滚环DNA扩增的原理、应用和展望

滚环DNA扩增 (rollingcircleDNAamplification ,RCA)是一种等温信号扩增方法 ,其线性扩增倍数为 1 0 ⁵,指数化扩增能力大于 10⁹,产生的扩增产物连接在固相支持物 (如玻片、微孔板等 )表面的DNA引物或抗体上。RCA是一种适合在芯片上 (on chip)进行信号扩增的新技术 ,它既能提供研究分析的敏感性和特异性 ,又能保持立体分析的多元性。RCA亦是一种痕量的分子检测方法 ,可用于极其微量的生物大分子和生物标志的检测与研究     

滚环扩增技术最新研究动态及展望

滚环扩增技术(rolling circle amplification,RCA)的建立模拟了自然界中环状病原生物DNA通过滚环模型方式自我复制的原理,经长期科学研究和实践应用,取得了诸多突破性成果。对最近几年在滚环扩增技术研究领域的最新动态进行了较全面的总结,其中包括了网状RCA、锁式探针RCA、目标成环RCA和跨越式RCA,也对滚环扩增中存在的问题进行了探讨,重点介绍了该技术在基础研究、实际检测、医疗诊断及纳米材料等方面的应用,最后对核酸等温扩增技术产业化的发展前景进行了展望。     

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。     

基于滚环扩增的鸭乙型肝炎病毒共价闭合环状DNA检测方法的建立

滚环扩增(RCA)是新近发展起来的一种能特异性扩增环形DNA的实验技术,自2008年以来被广泛用于HBV基因全长扩增及共价闭合环状DNA(cccDNA)耐药突变分析等研究。为了便于鸭乙型肝炎病毒(DHBV)cccDNA的分析,本研究建立了基于RCA的DHBV cccDNA的检测方法。通过针对DHBV高度保守序列设计的4对RCA硫化修饰引物,以血清DHBV DNA为阴性对照,从肝组织DHBV DNA标本中扩增得到DHBV cccDNA。然后用跨缺口引物扩增RCA产物测序替代限制性内切酶切分析进行DHBV cccDNA鉴定。应用该方法检测39份携带DHBV麻鸭肝组织与血清标本结果显示:全部肝组织标本均检出DHBV cccDNA,而全部血清标本则均无DHBV cccDNA检出,表明本研究建立的基于RCA的DHBV cccDNA检测法具有良好的特异性和灵敏性。该方法的建立为应用鸭乙型肝炎病毒动物模型研究cccDNA在病毒致病机制中的作用以及评价抗病毒疗效奠定了实验基础。     

滚环扩增技术在电化学生物传感器中的应用

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。     

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。     

MSAP技术在植物抗逆性方面的应用

DNA甲基化在植物的生长发育中起着重要的作用。近年来,随着对DNA甲基化研究的重视,基于PCR方法检测DNA甲基化水平的甲基化敏感扩增多态性技术(MSAP)得到广泛的应用。综述了MSAP技术在植物的生物与非生物胁迫研究方面的应用。     

0#柴油对斑马鱼基因组DNA和环境DNA遗传毒性的RAPD比较

研究利用随机扩增多态性DNA (Random Amplified Polymorphic DNA, RAPD)技术, 以斑马鱼基因组DNA和其养殖水体中的环境DNA (environmental DNA, eDNA)为模板, 检测0#柴油可溶性组分对斑马鱼(Danio rerio)遗传毒性的影响。结果显示, 通过基因组DNA和eDNA扩增的RAPD图谱均可检测到0#柴油对斑马鱼的遗传毒性。在未受到柴油暴露时, 斑马鱼基因组DNA和水环境中eDNA在96h内的RAPD图谱均无明显变化; 在不同浓度的柴油暴露下, 随着暴露时间(0、24h、48h、72h、96h)延长, 基因组DNA和eDNA的多态性位点减少, 模板稳定性降低; 随着柴油浓度(15%、50%、100%)的增加, 基因组DNA和eDNA的多态性位点也减少, 模板稳定性降低。这表明0#柴油对斑马鱼基因组DNA和eDNA的遗传毒性均呈现时间-效应和浓度-效应关系, 并且无论以斑马鱼基因组DNA还是eDNA为模板, 柴油暴露组和未进行暴露的对照组的RAPD扩增图谱条带变化趋势一致。研究结果为通过RAPD技术检测柴油对水生生物的遗传毒性提供了新的研究思路和技术手段。     

