重组酶聚合酶扩增技术研究进展

重组酶聚合酶扩增(recombinase polymerase amplification,RPA)技术是一种新兴的核酸恒温扩增技术,具有灵敏度高、特异性强、反应快速等特点。利用RPA技术可以对核酸拷贝数进行绝对定量并且可以同时检测多个靶标核酸序列,结合侧流层析试纸条或荧光信号监测装置等简易实验设备即可直接观察检测结果。就RPA技术的原理、发展、技术特点及其近年来在体外诊断、病原检测等领域的研究进展作一综述,旨在为该技术的深入研究和应用提供参考。     

CRISPR技术在生物传感中的应用——胞内核酸成像、体外核酸检测与胞内DNA记录

核酸分子是生命的遗传物质和生命信息载体。如何观察细胞内外特定核酸序列,快速检测核酸序列和记录检测的细胞活动过程?这些在分子生物学领域重要的科学问题需要强有力的技术来解决。如今,在基因组编辑领域赫赫有名的CRISPR技术开始涉足上述领域。重点介绍CRISPR相关蛋白在生物传感方面的应用,包括细胞内特定基因组序列和RNA的成像、体外核酸快速检测以及细胞内DNA记录。核酸成像与核酸检测是将特定核酸序列的信息,通过CRISPR蛋白的参与转化为易检测的信号(如荧光);而DNA记录则是将细胞的代际或所接触的信号转化为细胞内DNA序列进行储存。这些新兴研究方向的开发和发展将大大促进CRISPR技术在基础科学、合成生物学、分子诊断等领域的应用。     

PCR直接测序方法及其在肿瘤研究中的应用

PCR直接测序技术是PCR扩增与核酸测序技术相结合的一种方法.根据此技术的原理,建立了一种以PCR扩增引物为测序引物,α-³⁵S dATP直接掺入,Taq DNA聚合酶直接测序PCR扩增产物的方法.实验表明:该方法简便、快速、稳定.用此方法对人食管癌组织中的抗癌基因p53进行了突变测序分析,发现食管癌组织中p53存在点突变,插入、丢失移码突变.并用此方法对人和恒河猴的p53内含子序列进行了测定,发现猴第5内含子为81个核苷酸,第8内含子为92个核苷酸.     

多彩色荧光原位杂交技术原理及其应用

多彩色荧光原位杂交是一门新兴的分子细胞遗传学技术,它用几种不同颜色的荧光素单独或混合标记的探针,进行原位杂交,同时检测间期细胞或中期细胞中的几个特异核酸序列,为分析癌症遗传不稳定性提供了一种简便、快速、可靠的方法,并广泛应用于物理图谱绘制、致突变研究、肿瘤病理学和产前诊断.     

分子信标核酸检测技术研究进展

介绍了分子信标设计和分子信标核酸检测原理、技术特性和在基因突变大规模自动化检测中的应用. 分子信标是一种基于荧光共振能量转移现象设计的发卡型寡核苷酸探针,空间结构上呈茎环结构, 环序列是与靶核酸互补的探针,茎序列由与靶序列无关的互补序列构成,茎的一端连上荧光分子,另一端连上淬灭分子.通过空间结构改变决定分子信标发射荧光特性,从而对核酸进行定量检测. 分子信标技术具有操作简单、敏感、特异、可对核酸进行液相实时检测和对活体内核酸动态进行检测等特点,已应用于HIV辅助受体基因等基因突变的大规模自动化检测,是一种新型核酸定量检测技术.     

银染增强的纳米金标记探针对微量核酸的检测

本研究利用银染增强的纳米金技术建立了一种简单快速的核酸定量方法.该方法基于纳米金与烷巯基修饰的寡核苷酸分子共价键合作用,将纳米微粒报告基团标记在与靶核酸一端序列互补的寡核苷酸上,同时生物素化修饰另一端互补序列.靶核酸与两段寡核苷酸探针杂交后,借亲和素固定在酶标板孔内,通过纳米金催化的银染放大效应产生高灵敏的识别信号,适时记录其吸光度值从而实现核酸分子的定量.该检测方法检测单链核酸分子的灵敏度达0.1 fM,双链分子为10 fM.     

