滚环扩增技术的原理及其应用的研究

滚环扩增是近年来发展起来的一种恒温核酸扩增方法。这种方法不仅可以直接扩增DNA和RNA,还可以实现对靶核酸的信号放大,灵敏度达到一个拷贝的核酸分子,因此,RCA技术在全基因组扩增、单核苷酸多态性、DNA芯片、蛋白质芯片等方面检测中具有很大的应用价值和潜力。     

Signal amplification by rolling circle amplification on DNA microarrays

While microarrays hold considerable promise in large-scale biology on account of their massively parallel analytical nature, there is a need for compatible signal amplification procedures to increase sensitivity without loss of multiplexing. Rolling circle amplification (RCA) is a molecular amplification method with the unique property of product localization. This report describes the application of RCA signal amplification for multiplexed, direct detection and quantitation of nucleic acid targets on planar glass and gel-coated microarrays. As few as 150 molecules bound to the surface of microarrays can be detected using RCA. Because of the linear kinetics of RCA, nucleic acid target molecules may be measured with a dynamic range of four orders of magnitude. Consequently, RCA is a promising technology for the direct measurement of nucleic acids on microarrays without the need for a potentially biasing preamplification step.     

A protein detection technique by using surface plasmon resonance (SPR) with rolling circle amplification (RCA) and nanogold-modified tags

Surface plasmon resonance (SPR) can detect molecules bound to a surface by subtle changes in the SPR angle. By immobilizing probes onto the surface and passing analyte solution through the surface, changes in SPR angle indicate the binding between analyte and probes. Detection of analyte from solution can be achieved easily. By using rolling circle amplification (RCA) and nanogold-modified tags, the signals of analyte binding are greatly amplified, and the sensitivity of this technique is significantly improved. Furthermore, this technique has potentials for ultra-sensitive detection and microarray analysis. In this paper, this detection technique is introduced and shown to have great amplification capability. Using 5 nm nanogold with 30 min of RCA development time, this proposed protein detection technique shows over 60 times amplification of the original signal.     

SNP与基因检测及表达中的RCA技术

滚环扩增(rollingcircleamplification,RCA)技术是一种新的分子生物学检测方法。该方法不仅可以在体外等温条件下对核酸进行高度特异性的检测,而且还可通过线性或指数扩增来进行信号级联放大,其灵敏度能达到1个拷贝的核酸分子,因此,可用于痕量分子的检测。目前,滚环扩增技术广泛应用于全基因组DNA检测、核酸测序、单核苷酸多态性、DNA芯片及蛋白质芯片分析等领域。     

Multiplexed protein profiling on microarrays by rolling-circle amplification

Fluorescent-sandwich immunoassays on microarrays hold appeal for proteomics studies, because equipment and antibodies are readily available, and assays are simple, scalable, and reproducible. The achievement of adequate sensitivity and specificity, however, requires a general method of immunoassay amplification. We describe coupling of isothermal rolling-circle amplification (RCA) to universal antibodies for this purpose. A total of 75 cytokines were measured simultaneously on glass arrays with signal amplification by RCA with high specificity, femtomolar sensitivity, 3 log quantitative range, and economy of sample consumption. A 51-feature RCA cytokine glass array was used to measure secretion from human dendritic cells (DCs) induced by lipopolysaccharide (LPS) or tumor necrosis factor-alpha (TNF-alpha). As expected, LPS induced rapid secretion of inflammatory cytokines such as macrophage inflammatory protein (MIP)-1beta, interleukin (IL)-8, and interferon-inducible protein (IP)-10. We found that eotaxin-2 and I-309 were induced by LPS; in addition, macrophage-derived chemokine (MDC), thymus and activation-regulated chemokine (TARC), soluble interleukin 6 receptor (sIL-6R), and soluble tumor necrosis factor receptor I (sTNF-RI) were induced by TNF-alpha treatment. Because microarrays can accommodate approximately 1,000 sandwich immunoassays of this type, a relatively small number of RCA microarrays seem to offer a tractable approach for proteomic surveys.     

