第九章 核酸等温扩增技术
等温核酸扩增技术进展
等温核酸扩增技术进展等温核酸扩增技术已广泛应用于生物分析的体外核酸扩增，本文综合国内外报道，对等温核酸扩增技术的进展进行简要综述，包括依赖核酸序列型扩增（NASBA）、链置换扩增（SDA）、滚环扩增（RCA）、环介导等温扩增（LAMP）、单引物等温扩增（SPIA）、交叉引物等温扩增（CPA）、新型等温多自配引发扩增（IMSA）的原理、重要特性、应用及未来的展望。
核酸基于其生物特性被用来作为生物研究和医疗诊断的重要生物标志物。
聚合酶链式反应（PCR）是第一个也是最受欢迎的用于扩增及低丰度检测核酸扩增技术。
尽管PCR技术被广泛地应用到各个领域，但他需要反复的热循环及精密仪器的缺点限制了其在资源有限或实地分析中的应用。
近年来出现及迅猛发展的等温核酸扩增技术有望成为未来发展的新趋势，因为其只需恒温装置（例如：水浴锅），便能进行快速且高效的扩增反应。
从20世纪90年代始，很多等温核酸扩增技术被发展起来，多种等温扩增方法都具有高灵敏度，而且有部分技术已成功转向商业化[1]。
在众多的等温扩增技术中，环介导等温扩增技术应用范围较为广泛，其他的等温扩增技术还有依赖核酸序列型等温扩增、链置换等温扩增、滚环等温扩增、单引物等温扩增、交叉引物等温扩增。
本文综述了这些等温扩增技术及其在分子诊断的应用，并探讨等温扩增技术的发展及前景。
2 等温核酸扩增技术2.1 依赖核酸序列型扩增技术依赖核酸序列型扩增技术（nucleic acid sequence-based amplification，NASBA）[2]，是一种基于转录依赖扩增系统建立起来的等温扩增技术。
NASBA通过模拟逆转录病毒复制方式设计而成，用于扩增单链RNA序列。
NASBA的基本原理是模板RNA被反义引物识别，并在逆转录酶的RNA依赖型DNA聚合酶活性作用下形成互补DNA链，而RNA-DNA复合体被核糖核酸酶H（RNase H）处理，使得原来的RNA链被降解，接着在逆转录酶的DNA依赖型DNA聚合酶活性作用下，含T7启动子的特异引物（oligdNTP）识别新合成的单链DNA链，形成含T7启动子的双链DNA结构，该结构可作为随后循环扩增的底物。
核酸等温扩增技术及其应用
核酸等温扩增技术及其应用一、引言核酸等温扩增技术是一种新兴的分子生物学技术，其在生物医学研究、临床诊断和基因工程等领域具有重要应用价值。
本文将详细介绍核酸等温扩增技术的原理、方法和应用。
二、核酸等温扩增技术的原理核酸等温扩增技术是一种在恒温条件下进行的核酸扩增方法，通过利用逆转录酶和DNA聚合酶的活性，实现核酸的扩增。
其基本原理是通过逆转录酶将RNA模板转录成互补的DNA链，然后利用DNA聚合酶在恒温条件下合成新的DNA链。
这种等温扩增方法不需要复杂的温度变化，且具有较高的特异性和敏感性。
三、核酸等温扩增技术的方法核酸等温扩增技术主要包括RT-LAMP（逆转录环介导等温扩增法）和RPA（等温扩增法）。
RT-LAMP方法利用4-6个特异性的引物，在同一温度下通过逆转录酶和DNA聚合酶的协同作用，在短时间内扩增目标核酸。
RPA方法则利用DNA聚合酶和DNA单链结合蛋白在等温条件下，通过引物结合、DNA解旋、DNA聚合等步骤，实现核酸的扩增。
四、核酸等温扩增技术的应用1. 医学诊断：核酸等温扩增技术在医学诊断中有广泛应用。
例如，可以通过核酸等温扩增技术检测病毒、细菌和真菌等病原体，快速确认感染病原体的种类和数量，为临床治疗提供依据。
此外，核酸等温扩增技术还可以用于检测肿瘤标志物、遗传病突变等，为早期癌症和遗传病的筛查提供技术支持。
2. 食品安全检测：核酸等温扩增技术可以应用于食品安全检测领域。
例如，可以利用核酸等温扩增技术检测食品中的病原菌、转基因成分和食品中的传染性病毒等。
这种技术具有快速、灵敏和高效的特点，可以为食品安全监管提供重要依据。
3. 环境监测：核酸等温扩增技术在环境监测中也有广泛应用。
例如，可以利用核酸等温扩增技术检测水体、土壤和空气中的微生物，了解环境中的微生物多样性和污染程度。
此外，核酸等温扩增技术还可以用于环境污染源的追踪和监测。
4. 生物工程：核酸等温扩增技术在生物工程领域也有重要应用。
核酸扩增技术教学课件ppt
RT-PCR技术
总结词
转录、反转录、灵敏度高。
详细描述
RT-PCR技术是一种将RNA的逆转录反应和PCR反应联合应用的方法，具有高 灵敏度、高特异性、简单易行等特点，常用于检测细胞中基因表达水平。
qPCR技术
总结词
定量、高灵敏度、实时监测。
详细描述
qPCR技术即实时荧光定量PCR技术，是一种对特定DNA片段进行实时定量检测 的方法，具有高灵敏度、高特异性、可实时监测等特点，已广泛应用于基因表达 分析、病原体检测等领域。
详细介绍qPCR反应所需的各个组分及其作用，如 模板DNA、引物、dNTPs、DNA聚合酶、荧光染 料或探针等。
qPCR数据分析
介绍qPCR实验数据应该如何进行分析，包括扩增 曲线和熔解曲线的绘制和分析，以及Ct值的计算 和应用。
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核酸扩增技术的数据分析与解读
数据分析方法与技巧
描述性统计分析
对实验数据进行描述性统计，如均 值、标准差等，以了解数据的集中 趋势和离散程度。
通过检测特定基因的扩增或缺失，对疾病进 行诊断和预测。
生物制药
进化研究
利用核酸扩增技术生产重组蛋白、抗体等生 物药物，用于治疗和预防疾病。
研究物种间的基因序列差异，揭示物种进化 的历史和机制。
误差分析与质量控制
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误差来源
分析核酸扩增技术的误差来源，如试剂质量、操作流程、仪器设备等
。
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质量控制
制定质量控制标准，如重复实验、阳性对照等，以确保实验结果的准
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《核酸扩增技术教学课件 ppt》
目 录
Байду номын сангаас
• 核酸扩增技术概述 • 核酸扩增技术的种类与特点 • 核酸扩增技术实验方案与步骤 • 核酸扩增技术的数据分析与解读 • 核酸扩增技术的优化与改进建议 • 核酸扩增技术的未来发展趋势与展望
等温扩增技术的原理及应用
等温扩增技术的原理及应用等温扩增技术是一种新型的DNA扩增方法，它可以在等温条件下进行，无需对温度进行周期性变化，因此非常稳定，同时易于操作，成本也比传统的PCR方法低。
它的原理是利用一种特殊的DNA聚合酶，即Bst DNA聚合酶，在等温条件下，可将一条DNA模板扩增成数百万个拷贝。
其主要应用在医学诊断、生物工程、生物物质检测等领域。
1. 原理等温扩增技术的原理是利用Bst DNA聚合酶的内切割酶活性，在等温条件下，通过循环增强的DNA合成反应，将DNA模板快速扩增成数百万个拷贝。
具体而言，Bst DNA聚合酶可以通过在DNA链中寻找配对错误的碱基，并对其进行4’-5’链切断，然后在3’-5’方向上进行DNA聚合。
在等温条件下，反应温度一般为55-65℃，DNA聚合酶可以持续高效地工作，不需要多次升温降温，避免了反应条件的不稳定性，而且还可以进行高密度扩增，同时可以直接从样品中扩增出足够数量的DNA片段，避免了DNA的复制和纯化过程。
因此等温扩增技术的速度比传统PCR方法快，同时更加稳定，特别适合于用于快速DNA检测。
2. 应用(1) 医学诊断等温扩增技术可用于许多医学诊断领域，例如病毒和感染性疾病的快速检测和诊断。
病毒感染检测可用于检测脑膜炎病毒、细菌性脑膜炎、甲型H1N1流感等病毒感染。
这样，通过等温扩增技术，可以快速检测是否感染病毒或细菌，辅助诊断和治疗。
(2) 生物工程等温扩增技术可用于检测和筛选新的基因，进行DNA合成和基因编辑，生产转基因产品。
例如，科学家可以使用等温扩增技术扩增和检测工业微生物中的特定基因，在菌株发酵过程中对基因进行编辑和修饰，改良微生物发酵过程，提高产物质量和含量。
(3) 生物物质检测等温扩增技术还可用于生物物质检测领域。
例如，在食品安全检测中，可以使用等温扩增技术检测食品样本中的细菌、真菌、病毒等，判断其是否安全。
同样地，在水质检测领域，等温扩增技术可以帮助快速检测水中的大肠杆菌、肠炎沙门氏菌等细菌。
核酸提取及扩增技术简介(含等温扩增技术)-综述
核酸提取及扩增技术原理简介1核酸理化性质RNA和核苷酸的纯品都呈白色粉末或结晶，DNA则为白色类似石棉样的纤维状物。
除肌苷酸、鸟苷酸具有鲜味外，核酸和核苷酸都呈酸味。
DNA、RNA和核苷酸都是极性化合物，一般都溶于水，不溶于乙醇、氯仿等有机溶剂，它们的钠盐比游离酸易溶于水，RNA钠盐在水中溶解度可达40g/L。
DNA可达10g/L，呈黏性胶体溶液，在酸性溶液中，DNA、RNA易水解，在中性或弱碱性溶液中较稳定。
2细胞破碎大多数核酸分离与纯化的方法一般都包括了细胞裂解、核酸与其他生物大分子物质分离、核酸纯化等几个主要步骤。
每一步骤又可由多种不同的方法单独或联合实现。
根据原理不同，细胞破碎主要包含机械破碎法，化学试剂法，酶溶解法。
1）机械方法：包括低渗裂解、超声裂解、微波裂解、冻融裂解和颗粒破碎等物理裂解方法。
这些方法用机械力使细胞破碎，但机械力也可引起核酸链的断裂，因而不适用于高分子量长链核酸的分离。
有报道超声裂解法提取的核酸片段长度从< 500bp~>20kb之间，而颗粒匀浆法提取的核酸一般<10kb。
2）化学试剂法：经一定的pH 环境和变性条件下，细胞破裂，蛋白质变性沉淀，核酸被释放到水相。
