L mutant DNA and to determine a mutant allele frequency as low as 2.0%.展开更多
The fluorescence dye SYBR Green Ⅱ(SG Ⅱ)has been frequently used in rolling circle amplification(RCA)based analyses of nucleic acids.However,a good amount of inconsistencies have been reported in regards the quality ...The fluorescence dye SYBR Green Ⅱ(SG Ⅱ)has been frequently used in rolling circle amplification(RCA)based analyses of nucleic acids.However,a good amount of inconsistencies have been reported in regards the quality and reproducibility of RCA reactions.To properly examine this experimental issue,here we utilized a series of synthetic oligonucleotides and circular templates to investigate the impact of SG Ⅱ in RCA reactions.The results indicate that SG Ⅱ enables a strong fluorescence signal only when complexing with guanosine(G)residue.In RCA reactions,long single-stranded RCA products,enriched with G residues,result in higher fluorescence emission when compared with the addition of other nucleotide residues.These results suggest that the nucleotide composition of the reaction can affect the amplification results and,eventually,can lead to inconsistent fluorescence of the RCA products.This work indicates that particular attention should be given when circular templates are designed for the quantitative analysis of nucleic acids,to further allow the signal reproducibility of RCA-based experiments.展开更多
Nucleic acid(DNA and RNA)detection and quantification methods play vital roles in molecular biology.With the development of molecular biology,isothermal amplification of DNA/RNA,as a new molecular biology technology,c...Nucleic acid(DNA and RNA)detection and quantification methods play vital roles in molecular biology.With the development of molecular biology,isothermal amplification of DNA/RNA,as a new molecular biology technology,can be amplified under isothermal condition,it has the advantages of high sensitivity,high specificity,and high efficiency,and has been applied in various fields of biotechnology,including disease diagnosis,pathogen detection,food hygiene and safety detection and so on.This paper introduces the progress of isothermal amplification technology,including rolling circle amplification(RCA),nucleic acid sequence-dependent amplification(NASBA),strand displacement amplification(SDA),loop-mediated isothermal amplification(LAMP),helicase-dependent amplification(HDA),recombinase polymerase amplification(RPA),cross-priming amplification(CPA),and its principle,advantages and disadvantages,and application development are briefly summarized.展开更多
该文构建一种基于靶标诱导滚环扩增(rolling circle amplification,RCA)的无标记适配体快速检测赭曲霉毒素A(ochratoxin A,OTA)生物传感器。该生物传感器探针由RCA引物与OTA适配体两部分组成,在OTA存在的环境中,OTA适配体特异性识别靶标...该文构建一种基于靶标诱导滚环扩增(rolling circle amplification,RCA)的无标记适配体快速检测赭曲霉毒素A(ochratoxin A,OTA)生物传感器。该生物传感器探针由RCA引物与OTA适配体两部分组成,在OTA存在的环境中,OTA适配体特异性识别靶标,探针结构被打开,RCA引物与环状DNA模板(circular DNA template,CT)结合开启RCA反应,加入核酸染料SYBR Gold产生荧光信号。此生物传感器具有较高的特异性,检测限为6.6×10^(-2)nmol/L,线性检测范围为6.6×10^(-2)~660 nmol/L,可用于具体的分析检测。此生物传感器无需复杂化学修饰且操作简单,在食品安全检测中具有良好的应用前景。展开更多
建立一种不对称聚合酶链式反应结合滚环扩增信号放大(Rolling circle amplification,RCA)的技术,对牛肉中单增李斯特菌进行快速灵敏检测。通过改变上下游引物的浓度,扩增得到一条带有通用序列的单链DNA,进而引发RCA反应,产生大量的G-四...建立一种不对称聚合酶链式反应结合滚环扩增信号放大(Rolling circle amplification,RCA)的技术,对牛肉中单增李斯特菌进行快速灵敏检测。通过改变上下游引物的浓度,扩增得到一条带有通用序列的单链DNA,进而引发RCA反应,产生大量的G-四链体序列,硫黄素T嵌入G-四链体序列中产生荧光信号,从而实现对单增李斯特菌的检测。在最优的条件下,本研究所建立的方法对单增李斯特菌在PBS中的检测限为3.6×101 CFU/mL;在加标的牛肉中,其检测限达到了3.6×102 CFU/g。本研究所建立的方法可实现单增李斯特菌的快速检测,通过改变特异性的引物,该方法也可用于其他食源性致病菌的检测,具有较高的推广应用价值。展开更多
在电化学生物传感器的设计中,信号放大是实验环节中的重要步骤,特别是对靶标进行灵敏度分析时更是不可或缺。滚环扩增(rolling circle amplification,RCA)能够在短时间内得到大量产物,并在电极表面进行扩增或孵育,然后通过一定的设计使...在电化学生物传感器的设计中,信号放大是实验环节中的重要步骤,特别是对靶标进行灵敏度分析时更是不可或缺。滚环扩增(rolling circle amplification,RCA)能够在短时间内得到大量产物,并在电极表面进行扩增或孵育,然后通过一定的设计使电化学信号被快速放大。RCA技术具有高度的灵敏性和特异性,电化学生物传感器则可提供实时、快速、低成本的检测。为了更好的了解RCA,介绍了RCA环化的基本原理、RCA种类,重点总结了RCA与电化学生物传感器结合的不同技术类型及应用,并对未来相关研究领域的发展趋势进行了展望,旨在为RCA技术在电化学生物传感器中的进一步发展和应用提供参考。展开更多