鲮鱼DAF和RAPD的比较研究

鲮鱼( Cirrhina molitorella)为南方“四大家鱼” 之一,目前其产量约占两广地区池塘鱼总产量的20%左右,具有产量高、抗病力强、肉质鲜美等特点,也是驰名中外的加工淡水鱼类产品。在利用 RAPD开展鲮鱼的遗传多样性研究中,发现珠江水系不同江段鲮 RAPD 遗传多样性不甚丰富。CaetanoAnollés等学者在 1991 年利用 DNA扩增指纹(DNA Amplification Fingerprinting,DAF)开展了基因组分析研究,他是利用非常短的随机引物(7-8bp,甚至 5bp)对不同模板 DNA 的酶链式扩增,采用高分辨率的聚丙烯酰胺疑胶电泳和银染显色检测 DNA 的多态性,引物比 RAPD 更短,因而扩增产物更多,获得的谱带更丰富,目前已在生物遗传多样性和品种鉴定、性状标记等方面得到应用。       

线粒体COⅠ基因在昆虫DNA条形码中的研究与应用

自2003年DNA条形码(DNA barcodes)概念出现以来,DNA条形码技术(DNA barcoding)受到生物分类学领域普遍关注,线粒体细胞色素氧化酶亚基I(mtDNACOⅠ)被用作动物类群的主要条形码序列,基于该基因片段的昆虫条形码研究在国内外广泛开展。本文在概述DNA条形码、条形码技术及已开展的昆虫条形码研究计划的基础上,总结了昆虫mtDNACOⅠ条形码及其技术在发现和描述隐种、种类分子鉴定以及系统发育等方面的研究进展,分析了细胞核线粒体假基因(Numts)对mtDNACOⅠ条形码扩增的影响,提出检测和避免Numts的方法,并对DNA条形码技术的进一步研究和应用进行了讨论和展望。     

Detection of single base polymorphism in p53 gene by ligase detection reaction and rolling circle amplification on microarrays

We combined three modern technologies of single base polymorphism detection in human genome: ligase detection reaction, rolling circle amplification and IMAGE hydro-gel microarrays. Polymorphism in target DNA was tested by selective ligation on microarray. Product of the ligase reaction was determined in microarray gel pads by rolling circle amplification. Two different methods were compared. In first, selective ligation of short oligonucleotides immobilized on microarray was used with subsequent amplification on preformed circle probe ("common circle"). The circle probe was designed especially for human genome research. In second variant, allele-specific padlock probes that may be circularized by selective ligation were immobilized on microarray. Polymorphism of codon 72 in human p53 gene was used as a biological model. It was shown that LDR/RCA on microarray is a quantitative reaction and gives high discrimination of alleles. Principles and perspectives of selective ligation and rolling circle amplification are being discussed.     

滚环扩增技术及其在生物分子诊断中的应用

滚环扩增技术(RCA)是近年来发展起来的一种新型的核酸扩增技术.该技术是基于连接酶连接、引物延伸、与链置换扩增反应的一种等温核酸扩增方法.在恒温的条件下,可以产生大量的与环型探针互补的重复序列.与传统的核酸扩增方法相比,它具有扩增条件简单,特异性高,能在恒温条件下进行等特点.滚环扩增技术结合荧光、电化学、电化学发光等检...     

Cocaine detection via rolling circle amplification of short DNA strand separated by magnetic beads

A novel and sensitive fluorescence biosensor based on aptamer and rolling circle amplification for the determination of cocaine was developed in the present work. Here cocaine aptamers immobilized onto Au nanoparticles modified magnetic beads hybridized with short DNA strand. In the presence of cocaine, the short DNA strand was displaced from aptamer owing to cocaine specially binding with aptamer. Next, the short DNA strand was separated by magnetic beads and used to originate rolling circle amplification as primer. The end products of rolling circle amplification were detected by fluorescence signal generation upon molecular beacons hybridizing with the end products of rolling circle amplification. With rolling circle amplification and the separation by magnetic beads reducing the background signal, the new strategy was suitable for the detection of as low as 0.48 nM cocaine. Compared with reported cocaine sensors, our method exhibited excellent sensitivity. Our new strategy may provide a platform for numerous proteins and low molecular weight analytes to highly sensitively detect by DNA amplification.     