上述变性条件可通过加热、加入表面活性剂（SDS、Triton X-100、Tween 20、NP-40、CTAB、sar-cosyl、Chelex-100等）或强离子剂（异硫氰酸胍、盐酸胍、肌酸胍）而获得。
而pH环境则由加入的强碱（NaOH）或缓冲液（TE、STE 等）提供。
在一定的pH环境下，表面活性剂或强离子剂可使细胞裂解、蛋白质和多糖沉淀，缓冲液中的一些金属离子螯合剂（EDTA 等）螯合对核酸酶活性所必须的金属离子Mg2+ 、Ca2+ ，从而抑制核酸酶的活性，保护核酸不被降解。
3）酶解法：主要是通过加入溶菌酶或蛋白酶（蛋白酶K、植物蛋白酶或链酶蛋白酶）以使细胞破裂，核酸释放。
蛋白酶还能降解与核酸结合的蛋白质，促进核酸的分离。
核酸等温扩增ppt课件
• 产物相当复杂，无法进行后续的回收、鉴定、克 隆等基因工程操作。
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NASBA的优缺点
NASBA 是种 RNA 扩增法，因此其主要应用 是对 RNA 病毒的检测。
整个反应能在 42℃条件下进行，经过两个 小时的扩增可将模板 RNA放大至 109~1010 倍。
近年来，各国学者陆续利用RPA技术，对于DNA病毒、细菌等 进行核酸检测。
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RPA的原理
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四氢呋喃非碱基位点类似物
tetrahydrofuran abasic–site mimic (THF)
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结果观察：
Basic RPA
lateral-flow strip
exo RPA
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引物、探针设计
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LAMP的操作过程
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检测方法
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LAMP技术在病原体检测中的应 用
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LAMP技术小结
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LAMP技术的优点
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存在缺陷
• 所识别的靶位序列长度不得过大，一般在300 bp 以内。因此，无法进行长片段 DNA 的扩增；
• LAMP 技术具备高灵敏度，对操作要求严格分区， 否则极易受到污染而产生假阳性结果；
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SAT技术的原理
SAT技术（Simultaneous Amplification and Testing）是基于TMA （Transcription mediated amplification）恒温扩增技术发展起来的一项最新 核酸检测技术，是国内企业自主研发的专利技术
等温扩增原理
等温扩增原理
等温扩增（LAMP）是一种新型的核酸扩增技术，它具有高度特异性和高效性，能够在恒温条件下迅速扩增目标DNA序列。
等温扩增原理基于DNA聚合酶、反转录酶和DNA结合蛋白等多种酶的协同作用，通过一系列复杂的核酸反应过程，实现对目标DNA的高效扩增。
首先，等温扩增的原理涉及到DNA聚合酶的作用。
DNA聚合酶在等温条件下能够在目标DNA的两个特定区域上合成DNA链，形成环状结构。
这种环状结构具有特殊的功能，能够通过反复的DNA合成过程，迅速扩增目标DNA序列。
其次，反转录酶在等温扩增中也扮演着重要的角色。
反转录酶能够将RNA模板转录成互补的DNA链，进而参与到DNA合成的过程中。
这种RNA-DNA混合链的形成，为目标DNA的扩增提供了必要的前提条件。
此外，DNA结合蛋白在等温扩增中也发挥着重要的作用。
DNA结合蛋白能够在DNA的特定区域上结合并稳定DNA的结构，促进DNA 聚合酶和反转录酶的活性，从而保证等温扩增反应的顺利进行。
综上所述，等温扩增原理是基于DNA聚合酶、反转录酶和DNA
结合蛋白等多种酶的协同作用，通过一系列复杂的核酸反应过程，
实现对目标DNA的高效扩增。
这种技术具有操作简单、扩增速度快、特异性高等优点，被广泛应用于医学诊断、食品安全检测、环境监
测等领域。
相信随着技术的不断进步，等温扩增技术将在更多领域
展现出其巨大的应用潜力。
核酸恒温扩增技术(HDA)
EMBO Rep. 2004 August; 5(8): 795–800.PMCID: PMC1249482 Published online 2004 July 9. doi: 10.1038/sj.embor.7400200.Copyright© 2004, European Molecular Biology OrganizationScientific ReportHelicase-dependent isothermal DNA amplificationMyriam Vincent,1* Yan Xu,1* and Huimin Kong1a1New England Biolabs, 32 Tozer Road, Beverly, Massachusetts 01915, USAa Tel: +1 978 927 5054; Fax: +1 978 921 1350; E-mail: kong@*These authors contributed equally to this workReceived January 14, 2004; Revised May 24, 2004; Accepted June 14, 2004.This article has been cited by other articles in PMC.∙Other Sections▼o AbstractIntroductionResultsDiscussionMethodsSupplementary MaterialReferences AbstractPolymerase chain reaction is the most widely used method for in vitro DNA amplification. However, it requires thermocycling to separate two DNA strands. In vivo, DNA is replicated by DNA polymerases with various accessory proteins, including a DNA helicase that acts to separate duplex DNA. We have devised a new in vitro isothermal DNA amplification method by mimicking this in vivo mechanism. Helicase-dependent amplification (HDA) utilizes a DNA helicase to generate single-stranded templates for primer hybridization and subsequent primer extension by a DNA polymerase.HDA does not require thermocycling. In addition, it offers several advantages over other isothermal DNA amplification methods by having a simple reaction scheme and being a true isothermal reaction that can be performed at one temperature for the entire process. These properties offer a great potential for the development of simple portable DNA diagnostic devices to be used in the field and at the point-of-care.Keywords: DNA amplification, isothermal, helicase, DNA polymerase, UvrD∙Other Sections▼o AbstractIntroductionResultsDiscussionMethodsSupplementary MaterialReferences IntroductionThe polymerase chain reaction (PCR) revolutionized our capabilities to do biological research, and it has been widely used in biomedical research and disease diagnostics (Saiki et al, 1988). Hand-held diagnostic devices, which can