RCA combined nanoparticle-based optical detection technique for protein microarray: a novel approach

Developing a readily available biosensor with excellent performances is the main focus of many research groups. Recently, major breakthroughs in miniaturization of molecular analysis have produced DNA and protein microarrays. The aim of our group is to develop a sensitive technique for analyzing signals on protein microarray by applying the surface plasmon resonance (SPR) method. This new detection technique for specific molecular binding utilizes rolling circles amplification (RCA) post-signal processing method [Nat. Genet. 19 (1998) 225-232] and optical visualization by nanogold particle-labeled molecules on a micro-structured chip surface. By covalent bonding of the RCA primer to the detection antibody guarantees that the linkage between the analyte and the amplified RCA product is maintained during the assay. Experimental results show that RCA has significantly enhanced sensitivity compared to conventional methods. This combination of an easily detectable signal with chip technology should have the potential to become a successful commercial application.     

猪圆环病毒2型新疆株全基因组的滚环扩增与序列分析

滚环扩增技术是一种体外恒温DNA扩增方法,特异性好,敏感性强,已广泛应用于微生物学领域。首先采用鉴别PCR对来自新疆某地区的5份猪病料进行猪圆环病毒2型的检测,接着对检出的2份猪圆环病毒2型阳性DNA样品进行滚环扩增。滚环扩增产物经单一限制性内切酶(SacⅡ)酶切及琼脂糖凝胶电泳鉴定,结果显示2份样品都出现了猪圆环病毒2型基因组大小的条带。对目的条带进行回收、克隆与测序,结果表明2株新疆株猪圆环病毒2型的全基因组大小皆为1768bp。遗传进化分析显示2株的基因型为PCV2a和PCV2e。     

利用滚环扩增鉴别同源家族miRNAs

滚环扩增（rolling circle amplification, RCA）是一种基于病毒DNA复制而发明的新技术。近些年，RCA技术已经被广泛应用于微小核糖核酸（micro ribonucleic acid, miRNA）的检测。在miRNA检测研究领域中，鉴别高度同源的家族miRNAs成为该研究领域的瓶颈。本研究引入新型的RCA技术来增加鉴别的灵敏度和特异性，进一步提高家族miRNA鉴别的灵敏度，滚环扩增的程度用相对荧光强度来表示。研究结果显示，T4 RNA连接酶2可在RCA的环化过程中实现最大的环化效率，从而提高RCA的检测特异性。本文利用优化的RCA技术，实现对let 7高度同源的家族miRNAs高灵敏度的鉴别，灵敏度可达5 fmol。let 7a的滚环探针对Let 7a这一miRNA扩增后的相对荧光强度为1 550，而对其他的家族miRNA相对荧光强度仅为260。其他的家族miRNA探针在鉴别时相对荧光强度也显示了较大的差异。而依靠传统的RT-qPCR方法的鉴别灵敏度是4 pmol，与本研究相比，灵敏度低了近1 000倍。本研究的结果表明，利用RCA技术鉴别高度同源性miRNAs是高效灵敏的，此前未见相关研究的报道。RCA技术可能被应用于miRNA高灵敏度检测和鉴别的相关研究中。     

Molecular Zipper: a fluorescent probe for real-time isothermal DNA amplification

Rolling-circle amplification (RCA) and ramification amplification (RAM, also known as hyperbranched RCA) are isothermal nucleic acid amplification technologies that have gained a great application in in situ signal amplification, DNA and protein microarray assays, single nucleotide polymorphism detection, as well as clinical diagnosis. Real-time detection of RCA or RAM products has been a challenge because of most real-time detection systems, including Taqman and Molecular Beacon, are designed for thermal cycling-based DNA amplification technology. In the present study, we describe a novel fluorescent probe construct, termed molecular zipper, which is specially designed for quantifying target DNA by real-time monitoring RAM reactions. Our results showed that the molecular zipper has very low background fluorescence due to the strong interaction between two strands. Once it is incorporated into the RAM products its double strand region is opened by displacement, therefore, its fluorophore releases a fluorescent signal. Applying the molecular zipper in RAM assay, we were able to detect as few as 10 molecules within 90 min reaction. A linear relationship was observed between initial input of targets and threshold time (R² = 0.985). These results indicate that molecular zipper can be applied to real-time monitoring and qualification of RAM reaction, implying an amenable method for automatic RAM-based diagnostic assays.     