多重核酸检测技术研究进展

核酸检测技术因其快速、灵敏、特异、准确等优点,被广泛应用于细菌、真菌、病毒、寄生虫的快速检测和鉴定,以及疾病的早期筛查与诊断中。随着生物检测技术的发展,基于核酸的多重检测技术在核酸诊断领域发挥了越来越重要的作用,主要包括以多重PCR、核酸等温扩增、基因芯片为基础的多重核酸检测技术,这些技术可对多个靶标进行同时检测,具有快速、高通量、样品消耗少等特点。本文扼要介绍这些技术的原理,及其在病原检测、疾病诊断等方面的应用。     

利用高通量测序快速检测疑似感染患者体内未知病原体

采用第二代高通量测序技术从复杂的疑似感染患者组织标本中直接快速检测未知病原体.方法:取临床和实验室均不能确诊的病人血液和淋巴组织,进行病毒DNA和RNA的提取,直接用于第二代高通量测序,将测序结果用于生物信息学分析,与本地化的病毒核酸数据库进行比对,寻找潜在的病毒序列.结果:生物信息学分析显示,3901条序列reads与人内源性病毒K113同源;当使用K113特异性引物对经过Dnasel处理的患者和正常个体进行荧光定量RT - PCR时,发现在患者体内K113病毒基因的表达远远高于正常个体,提示该患者可能存在K113病毒的感染.结论:第二代高通量测序可能用于快速检测未知病原体.     

基于环介导等温扩增技术对人乳头瘤病毒的分型检测

目的:利用环介导等温扩增技术(LAMP)对人乳头瘤病毒(HPV)进行快速分型检测。方法:以HPV59、HPV66和HPV68等3种病毒亚型为实验对象,选择合适的特异性核酸序列作为检测的靶序列并构建质粒,针对每个待检靶序列设计若干组扩增引物,并利用构建的质粒筛选出特异性强、灵敏度高的引物组合;将筛选成功的引物加至微流控芯片上,实现人宫颈脱落上皮细胞样本内HPV的快速核酸分型检测。结果:针对HPV59、HPV66和HPV68这3种病毒亚型,设计筛选出具有一定灵敏度的特异性引物,结合微流控芯片,无需对样本进行复杂的核酸提取纯化操作,实现了对临床样本的直接快速检测,整个检测过程在1.5 h内即可完成。结论:LAMP技术可用于临床样本中HPV的分型检测,与微流控芯片结合后操作简单、检测时间短,在临床上有较好的应用前景。     

绿豆Ty1/Copia类反转录转座子逆转录酶序列的克隆和分析

利用Ty1/copia类反转录转座子的保守位点设计简并引物,从绿豆(Vigna radiata(L.)Wilczek)基因组中扩增得到了反转录转座子的逆转录酶序列.对扩增得到的约270bp的片段进行分离和克隆,并随机挑选了40个克隆进行测序,结果得到了36个单独的核酸序列,其中18个含有移码突变或终止子.根据序列比对,这些克隆可分为9组以及单个的9种.这40个克隆中,核酸序列相似性从8%到100%,显示出其核酸序列的高度异质性.将这些克隆的核酸序列与来自其他种植物的相应序列进行谱系分析,发现有些克隆与来自其他种植物的相应序列的亲缘关系比这些克隆之间更为接近.斑点杂交显示Ty1/copia反转录转座子约占绿豆基因组的9.3%.     

Polymerizing immobilization of acrylamide-modified nucleic acids and its application

The immobilization of nucleic acids on solid supports has been widely used in the detection of DNA and other biomolecules in sensor technology. Because three dimensional (3-D) hydrogel matrixes offer significant advantages for capturing probes over more conventional two dimensional (2-D) rigid substrates and the ability to provide a solution-mimicking environment, they are becoming increasingly attractive as desired supports for bio-analysis. Acrylamide-modified nucleic acids and acrylamide monomers being polymerized directly to immobilize nucleic acids is only one-step chemical process which is not interfered by exterior surroundings, and the 3-D polyacrylamide gel fabricated by this method is not required to be activated by some labile chemical treatments. Moreover, the attachment is extremely stable to withstand the cycling process involved in the polymerase chain reaction (PCR). In this paper, the development of polymerizing immobilization of acrylamide-modified nucleic acids is reviewed, and its applications in DNA sequence high-throughput analysis including mutation analysis and the whole genome sequencing are summarized.     

Nanopores and nucleic acids: prospects for ultrarapid sequencing

DNA and RNA molecules can be detected as they are driven through a nanopore by an applied electric field at rates ranging from several hundred microseconds to a few milliseconds per molecule. The nanopore can rapidly discriminate between pyrimidine and purine segments along a single-stranded nucleic acid molecule. Nanopore detection and characterization of single molecules represents a new method for directly reading information encoded in linear polymers. If single-nucleotide resolution can be achieved, it is possible that nucleic acid sequences can be determined at rates exceeding a thousand bases per second.     