be used to detect pathogens in the field and at point-of-care, are demanded currently. However, the need for power-hungry thermocycling limits PCR application in such a situation. Several isothermal target amplification methods have been developed (Andras et al, 2001).Strand-displacement amplification (SDA) combines the ability of a restriction endonuclease to nick the unmodified strand of its target DNA and the action of an exonuclease-deficient DNA polymerase to extend the 3′ end at the nick and displace the downst ream DNA strand (Walker et al, 1992). Transcription-mediated amplification (TMA) uses an RNA polymerase to make RNA from a promoter engineered in the primer region, a reverse transcriptase to produce complementary DNA from the RNA templates and RNase H to remove the RNA from cDNA (Guatelli et al, 1990). In the rolling circle amplification (RCA), a DNA polymerase extends a primer on a circular template, generating tandemly linked copies of the complementary sequence of the template (Fire & Xu, 1995). However, these isothermal nucleic acid amplification methods also have their limitations. Most of them have complicated reaction schemes. In addition, they are incapable of amplifying DNA targets of sufficient length to be useful for many research and diagnostic applications.In living organisms, a DNA helicase is used to separate two complementary DNA strands during DNA replication (Kornberg & Baker, 1992). We have devised a new isothermal DNA amplification technology,helicase-dependent amplification (HDA), by mimicking nature. HDA uses a DNA helicase to separate doublestranded DNA (dsDNA) and generatesingle-stranded templates for primer hybridization and subsequent extension. As the DNA helicase unwinds dsDNA enzymatically, the initialheat denaturation and subsequent thermocycling steps required by PCR can all be omitted. Thus, HDA provides a simple DNA amplification scheme: one temperature from the beginning to the end of the reaction. In this study, we present the Escherichia coli UvrD-based HDA system, which can achieve over a million-fold amplification.Other Sections▼o AbstractIntroductionResultsDiscussionMethodsSupplementary MaterialReferences ResultsHDA designThe fundamental reaction scheme of HDA is shown in Fig 1.In this system, strands of duplex DNA are separated by a DNA helicase and coated by singlestranded DNA (ssDNA)-binding proteins (SSBs; Fig1, step 1). Two sequencespecific primers hybridize to eachborder of the target DNA (Fig 1, step 2). DNA polymerases extend the primers annealed to the templates to produce a dsDNA (Fig1, step 3). The two newly synthesized dsDNA products are then used as substrates by DNA helicases, entering the next round of thereaction (Fig 1, step 4). Thus, a simultaneous chain reaction proceeds resulting in exponential amplification of the selected target sequence.Figure 1Schematic diagram of HDA. Two complementary DNA strands are shown as twolines: the thick one is the top strand and the thin one is the bottom strand. 1: Ahelicase (black triangle) separates the two complementary DNA strands, which arebound by SSB (grey (more ...)E. coli UvrD helicase was chosen as the DNA helicase for our first HDA system because it can unwind blunt-ended DNA fragments (Runyon & Lohman, 1989). The SSB in the HDA reaction is either bacteriophage T4 gene 32 protein (Casas-Finet & Karpel, 1993) or RB 49 gene 32 protein (Desplats et al, 2002).Amplification of a target sequence from plasmid DNATwo M13/pUC19 universal primers (1224 and 1233) were used in an HDA reaction to amplify selectively a 110 base pair (bp) target sequence from a derivative of pUC19 plasmid. In a first step, substrate DNA was mixed with the primers for heat denaturation and subsequent annealing. The component B mixture containing key enzymes, such as E. coli UvrD helicase plus its accessory protein MutL, phage T4 gene 32 protein and the exo−Klenow fragment of DNA polymerase I, was then added into component A. After a 1 hr incubation period at 37°C, a 110-bp amplification product wasobserved on a 2% agarose gel (Fig 2, lane 1). Sequencing results confirmed that it matched the target DNA sequence.Figure 2 Electrophoresis