A DNAzyme based label-free detection system for miniaturized assays

Sensitive detection assays are a prerequisite for the analysis of small amounts of samples derived from biological material. There is a great demand for highly sensitive and robust detection techniques to analyze biomolecules. The combination of catalytic active DNA (DNAzyme) with a peroxidase activity with rolling circle amplification (RCA) is a promising alternative to common detection systems. The rolling circle amplification leads to a product with tandemly linked copies of DNAzymes. The continuous signal generation of the amplified DNAzymes results in an increased sensitivity. The combination of two amplification reactions, namely RCA and DNAzymes, results in increased signal intensity by a factor of 10(6). With this approach the labeling of samples can be avoided. The advantage of the introduced assay is the usage of nucleic acids as biosensors for the detection of biomolecules. Coupling of the analyte molecule to the detection molecules allows the direct detection of the analyte molecule. The described label-free hotpot assay has a broad potential field of applications. The hotpot assay can be adapted to detect and analyze RNA, DNA and proteins down to femtomolar concentrations in a miniaturized platform with a total reaction solution of 50 nl. The applicability of the assay for diagnostics and research will be shown with a focus on high throughput systems using a nano-well platform.     

Highly specific quantification of microRNA by coupling probe–rolling circle amplification and Förster resonance energy transfer

MicroRNA (miRNA) plays vital roles in various biological processes. In general, sensitivity and specificity are the major parameters for the quantification of miRNA. In this study, padlock probe–rolling circle amplification and Förster resonance energy transfer (pRCA–FRET) were coupled for specific and quantitative detection of miRNA. pRCA–FRET showed superior specificity to differentiate single-base mismatch and excellent sensitivity with a detection limit of 103 aM. The current method has the potential to quantify low amounts of miRNA in the same family for studies on their biological functions.     

滚环复制技术的建立及在RNA病毒基因检测中的初步应用

滚环复制是噬菌体繁殖所采取的一种基因复制方式,这种方式可使单链的环形分子在聚合酶和引物的作用下进行体外自我扩增。本文中用可特异性连接环化的寡核苷酸链作为探针,分别进行了1份细胞培养的禽流感病毒H5N1亚型样品、1份细胞培养的SARS病毒样品和4份丙型肝炎病毒阳性血清样品的检测。检测原理是探针与靶序列杂交后便可在T4DNA连接酶的作用下形成滚环复制中的环化单链分子,该分子在同温下可被特异性引物滚动复制和支链扩增。本文还利用按禽流感病毒NA1基因区序列合成的模拟DNA分子对该检测方法的灵敏度进行了测试。结果显示：利用固相RCA技术成功检测到三种RNA病毒的基因,该方法的灵敏度可达到能检测10^3拷贝模式DNA分子的水平。与传统的PCR方法敏感性的比较尚待进一步研究。     

Immuno-rolling circle amplification using a multibinding fusion protein

Ultrasensitive detection of specific, low level proteins in body fluids is particularly challenging. Owing to the extreme sensitivity of the polymerase chain reaction step, the requirements for immuno-rolling circle amplification (immuno-RCA) are much more stringent than for conventional ELISA. Here, we report the development of a rolling circle amplification procedure using multibinding fusion protein to enhance signals of immuno-RCA to detect a cancer biomarker, α-fetoprotein (AFP). We successfully avoid the covalent linkage between antibody and DNA or antibody and biotin/streptavidin by introducing a new genetically engineered fusion protein which contains the C2 domain of protein G and biotin acceptor peptide (BAP) which is intended to maintain the biological activity of the antibody. The purified fusion protein retained its binding affinity with IgG and streptavidin after efficient expression in Escherichia coli. Immuno-RCA in combination with BAP-C2 specifically and sensitively detected AFP in a microplate format. Therefore, the sensitivity and convenient nature of this method should contribute to effective signal enhancement in immunoassays for cancer biomarker detection.     

Attomole DNA detection assay via rolling circle amplification and single molecule detection

A bead-based assay was developed for highly sensitive single molecule DNA detection. Rolling circle amplification (RCA), an isothermal amplification technique that creates tandem repeated sequences, was used in combination with a fluorescent complementary DNA to create dense clusters of fluorescence. These clusters, each corresponding to a single target molecule, can be detected unambiguously due to their high signal/noise ratios. The limit of detection of this assay is approximately 1 amol. This simple single molecule assay allows high detection sensitivity without the use of complex equipment.