L-RCA (ligation-rolling circle amplification): a general method for genotyping of single nucleotide polymorphisms (SNPs)

A flexible, non-gel-based single nucleotide polymorphism (SNP) detection method is described. The method adopts thermostable ligation for allele discrimination and rolling circle amplification (RCA) for signal enhancement. Clear allelic discrimination was achieved after staining of the final reaction mixtures with Cybr-Gold and visualisation by UV illumination. The use of a compatible buffer system for all enzymes allows the reaction to be initiated and detected in the same tube or microplate well, so that the experiment can be scaled up easily for high-throughput detection. Only a small amount of DNA (i.e. 50 ng) is required per assay, and use of carefully designed short padlock probes coupled with generic primers and probes make the SNP detection cost effective. Biallelic assay by hybridisation of the RCA products with fluorescence dye-labelled probes is demonstrated, indicating that ligation-RCA (L-RCA) has potential for multiplexed assays.     

柑桔溃疡病菌滚环扩增检测体系的建立

根据柑桔溃疡病菌(Xanthomonas axonopodis pv.citri,Xac)独有的蛋白基因序列和锁式探针公共连接序列分别设计特异性的锁式探针及其扩增引物,优化系列反应条件,建立了特异性的柑桔溃疡病菌滚环扩增体系.初步检测结果表明该体系能够特异性地检出Xac的菌体细胞及其DNA,而检测不出供试的其它植物病原细菌和柑桔叶面常见的多种附生细菌;对Xac靶片段克隆质粒DNA的检测灵敏度为10 2 copy/μL,对Xac菌悬液的检测灵敏度为20 cfu/μL,比常规PCR的检测灵敏度稍高.用滚环扩增技术和常规PCR技术对田间采集的实际样品进行了检测,两种方法的检测结果没有显著差异(P＞0.01).由于锁式探针的公共连接序列对扩增的条件要求一致,本体系的建立可以为植物病原微生物多靶标检测和病害检疫检验提供新的技术支撑.     

利用滚环扩增鉴别同源家族miRNAs

滚环扩增（rolling circle amplification, RCA）是一种基于病毒DNA复制而发明的新技术。近些年,RCA技术已经被广泛应用于微小核糖核酸（micro ribonucleic acid, miRNA）的检测。在miRNA检测研究领域中,鉴别高度同源的家族miRNAs成为该研究领域的瓶颈。本研究引入新型的RCA技术来增加鉴别的灵敏度和特异性,进一步提高家族miRNA鉴别的灵敏度,滚环扩增的程度用相对荧光强度来表示。研究结果显示,T4 RNA连接酶2可在RCA的环化过程中实现最大的环化效率,从而提高RCA的检测特异性。本文利用优化的RCA技术,实现对let 7高度同源的家族miRNAs高灵敏度的鉴别,灵敏度可达5 fmol。let 7a的滚环探针对Let 7a这一miRNA扩增后的相对荧光强度为1 550,而对其他的家族miRNA相对荧光强度仅为260。其他的家族miRNA探针在鉴别时相对荧光强度也显示了较大的差异。而依靠传统的RT-qPCR方法的鉴别灵敏度是4 pmol,与本研究相比,灵敏度低了近1 000倍。本研究的结果表明,利用RCA技术鉴别高度同源性miRNAs是高效灵敏的,此前未见相关研究的报道。RCA技术可能被应用于miRNA高灵敏度检测和鉴别的相关研究中。     

Sensitive detection of proteins using assembled cascade fluorescent DNA nanotags based on rolling circle amplification