of HDA products amplified from plasmid DNA. A two-step HDAreaction, with a 1 h incubation at 37°C, was performed in the presence of allcomponents (lane 1) including a pUC19-derived plasmid DNA (0.035 pmol),primer-1224 (10 pmol) (more ...)To determine the essential elements in the HDA reaction, each key component was omitted from the reaction. In the absence of UvrD helicase, no amplification was observed (Fig 2, lane 2), confirming that helicase is required for the amplification. In the absence of accessory protein MutL, no amplification product was observed (Fig 2, lane 3), suggesting that UvrD helicase mediated-amplification requires MutL. In vivo , MutL, the mastercoordinator of mismatch repair, recruits UvrD helicase to unwind the DNA strand containing the replication error (Lahue et al , 1989). MutLstimulates UvrD helicase activity more than tenfold by loading it onto the DNA substrate (Mechanic et al, 2000). In the absence of T4 gene 32 protein, again no amplification product was observed (Fig2A, lane 4), indicating that SSB is required in this reaction, probably to prevent reassociation of the complementary ssDNA templates at 37°C. In the abs ence of ATP, no amplification product was detected, indicating that the helicase cofactor is essential for HDA. Target sequences up to 400 bp can be efficiently amplified from plasmid DNA, beyond which the yield drops markedly (data not shown). Amplification of target sequences from genomic DNATo test whether HDA can be used to amplify a specific sequence from more complex DNA samples, such as bacterial genomic DNA, the E. coli UvrD-based HDA system was used to amplify a 123-bp fragment from an oral pathogen, Treponema denticola. A restriction endonuclease gene encoding a homologue of earIR(GenBank accession number: TDE0228) was chosen as the target gene. The amplification power of the current HDA system was also determined by decreasing the amount of T. denticola genomic DNA. The amount of template was varied from 107 to 103 copies of the T. denticola genome. In general, the intensities of the HDA product decreased as the initial copy numberwas lowered (Fig 3A). With 103 copies of initial target, about 10 ng of products were generated, which corresponds to 1010molecules of the 123-bp fragment. Thus, the current HDA system described here is capable of achieving over ten million-fold amplification. The negative control, containing no T. denticola genomic DNA, showed no trace of amplified products, proving the specificity and reliability of HDA.Figure 3Electrophoresis of HDA products amplified from bacterial genomic DNA. (A)Amplification of a 123-bp target sequence from T. denticola genomic DNA. Atwo-step HDA reaction, with a 3 h incubation at 37°C, was performed in thepresence of primer (more ...)In addition to T. denticola, the E. coli UvrD-based HDA system can amplify target sequences from various genomic DNAs isolated from Helicobacter pylori, E. coli, Neisseria gonorrhoeae, Brugia malayi and human cells (data not shown).One temperature HDAAs helicases are able to unwind duplex DNA enzymatically, we tested whether the entire HDA reaction could be carried out at one temperature without prior heat denaturation. Another region (102 bp) of the earIR homologue gene was chosen as target. Component B was added to A either immediately or after a denaturation step. The yield of the one-step HDA amplification was about 40–60% of the two-step HDA reaction. Nevertheless, enough product is generated to be detected (Fig3B). This demonstrates that HDA is able to amplify a target sequence from bacterial genomic DNA at one temperature for the entire process.Amplification of a target sequence from T. denticola cellsTo test whether HDA canbe used on crude samples, the reaction was carried out directly on bacterial cells. A 111-bp sequence within T. denticola glycogen phosphorylase gene (GenBank accession number: TDE2411) was chosen as target. A specific product was obtained when using 107 to 104cells as template (Fig 3C). As the initial cell number was lowered, the intensity of the HDAspecific product decreased and other products of lower molecular weight were observed. These products