文摘Cyclic polyolefin (COP) is an inexpensive hydrophobic material with low auto-fluorescence, high light transmittance and thermal stability, broad chemical resistance and no non-specific protein binding. Here, the hydrophobic alkane COP was modified to have carbonyl functionalities through oxygen plasma and chemical etching treatments to increase usefulness for chemical and biochemical applications. Then, biotin-hydrazide was used to create biotinylated surfaces that bound streptavidin. A biotinylated target oligonucleotide was subsequently bound to the immobilized biotin-streptavidin and ligation mediated rolling circle amplification-based (L-RCA) SNP detection was demonstrated.
基金supported by the National Natural Science Foundation of China(Nos.60501010,60701008 and 60771024)
文摘为微数组上的 DNA 有约束力的蛋白质的质的察觉的一条敏感途径被开发。一个部分双区域从一份 biotin 教材和圆单身者海滨 DNA (ssDNA ) 在被形成的 DNA 建筑群在一个微数组上被看到。endonuclease 识别地点(ERS ) 和 DNA 有约束力的地点(DBS ) 在双区域以内并排被安排。察觉系统的工作原则如下被描述：什么时候 DNA 有约束力的蛋白质俘获 DBS， endonuclease 不能在 DNA 建筑群属于 ERS，和使不能调动的教材能被卷圆扩大(RCA ) 沿着圆 ssDNA 扩大。当没有蛋白质保护 DBS 时， ERS 能被 endonuclease 攻击，随后，没有滚动的圆扩大发生。从而，我们能检测顺序特定的 DNA 有约束力的活动与由于 RCA 的信号扩大高敏感。
文摘Banana (Musa sp.) is a popular and important crop among many communities in East Africa. Banana production is however threatened by the wide-spread banana streak disease (BSD), caused by Banana streak virus (BSV). The success of BSV management is inherently coupled to the availability of a sensitive indexing method. In this study, the sensitivity of three BSV detection techniques: rolling circle amplification (RCA), immunocapture PCR (with degenerate and Gold finger primers) and standard PCR was compared. A set of 32 BSD-asymptomatic samples were used to compare the techniques. Analysis of variance (ANOVA) for comparison of the four techniques showed that there were significant differences (P Musa tissues for BSV. This study unveils a more reliable BSV detection method, a need that has remained unaddressed for a long while.
基金financially supported by Hainan Provincial Natural Science Foundation of China(No.822CXTD514)Hainan Province Science and Technology Special Found(No.ZDYF2022SHFZ123)。
文摘Compared with other types of breast cancer,triple-negative breast cancer(TNBC)has the characteristics of a high degree of malignancy and poor prognosis.Early diagnosis of TNBC through biological markers and timely development of effective treatment methods can reduce its mortality.Many Research experiments have confirmed that some specific mi RNA expression profiles in TNBC can used as markers for early diagnosis.However,detecting the expression profiles of multiple groups of miRNAs according to traditional detection methods is complicated and consumes many samples.To address this issue,we developed a method for high-throughput,high-sensitivity quantitative detection of multiple sets of miRNAs(including mi R-16,mi R-21,mi R-92,mi R-199,and mi R-342)specifically expressed in TNBC by rolling circle amplification(RCA)on fluorescence-encoded microspheres.Through the optimization of reaction system conditions,the developed method showed an extensive linear dynamic range and high sensitivity for all five miRNAs with the lowest limit of detection of 2 fmol/L.Meanwhile,this high-throughput detection method also appeared reasonable specificity.Only in the presence of a specific target miRNA,the fluorescence signal on the correspondingly encoded microspheres is significantly increased,while the fluorescence signal on other non-correspondingly encoded microspheres is almost negligible.Furthermore,this process exhibited good recovery and reproducibility in serum.The advantages of this method allow us to more conveniently obtain the expression profiles of multiple groups of TNBC-associated mi RNAs,which is beneficial for the early detection of TNBC.