A novel cascade fluorescence signal amplification strategy based on the rolling circle amplification (RCA)-aided assembly of fluorescent DNA nanotags as fluorescent labels and multiplex binding of the biotin-streptavidin system was proposed for detection of protein target at ultralow concentration. In the strategy, fluorescent DNA nanotags are prepared relying on intercalating dye arrays assembled on nanostructured DNA templates by intercalation between base pairs. The RCA product containing tandem-repeat sequences could serve as an excellent template for periodic assembly of fluorescent DNA nanotags, which were presented per protein recognition event to numerous fluorescent DNA nanotags for assay readout. Both the RCA and the multiplex binding system showed remarkable amplification efficiency, very little nonspecific adsorption, and low background signal. Using human IgG as a model protein, the designed strategy was successfully demonstrated for the ultrasensitive detection of protein target. The results revealed that the strategy exhibited a dynamic response to human IgG over a three-decade concentration range from 1.0 pM to 1.0 fM with a limit of detection as low as 0.9 fM. By comparison with the assay of multiple labeling antibodies with the dye/DNA conjugate, the limit of detection was improved by 4 orders. The designed signal amplification strategy would hold great promise as a powerful tool to be applied for the ultrasensitive detection of target protein in immunoassay.     

Ferrocenemonocarboxylic-HRP@Pt nanoparticles labeled RCA for multiple amplification of electro-immunosensing

A multiple amplification immunoassay was proposed to detect alpha-fetoprotein (AFP), which was based on ferrocenemonocarboxylic-HRP conjugated on Pt nanoparticles as labels for rolling circle amplification (RCA). Firstly, the capture antibody (anti-AFP) was immobilized on glass carbon electrode (GCE) deposited nano-sized gold particles. After a typical immuno-sandwich protocol, primary DNA was immobilized by labeling secondary antibody, which acted as a precursor to initiate RCA. The products of RCA provide large amount of sites to link detection DNAs, which were labeled by signal probes (ferrocenemonocarboxylic) and horseradish peroxidase (HRP). Moreover, the enzymatic amplification signals could be produced by the catalysis of HRP and Pt nanoparticles with the addition of H?O?. These lead to multiple amplification signals monitoring by electrochemical instrument and further resulted in high sensitivity of the immunoassay with the detection limit of 1.7 pg/mL.     

Improvements of rolling circle amplification (RCA) efficiency and accuracy using Thermus thermophilus SSB mutant protein

Rolling circle amplification (RCA) of plasmid or genomic DNA using random hexamers and bacteriophage phi29 DNA polymerase has become increasingly popular in the amplification of template DNA in DNA sequencing. We have found that the mutant protein of single-stranded DNA binding protein (SSB) from Thermus thermophilus (Tth) HB8 enhances the efficiency of amplification of DNA templates. In addition, the TthSSB mutant protein increased the specificity of phi29 DNA polymerase. We have overexpressed the native and mutant forms of TthSSB protein in Escherichia coli and purified them to homogeneity. In vitro, these proteins were found to bind specifically to single-stranded DNA. Addition of TthSSB mutant protein to RCA halved the elongation time required for phi29 DNA polymerase to synthesize DNA fragments in RCA. Furthermore, the presence of the TthSSB mutant protein essentially eliminates nonspecific DNA products in RCA reactions.     

Enzymatic preparation of multimilligram amounts of pure single-stranded DNA samples for material and analytical sciences

We present a method for high-yield production of multimilligram amounts of pure single-stranded DNA employing rolling circle amplification (RCA) and processing by restriction enzymes. Pure and homogeneous samples are produced with minimal handling time, reagents, and waste products. The RCA method is more than twice as efficient in dNTP incorporation than conventional polymerase chain reaction in producing end product. The validity and utility of the method are demonstrated in the production of a uniformly ¹³C/¹⁵N-labeled 38-nt cocaine aptamer DNA used in nanosensing devices.     

Amplification of fluorescence with packed beads to enhance the sensitivity of miniaturized detection in microfluidic chip