arenon-target specific as they could also be detected for the negative control. They result from a nonspecific amplification and are most probably derivates of primer-dimers. Primer-dimers can be generated by the HDA reaction when the template amount is very low; they also occur in the PCR reaction (Brownie et al, 1997). Nevertheless, the negative control allows us to distinguish the targetspecific from thenon-target-specific products. The current HDA system can work on crude samples, such as whole bacterial cells with only a tenfold loss of sensitivity compared with the purified genomic DNA (Fig3B).Detection of B. malayi DNA in bloodTo test the possibility of using HDA on real samples, a pathogen's DNA sequence was amplified in the presence of human blood. A 99-bp fragment of the Hha I repeat of the filarial parasite B. malayi was chosen as target. First reported to comprise 10–12% (McReynolds et al, 1986), and then 1% of the Brugia genome (Ghedin et al, 2004), this highly repeated sequence became a target of choice for the detection of B. malayi(Rao et al, 2002). Decreasing amounts of B. malayi genomic DNA were added to human blood samples. After extraction and dialysis, the samples were used as templates for HDA reactions. A specific product was detected for samples containing as low as 5 pg of B. malayi DNA, which corresponds to 500 copies of thegenome (Fig 4). These results demonstrate the feasibility of usingHDA to detect a pathogen in a real sample.Figure 4Electrophoresis of 99-bp HDA products amplified from B. malayi genomic DNA in human blood samples. A 0.1–1,000 ng portion of B. malayi genomic DNA was added to 200 μl of human blood samples. After processing, 1 μl of each sample (more ...)Real-time HDAWe have developed a real-time detection system using a LUX™ primer specific to the earIR homologue gene in T. denticola. Two identical HDA reactions (curves 1 and 2) along with a negative control (curve 3) wereperformed (Fig 5A). After 35 min, product accumulation generated a typical sigmoid curve. A semilogarithmic plot of the increase in fluorescence in the early phase of the reaction revealed an initial first-order reaction with a rate of amplification (V) of 0.23 RFU/min,which corresponds to a doubling time of 3 min (Fig 5B). Following the log-linear phase, the reaction slowed, entering a transition phase (between 45 and 80 min), eventually reaching the plateauphase (Fig 5A). Curves 1 and 2 derived from two identical reactions were very similar, suggesting that the real-time HDA reaction has a good reproducibility. In the negative control, the fluorescentsignal remained below the T t(time of threshold) line (Fig 5A,curve 3) and no amplified DNA was observed on the agarose gel (Fig5C, lane 3).Figure 5Real-time HDA. A 97-bp fragment from T. denticola genomic DNA was amplifiedusing a LUX primer. (A ) Amplification products were detected in real time bymeasuring fluorescent signals (relative fluorescence unit (RFU)). Curves 1 and 2:two identical reactions (more ...)Other Sections▼ o AbstractIntroductionResultsDiscussionMethodsSupplementary MaterialReferences DiscussionIn this study, we report a new isothermal DNA amplification technique, named HDA. It has a significant advantage over PCR in that it eliminates the need for an expensive and power-hungry thermocycler. HDA also offers several advantages over existing isothermal DNA amplification methods. First, it has a simple reaction scheme, in which a target sequence can beamplified by two flanking primers, similar to PCR (Fig 1).In contrast, other isothermal DNA amplification techniques have complicated reaction mechanisms and experimental designs. For example, SDA uses four primers to generate initial amplicons and modified deoxynucleotides to provide strandspecific nicking (Walker et al, 1992). TMA needs three different enzymatic steps (transcription/cDNA synthesis/RNA degradation) to accomplish an isothermal RNA amplification (Guatelli et al, 1990). This complexity and the inefficiency in amplifying long targets limit their use in biomedical research. As a result, these isothermal amplification techniques are primarily used in specifically designed diagnostic