基金supported by the National Key Research Program(grant no.2019YFA0905800)the NSFC Program(grant no.22090053)the Natural Science Foundation of Hunan Province(grant no.2021JJ40040).
文摘Exosomes secreted by tumor cells carry abundant molecular biomarkers that reflect the status of their originating cells.These tumor-derived exosomes(TDEs)have emerged as attractive diagnostic targets.However,the identification and characterization of highly heterogeneous TDEs remain practically challenging.Here,we report a dual rolling circle amplification(DRCA)-assisted approach for the selective encapsulation of single TDEs for fluorescence microscopic and flow cytometric analysis.TDEs have been targeted by aptamers that recognized their surface tumor marker and exosomal marker CD63,following DRCA that produced entangling polymeric DNA chains,resulting in facile particle enlargement that allows single-particle fluorescence profiling of exosome heterogeneity.We have demonstrated the use of a dual-marker positive ratio for exosome differentiation and applied division and multiplication operations for normalized andmagnified marker heterogeneity analysis.We further applied this assay to distinguish lung adenocarcinoma and pulmonary nodule patients and found an accuracy of 90%.We anticipate promising transformations of this straightforward assay into clinically implantable diagnostic methods.
基金This work was supported by grant from the National Natural Science Foundation of China(Nos.21105110&21103219)and the Knowledge Innovation Program of Chinese Academy of Sciences.
文摘During the development of structural DNA nanotechnology,the emerging of scaffolded DNA origami is marvelous.It utilizes DNA double helix inherent specificity of Watson-Crick base pairing and structural features to create self-assembling structures at the nanometer scale exhibiting the addressable character.However,the assembly of DNA origami is disorderly and unpredictable.Herein,we present a novel strategy to assemble the DNA origami using rolling circle amplification based DNA nanoribbons as the linkers.Firstly,long single-stranded DNA from Rolling Circle Amplification is annealed with several staples to form kinds of DNA nanoribbons with overhangs.Subsequently,the rectangle origami is formed with overhanged staple strands at any edge that would hybridize with the DNA nanoribbons.By mixing them up,we illustrate the one-dimensional even two-dimensional assembly of DNA origami with good orientation.
基金supported by the Specialized Research Fund for the Doctoral Program of Higher Education of China (20070075003, 20091301120003)the Natural Science Foundation of Hebei Province (B2009000170)
文摘A novel, homogeneous and sensitive assay for the detection of single nucleotide polymorphisms (SNPs) by integration of rolling circle amplification (RCA) and cationic conjugated polymer (CCP) has been developed and tested. Mutant DNA serves as the template for specifically circularizing a padlock probe (PLP) with a sequence that is complementary to the mutant DNA. Afterwards, the mutant DNA directly acts as the primer to initiate the RCA reaction in the presence of phi29 DNA polymerase that generates a long, tandem single-strand DNA product. During the RCA reaction, fluorescein-labeled dUTPs are incorporated into the RCA products. When the CCP is introduced, efficient FRET from CCP to fluorescein occurs as a result of the strong electrostatic interactions between the CCP and the DNA produced by RCA. The wild-type DNA contains a single base mismatch with PLP with the result that the PLP is not circularized, RCA is not triggered and inefficient FRET results. By measuring the change of the emission intensities of CCP and fluorescein, it was possible to detect the SNP in a homogeneous manner. The method is sensitive and specific enough to detect 0.1 pmol/L mutant DNA and to determine a mutant allele frequency as low as 2.0%.
基金Supported by the National Natural Science Foundation of China(No.31670821).