This paper reports the pre-concentration of C-reactive protein (CRP) antigen with packed beads in a microfluidic chamber to enhance the sensitivity of the miniaturized fluorescence detection system for portable point-of-care testing devices. Although integrated optical systems in microfluidic chips have been demonstrated by many groups to replace bulky optical systems, the problem of low sensitivity is a hurdle for on-site clinical applications. Hence we integrated the pre-concentration module with miniaturized detection in microfluidic chips (MDMC) to improve analytical sensitivity. Cheap silicon-based photodiodes with optical filter were packaged in PDMS microfluidic chips and beads were packed by a frit structure for pre-concentration. The beads were coated with CRP antibodies to capture antigens and the concentrated antigens were eluted by an acid buffer. The pre-concentration amplified the fluorescence intensity by about 20-fold and the fluorescence signal was linearly proportional to the concentration of antigens. Then the CRP antigen was analyzed by competitive immunoassay with an MDMC. The experimental result demonstrated that the analytical sensitivity was enhanced up to 1.4 nM owing to the higher signal-to-noise ratio. The amplification of fluorescence by pre-concentration of bead-based immunoassay is expected to be one of the methods for portable fluorescence detection system.     

Quantitative determination of free-DNA uptake in river bacteria at the single-cell level by in situ rolling-circle amplification

Detection of plasmid DNA uptake in river bacteria at the single-cell level was carried out by rolling-circle amplification (RCA). Uptake of a plasmid containing the green fluorescent protein gene (gfp) by indigenous bacteria from two rivers in Osaka, Japan, was monitored for 506 h using this in situ gene amplification technique with optimized cell permeabilization conditions. Plasmid uptake determined by in situ RCA was compared to direct counts of cells expressing gfp under fluorescence microscopy to examine differences in detection sensitivities between the two methods. Detection of DNA uptake as monitored by in situ RCA was 20 times higher at maximum than that by direct counting of gfp-expressing cells. In situ RCA could detect bacteria taking up the plasmid in several samples in which no gfp-expressing cells were apparent, indicating that in situ gene amplification techniques can be used to determine accurate rates of extracellular DNA uptake by indigenous bacteria in aquatic environments.     

Isothermal strand-displacement amplification applications for high-throughput genomics

Amplification of source DNA is a nearly universal requirement for molecular biology applications. The primary methods currently available to researchers are limited to in vivo amplification in Escherichia coli hosts and the polymerase chain reaction. Rolling-circle DNA replication is a well-known method for synthesis of phage genomes and recently has been applied as rolling circle amplification (RCA) of specific target sequences as well as circular vectors used in cloning. Here, we demonstrate that RCA using random hexamer primers with 29 DNA polymerase can be used for strand-displacement amplification of different vector constructs containing a variety of insert sizes to produce consistently uniform template for end-sequencing reactions. We show this procedure to be especially effective in a high-throughput plasmid production sequencing process. In addition, we demonstrate that whole bacterial genomes can be effectively amplified from cells or small amounts of purified genomic DNA without apparent bias for use in downstream applications, including whole genome shotgun sequencing.     

A sequence-independent strategy for detection and cloning of circular DNA virus genomes by using multiply primed rolling-circle amplification

The discovery of novel viruses has often been accomplished by using hybridization-based methods that necessitate the availability of a previously characterized virus genome probe or knowledge of the viral nucleotide sequence to construct consensus or degenerate PCR primers. In their natural replication cycle, certain viruses employ a rolling-circle mechanism to propagate their circular genomes, and multiply primed rolling-circle amplification (RCA) with phi29 DNA polymerase has recently been applied in the amplification of circular plasmid vectors used in cloning. We employed an isothermal RCA protocol that uses random hexamer primers to amplify the complete genomes of papillomaviruses without the need for prior knowledge of their DNA sequences. We optimized this RCA technique with extracted human papillomavirus type 16 (HPV-16) DNA from W12 cells, using a real-time quantitative PCR assay to determine amplification efficiency, and obtained a 2.4 x 10(4)-fold increase in HPV-16 DNA concentration. We were able to clone the complete HPV-16 genome from this multiply primed RCA product. The optimized protocol was subsequently applied to a bovine fibropapillomatous wart tissue sample. Whereas no papillomavirus DNA could be detected by restriction enzyme digestion of the original sample, multiply primed RCA enabled us to obtain a sufficient amount of papillomavirus DNA for restriction enzyme analysis, cloning, and subsequent sequencing of a novel variant of bovine papillomavirus type 1. The multiply primed RCA method allows the discovery of previously unknown papillomaviruses, and possibly also other circular DNA viruses, without a priori sequence information.