assays, and PCR remains the only protocol used by researchers to amplify specific targets of DNA.Second, HDA is a true isothermal DNA amplification method. As DNA helicase can melt double-stranded target DNA at the beginning of the reaction, the entire HDA reaction can be performed at one temperature (Fig3B). In contrast, other isothermal methods, such as SDA, still need an initial heat denaturation step at a high temperature followed by amplification at a lower temperature (Walker et al, 1992). Third, HDA is at its early development stage. The current UvrD system can achieve over a million-fold amplification. A pathogen genomic DNA can even be detected in a human blood sample. This demonstrates that HDA can be performed on crude samples and has the potential to be used as a diagnostic tool. E. coli UvrD helicase, a repair helicase, was chosen as our model system because it is a well-studied helicase and it unwinds blunt-end substrates. However, its speed (20 bp/s) and processivity (less than 100 bp per binding) are limited (Ali et al, 1999). MutL can stimulate UvrD unwinding activity but fails to increase its processivity (Mechanic et al, 2000). This may explain as to why the current UvrD HDA system is inefficient at amplifying long target sequences. The performance of anHDA system may be further improved by testing different helicases. DNA helicases are found in all organisms and participate in major cellular DNA metabolisms including replication, repair and recombination (Kornberg & Baker, 1992; Caruthers & McKay, 2002). In a recent experiment, we were able to amplify a 2.5-kb target from a plasmid DNA by using a processive replicative helicase, T7 gene 4 protein (Y. Xu and H. Kong, unpublished data), which unwinds DNA at a rate of 300 bp/s and with high processivity (Kornberg & Baker, 1992).Optimization of current HDA systems involves identifying rate-limiting steps. In the HDA reaction, the unwinding, primer-annealing and extension steps must be coordinated. One of the rate-limiting steps could be the coordination between the helicase and the DNA polymerase. The exo−Klenow fragment can be substituted by other polymerases such as T7 sequenase (USB) or Klenow fragment, but none of these polymerases improved the reaction (data not shown). A DNA polymerase, which can move with the DNA helicase in a coordinated way, would be an ideal combination. This kind of coordination can be found at the in vivo replication fork where DNA polymerase III interacts with the DnaB helicase (Kornberg & Baker, 1992). One way to achieve this kind of coordination is to use ahelicase/polymerase pair that works together naturally. Anotherrate-limiting step could be the interaction between SSB and DNA. The essential role of SSB in the HDA reaction is probably to prevent the reassociation of the separated DNA strands. Indeed, no DNA amplification was observed in the absence of SSB. Both T4 gene 32 protein (Lohman, 1984) and RB49 gene 32 protein (Desplats et al, 2002) can efficiently support the HDA reaction. They can be substituted by E. coli SSB (Bujalowski & Lohman, 1989) or T7 gene 2.5 SSB (Nakai & Richardson, 1988), but the yield of amplification is lower (data not shown).Future experiments will be directed towards improving the efficiency of HDA by testing different helicases/polymerases and by optimizing the existing HDA systems by varying the ratio and concentration of each key component. Indeed, the concentration of each protein in an HDA reaction has significant effects on the outcome of the reaction. Deviation from the optimal concentration results in a decrease in the yield and, eventually, failure of the amplification process. The simplicity and true isothermal nature of the HDA platform offer great potential for the development of hand-held DNA diagnostic devices that could be used to detect pathogens at point-of-care or in the field.Other Sections▼o AbstractIntroductionResultsDiscussionMethodsSupplementary MaterialReferences MethodsMaterialT4 gene 32 protein was purchased from Roche Applied Science. Adenosine 5′-triphosphate (ATP) was purchased from Amersham Biosciences.Primer-175-LUX was purchased from Invitrogen. All other