文摘The fluorescence dye SYBR Green Ⅱ(SG Ⅱ)has been frequently used in rolling circle amplification(RCA)based analyses of nucleic acids.However,a good amount of inconsistencies have been reported in regards the quality and reproducibility of RCA reactions.To properly examine this experimental issue,here we utilized a series of synthetic oligonucleotides and circular templates to investigate the impact of SG Ⅱ in RCA reactions.The results indicate that SG Ⅱ enables a strong fluorescence signal only when complexing with guanosine(G)residue.In RCA reactions,long single-stranded RCA products,enriched with G residues,result in higher fluorescence emission when compared with the addition of other nucleotide residues.These results suggest that the nucleotide composition of the reaction can affect the amplification results and,eventually,can lead to inconsistent fluorescence of the RCA products.This work indicates that particular attention should be given when circular templates are designed for the quantitative analysis of nucleic acids,to further allow the signal reproducibility of RCA-based experiments.
基金supported by grants from Jiangsu Higher Education Institution Innovative Research Team for Science and Technology(2021),the Key Technology Program of Suzhou People’s Livelihood Technology Projects(Grant Nos.SKY2021029,SZS2020311)the Open Project of Jiangsu Biobank of Clinical Resources(TC2021B009)the Qing-Lan Project of Jiangsu Province in China(2021,2022).
文摘Nucleic acid(DNA and RNA)detection and quantification methods play vital roles in molecular biology.With the development of molecular biology,isothermal amplification of DNA/RNA,as a new molecular biology technology,can be amplified under isothermal condition,it has the advantages of high sensitivity,high specificity,and high efficiency,and has been applied in various fields of biotechnology,including disease diagnosis,pathogen detection,food hygiene and safety detection and so on.This paper introduces the progress of isothermal amplification technology,including rolling circle amplification(RCA),nucleic acid sequence-dependent amplification(NASBA),strand displacement amplification(SDA),loop-mediated isothermal amplification(LAMP),helicase-dependent amplification(HDA),recombinase polymerase amplification(RPA),cross-priming amplification(CPA),and its principle,advantages and disadvantages,and application development are briefly summarized.
文摘该文构建一种基于靶标诱导滚环扩增(rolling circle amplification,RCA)的无标记适配体快速检测赭曲霉毒素A(ochratoxin A,OTA)生物传感器。该生物传感器探针由RCA引物与OTA适配体两部分组成,在OTA存在的环境中,OTA适配体特异性识别靶标,探针结构被打开,RCA引物与环状DNA模板(circular DNA template,CT)结合开启RCA反应,加入核酸染料SYBR Gold产生荧光信号。此生物传感器具有较高的特异性,检测限为6.6×10^(-2)nmol/L,线性检测范围为6.6×10^(-2)~660 nmol/L,可用于具体的分析检测。此生物传感器无需复杂化学修饰且操作简单,在食品安全检测中具有良好的应用前景。
文摘建立一种不对称聚合酶链式反应结合滚环扩增信号放大(Rolling circle amplification,RCA)的技术,对牛肉中单增李斯特菌进行快速灵敏检测。通过改变上下游引物的浓度,扩增得到一条带有通用序列的单链DNA,进而引发RCA反应,产生大量的G-四链体序列,硫黄素T嵌入G-四链体序列中产生荧光信号,从而实现对单增李斯特菌的检测。在最优的条件下,本研究所建立的方法对单增李斯特菌在PBS中的检测限为3.6×101 CFU/mL;在加标的牛肉中,其检测限达到了3.6×102 CFU/g。本研究所建立的方法可实现单增李斯特菌的快速检测,通过改变特异性的引物,该方法也可用于其他食源性致病菌的检测,具有较高的推广应用价值。
文摘在电化学生物传感器的设计中,信号放大是实验环节中的重要步骤,特别是对靶标进行灵敏度分析时更是不可或缺。滚环扩增(rolling circle amplification,RCA)能够在短时间内得到大量产物,并在电极表面进行扩增或孵育,然后通过一定的设计使电化学信号被快速放大。RCA技术具有高度的灵敏性和特异性,电化学生物传感器则可提供实时、快速、低成本的检测。为了更好的了解RCA,介绍了RCA环化的基本原理、RCA种类,重点总结了RCA与电化学生物传感器结合的不同技术类型及应用,并对未来相关研究领域的发展趋势进行了展望,旨在为RCA技术在电化学生物传感器中的进一步发展和应用提供参考。