enzymes and reagents including exo− Klenow fragment, pTYB1, pTYB3, pTXB1, dNTPs and oligodeoxynucleotides were from New England Biolabs.Cloning and purification of UvrD helicase and MutL from E. coliuvrD (Swissprot accession number: P03018) and mutL (Swissprot accession number: P23367) genes were amplified from E. coli K12 genomic DNA using PCR and cloned into the Nco I and Sap I sites of pTYB3 and Nde I and Sap I sites of pTYB1, respectively, to construct C-terminal fusions with a self-cleavable affinity tag (Impact™ system, NEB) (Chong et al, 1998).See supplementary information online for details on purification.Cloning and purification of gene protein 32 from bacteriophage RB 49 Gene 32 (GenBank accession number: NP_891812) was amplified from RB49 genomic DNA using PCR and cloned into the Nde I and Sap I sites of pTXB1 to construct C-terminal fusions with a self-cleavable affinity tag(Impact™ system, NEB) (Chong et al, 1998). See supplementary information online for details on purification.HDA reactions for amplifying target sequenceTwo HDA buffers were prepared. The 10 × HDA buffer A contains 350 mM Tris-acetate (pH 7.5) and 100 mM dithiothreitol and the 10 × HDA buffer B contains 10 mM Tris-acetate (pH 7.5), 1 mg/ml bovine serum albumin and 100 mM magnesium acetate. HDA reaction component A (30 μl) was prepared by combining 5 μl of 10 × HDA buffer A, template (plasmid DNA, genomic DNA, cells, processed human blood sample (see supplementary information online for information on the preparation of the reconstituted human blood sample)), 10–20 pmol of each target-specific primer (see supplementaryO. information online for details on the HDA primers), 20 nmol dNTPs and dH2 The reaction component A was heated for 2–10 min at 95°C to denature the template and 1–4 min at 37°C. Reaction component B (20 μl) was freshly prepared by mixing 5 μl of 10 × HDA buffer B, 150 nmol ATP, 5 U exo− Klenow fragment, 100 ng UvrD helicase, 400–800 ng MutL protein, 4.5 μg T4 gp32 or 5.8 μg RB49 gp32, and dHO. Component B was then added2to component A. The reaction was continued for 1–3 h at 37°C and was then terminated by addition of 12.5 μl of stop buffer (0.1% sodium dodecyl EDTA, 15% Ficoll and 0.2% orange G). Reaction productssuphate, 50 mM Na2were analysed on a 2% GPG LMP agarose gel containing ethidium bromide. The HDA reaction without heat denaturation was set up by combining all the elements mentioned above in the same tube and incubating directly for 2 h at 37°C. To monitor HDA in real time, fluorescent primers were used (primer-175-LUX). The amplification products were detected by measuring fluorescent signals at 490 nM at 5 min intervals using an iCyler (Bio-Rad). Supplementary information is available at EMBO reports online().Supplementary MaterialSupplementary informationWe thank L. Higgins, D. Robinson and M. Dalton for assistance in protein purification and R. Kucera for the plasmid substrate. We are grateful to Dr Krisch for providing the RB49 genomic DNA and J. Foster for the B. malayi microfilariae. We thank E. Raleigh, L. McReynolds and G. Tzertzinis for helpful discussions and are grateful to R. Roberts and W. Jack for critical reading of the manuscript. We thank D. Comb for his support.。
等温扩增技术实验报告(3篇)
第1篇一、实验目的1. 掌握等温扩增技术的原理和操作步骤。
2. 学习利用等温扩增技术对特定靶标进行扩增和检测。
3. 了解等温扩增技术在分子生物学研究中的应用。
二、实验原理等温扩增技术（Isothermal Amplification Technology）是一种在恒定温度下进行核酸扩增的技术。
与传统的PCR技术相比，等温扩增技术具有操作简便、快速、成本低、特异性高等优点。
其原理是利用特定设计的引物和聚合酶在恒定温度下进行核酸扩增，无需热循环，从而实现快速、高效、特异的核酸扩增。
三、实验材料1. 样本：含有靶标基因的DNA样本。
2. 引物：针对靶标基因设计的引物。
3. 聚合酶：等温扩增用聚合酶。
4. 反应体系：缓冲液、dNTPs、引物、聚合酶等。
5. 实验器材：PCR仪、离心机、电泳仪、凝胶成像系统等。
四、实验步骤1. 引物设计：根据靶标基因的序列设计引物，确保引物特异性高、Tm值相近。
2. 反应体系配置：按照实验要求，配置反应体系，包括缓冲液、dNTPs、引物、聚合酶等。
3. 反应：将配置好的反应体系加入PCR管中，放入PCR仪进行等温扩增。
反应温度根据所使用的聚合酶和引物进行优化。
4. 扩增产物检测：将扩增产物进行电泳分析，观察扩增结果。
5. 结果分析：根据电泳结果，判断靶标基因是否存在，并进行定量分析。
五、实验结果与分析1. 扩增结果：根据电泳结果，观察到扩增产物条带，说明等温扩增成功。
2. 特异性分析：通过设置阴性对照和阳性对照，验证扩增结果的特异性。
3. 定量分析：通过比较扩增产物条带的亮度，对靶标基因进行定量分析。
六、实验讨论1. 引物设计：引物设计是等温扩增技术成功的关键。
引物设计时应考虑引物长度、Tm值、GC含量等因素，以确保扩增结果的特异性。
2. 反应体系优化：反应体系中的各种成分比例对扩增结果有较大影响。
在实验过程中，应优化反应体系，以提高扩增效率和特异性。
3. 扩增温度优化：不同聚合酶对温度的敏感度不同，因此在实验过程中，需要根据所使用的聚合酶和引物优化扩增温度。
详解核酸等温扩增技术
详解核酸等温扩增技术近年新发展起来的核酸等温扩增技术，无论是在实际操作还是仪器要求方面，都比PCR 技术更为简单方便，它摆脱了对精良设备的依赖，在临床和现场快速诊断中显示了其良好的应用前景。
在众多等温扩增技术中，环介导等温扩增目前已在一定范围内得到了应用，其他一些新发展起来的等温扩增技术，如链替代等温扩增、滚环等温扩增、依赖解旋酶等温扩增、依赖核酸序列等温扩增、单引物等温扩增和核酸快速等温检测放大等技术，也在不断发展与完善之中。
为了更好地有选择地开发利用这方面技术，现就这些等温扩增技术的原理、特点及应用进行简要总结。
环介导等温扩增(Loop-mediated isothermalamplification，LAMP )环介导等温扩增(LAMP)是Notomi等于2000年首先提出来的一种新的核酸扩增技术，其原理主要是基于靶基因3'和5'端的6个区域设计3对特异性引物，包括1对外引物、1对环状引物和1对内引物，3种特异引物依靠链置换BstDNA聚合酶，使得链置换DNA合成不停地自我循环，从而实现快速扩增。
反应1h后可根据扩增副产物焦磷酸镁沉淀形成的浊度或者荧光染料进行判断扩增情况。
此反应先形成哑铃状模板，进入循环扩增阶段，再进行伸长、循环扩增，共3个阶段。
Loop-mediated isothermal amplification(LAMP，Fig1)依赖核酸序列的扩增(Nucleic acid sequence-based amplification，NASBA)依赖核酸序列的扩增技术 (NASBA) 是 1991 年由加拿大 Can －gene 公司首次介绍。
它是一项以核酸序列中RNA为模板，由两个引物介导的、连续均一的特异性体外等温扩增核苷酸序列的酶促过程。
整个反应由非循环相和循环相组成: 首先进行非循环相，在AMV 逆转录酶的作用下，引物I 与模板RNA 退火后合成cDNA，形成RNA/DNA 杂合体，随即RNaseH 降解 RNA，引物Ⅱ与 cDNA 退火，合成第二条 DNA 互补链。
第九章 核酸等温扩增技术
品质量安全方面的第一部专门法律，该 法明确提出了转基因农产品标识制度， 对转基因农产品安全问题的重视可见一 斑。
• 早在 2001 年，我国就建立了转基因农产品 的标识制度。当年颁布的《农业转基因生 物安全管理条例》及其 2002 年的两部配套 规章《农业转基因生物标识管理办法》和 《农业转基因生物标识审查认可程序》对 这一制度作出了较为详细的规定。此外， 同年颁布的《进出境转基因产品检验检疫 管理办法》和 2002 年颁布的《转基因食品 卫生管理办法》也对这一制度作出了规定。
• B3引物：下游外部引物(Backward Outer Primer )，由 B3区域组成，和靶基因的B3c区域互补。
扩增原理
• 60—65℃是双链DNA复性及延伸的中间 温度，DNA在65℃左右处于动态平衡状 态。因此，DNA在此温度下合成是可能 的。利用4种特异引物依靠一种高活性链 置换DNA聚合酶。使得链置换DNA合成 在不停地自我循环。
中国的转基因标识政策
• 根据《农业转基因生物安全管理条例》 和《农业转基因生物标识管理办法》规定， 中国采用有标识目录的定性强制性标识制 度。2002年公布的标识目录包括5大类17种。 表示方法分3种，包括“转基因xx”、“含 有转基因xx”、“由转基因xx原料加工， 但已不含有转基因成分”。关于阴性标识， 未作相关规定。
• 《进出境转基因产品检验检疫管理办法》 则规定了进出境转基因产品标识制度。
中国转基因标识现状及建议
现状：
我国的转基因标识制度目前还比较粗糙和单薄， 主要见于《农业转基因生物安全管理条例》及其 配套规章《农业转基因生物标识管理办法》和 《农业转基因生物标识审查认可程序》 。另外 《进出境转基因产品检验检疫管理办法》和《转 基因食品卫生管理办法》也对这一制度作出了规 定。这些法律规定仍有很多不足之处，不能适应 规制转基因生物安全的要求，也不能满足消费者 知情权的需要。
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扩增原理
• 60—65℃是双链DNA复性及延伸的中间 温度，DNA在65℃左右处于动态平衡状 态。因此，DNA在此温度下合成是可能 的。利用4种特异引物依靠一种高活性链 置换DNA聚合酶。使得链置换DNA合成 在不停地自我循环。
第2阶段
• 是扩增循环阶段。以茎环状结构为模板，FIP 与茎环的F2c区结合。开始链置换合成，解离 出的单链核酸上也会形成环状结构。迅速以3’ 末端的B1区段为起点，以自身为模板。进行 DNA合成延伸及链置换．形成长短不一的2条 新茎环状结构的DNA，BIP引物上的B2与其杂 交。启动新一轮扩增。且产物DNA长度增加一 倍。在反应体系中添加2条环状引物LF和LB， 它们也分别与茎环状结构结合启动链置换合成， 周而复始。扩增的最后产物是具有不同个数茎 环结构、不同长度DNA的混合物。且产物DNA 为扩增靶序列的交替反向重复序列。
• F3引物：上游外部引物(Forward Outer Primer)，由F3 区组成，并与靶基因的F3c区域互补。
• BIP引物：下游内部引物(Backward Inner Primer )，由 B1C和B2区域组成，B2区与靶基因3’端的B2c区域互 补，B1C域与靶基因5’端的BlP克服了传统PCR反应需要通过反 复热变性获得单链模板的缺点，避免了 反复升降温的过程，实现了恒温条件下 的连续快速扩增，具有更高的灵敏度和 扩增效率。
• 15-60min内可扩增出109-1010倍靶序列拷 贝，得到500ug/ml的目的DNA。具有简 单、快速、特异性强的特点。
LAMP技术的原理
LAMP主要是利用4种不同的特异性引物识 别靶基因的6个特定区域，在等温条件进行 扩增反应。基因的扩增和产物的检测可一步 完成，所有靶基因序列的检测可只通过扩增 产物的有、无来判别。有、无扩增反应是利 用荧光定量PCR仪检测反应的荧光强度或利 用核酸扩增过程中产生的焦磷酸镁沉淀反应 用浊度仪检测沉淀浊度来判定。
LAMP引物设计
• 主要是针对靶基因的六个不同的区域，基于靶基因3’ 端的F3c、F2c和Flc区以及5’ 端的Bl、B2和B3区等6个 不同的位点设计4种引物。
• FIP(Forward Inner Primer)：上游内部引物，由F2区和 F1C区域组成，F2区与靶基因3’端的F2c区域互补， F1C区与靶基因5’端的Flc区域序列相同。
环介导等温扩增技术（LAMP）
• 环介导等温扩增技术（loop-mediated isothermal amplification,LAMP）是2000年日本 研究人员Notomi等发明的一种新型体外等温 扩增特异性核酸片段的技术。
• 该技术主要利用两对特殊设计的引物和具有 链置换活性的DNA聚合酶，使反应中在模板 两端引物结合处循环出现环状单链结构，从 而保证引物可以在等温条件下顺利与模板结 合，并进行链置换扩增反应。
产物检测的原理
• 电泳分析 • 荧光检测 • 浑浊度检测
LAMP扩增产物电泳分析
肉眼观察LAMP结果
DNA与SYBR Green结合现示绿色
肉眼观察显白色沉淀
LAMP的优点
• 操作简单，LAMP核酸扩增是在等温条件下进行，产 物检测用肉眼观察或浊度仪检测沉淀浊度即可判断。 对于RNA的扩增只需要在反应体系中加入逆转录酶就 可同步进行(RT-LAMP)，不需要特殊的试剂及仪器。
第九章 核酸等温扩增技术
核酸等温扩增技术的定义及特点
• 核酸等温扩增技术是一类分子生物 学技术的总称，它们能在某一特定 的温度下扩增特定的DNA或者RNA。
• 与PCR技术相比核酸等温扩增对仪 器的要求大大简化，反应时间大大 缩短，更能满足快速简便的需求。
核酸等温扩增技术的分类
– 环介导等温扩增技术（LAMP） – 依赖于核酸序列的扩增技术（NASBA） – 滚环扩增技术（RCA） – 单引物等温扩增技术（SPIA） – 依赖于解旋酶的等温扩增技术（HAD） – 链替代扩增技术（SDA） – 快速等温检测放大技术（RIDA） – 切刻内切酶核酸恒温扩增技术（NEMA）
• 快速高效，因为不需要预先的双链DNA热变性．避免 了温度循环而造成的时间损失．核酸扩增在l h内均可 完成。
• 高特异性，由于是针对靶序列6个区域设计的4种特异 性引物。6个区域中任何区域与引物不匹配均不能进 行核酸扩增。故其特异性极高。
• 高灵敏度，对于病毒扩增模板可达几个拷贝，比PCR 高出数量级的差异。
扩增分两个阶段
• 第1阶段为起始阶段，任何一个引物向双链DNA的 互补部位进行碱基配对延伸时，另一条链就会解 离，变成单链。上游内部引物FIP的F2序列首先与 模板F2c结合。在链置换型DNA聚合酶的作用下向 前延伸启动链置换合成。外部引物F3与模板F3c结 合并延伸，置换出完整的FIP连接的互补单链。FIP 上的F1c与此单链上的Fl为互补结构。自我碱基配 对形成环状结构。以此链为模板。下游引物BIP与 B3先后启动类似于FIP和F3的合成，形成哑铃状结 构的单链。迅速以3’末端的Fl区段为起点．以自身 为模板，进行DNA合成延伸形成茎环状结构。该 结构是LAMP基因扩增循环的起始结构。
依赖于核酸序列的扩增
• 依赖于核酸序列的扩增（nucleic-acid sequence-based amplification, NASBA)是一项 以RNA为模板的快速等温扩增技术。
• 由加拿大Can-gene公司1991年首先发明。 • 主要用于RNA检测，具有高度敏感性和特异
LAMP的缺点
•由于LAMP扩增是链置换合成，靶序列长度最 好在300 bp以内。>500 bp则较难扩增。故不能 进行长链DNA的扩增。 •由于灵敏度高。极易污染而产生假阳性结果。 故要特别注意严谨操作。 •在产物的回收鉴定、克隆、单链分离方面均 逊色于传统的PCR方法。 •引物设计复杂。