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。     

单分子测序技术及应用研究进展

从DNA双螺旋结构的发现开始,生命科学研究进入分子水平,在20世纪70年代出现的测序技术为破译遗传密码作出了巨大贡献.近几年出现的单分子测序技术,可以在单个分子水平读取核苷酸序列,也被称为第三代测序技术,主要代表有HeliScope、Nanopore和PacBio等.与传统的第一代和第二代测序技术相比,第三代测序能够产生更长的碱基读长,能直接对RNA进行测序,无需逆转录,测序速度极快,同时其中某些技术所涉及的设备可以小型化,可便携至野外现场测序.第三代测序技术在生命科学基础理论研究及生物医学临床实践中,具有广泛的应用.本文重点介绍了各种单分子测序技术的原理、优缺点,及其应用研究进展.     

Real-time detection of <Emphasis Type="Italic">BRAF</Emphasis> V600E mutation from archival hairy cell leukemia FFPE tissue by nanopore sequencing

The MinION is a miniaturized high-throughput next generation sequencing platform of novel conception. The use of nucleic acids derived from formalin-fixed paraffin-embedded samples is highly desirable, but their adoption for molecular assays is hurdled by the high degree of fragmentation and by the chemical-induced mutations stemming from the fixation protocols. In order to investigate the suitability of MinION sequencing on formalin-fixed paraffin-embedded samples, the presence and frequency of BRAF c.1799T?>?A mutation was investigated in two archival tissue specimens of Hairy cell leukemia and Hairy cell leukemia Variant. Despite the poor quality of the starting DNA, BRAF mutation was successfully detected in the Hairy cell leukemia sample with around 50% of the reads obtained within 2 h of the sequencing start. Notably, the mutational burden of the Hairy cell leukemia sample as derived from nanopore sequencing proved to be comparable to a sensitive method for the detection of point mutations, namely the Digital PCR, using a validated assay. Nanopore sequencing can be adopted for targeted sequencing of genetic lesions on critical DNA samples such as those extracted from archival routine formalin-fixed paraffin-embedded samples. This result let speculating about the possibility that the nanopore sequencing could be trustably adopted for the real-time targeted sequencing of genetic lesions. Our report opens the window for the adoption of nanopore sequencing in molecular pathology for research and diagnostics.     

A Rapid Method for Manual or Automated Purification of Fluorescently Labeled Nucleic Acids for Sequencing,Genotyping, and Microarrays

Fluorescent dyes provide specific, sensitive, and multiplexed detection of nucleic acids. To maximize sensitivity, fluorescently labeled reaction products (e.g., cycle sequencing or primer extension products) must be purified away from residual dye-labeled precursors. Successful high-throughput analyses require that this purification be reliable, rapid, and amenable to automation. Common methods for purifying reaction products involve several steps and require processes that are not easily automated. Prolinx^®, Inc. has developed RapXtract^™ superparamagnetic separation technology, affording rapid and easy-to-perform methods that yield high-quality product and are easily automated. The technology uses superparamagnetic particles that specifically remove unincorporated dye-labeled precursors. These particles are efficiently pelleted in the presence of a magnetic field, making them ideal for purification because of the rapid separations that they allow. RapXtract-purified sequencing reactions yield data with good signal and high Phred quality scores, and they work with various sequencing dye chemistries, including BigDye and near-infrared fluorescence IRDyes. RapXtract technology can also be used to purify dye primer sequencing reactions, primer extension reactions for genotyping analysis, and nucleic acid labeling reactions for microarray hybridization. The ease of use and versatility of RapXtract technology makes it a good choice for manual or automated purification of fluorescently labeled nucleic acids.     

纳米孔测序技术应用于食品微生物检测的可行性分析

近些年来DNA测序技术发展迅速,已经从第一代生化测序发展到第三代单分子测序。作为第三代测序技术中的一种不同于当前流行的其他测序技术,纳米孔测序技术是基于电信号的一种物理方法测序。许多研究者通常将高通量测序技术应用于食品微生物的研究,但是将纳米孔测序技术应用于食品中微生物的检测却鲜有报道。Oxford Nanopore Technologies(牛津纳米孔科技公司)研发的DNA测序仪MinION,是世界首例用于商业测序的纳米孔测序仪,经过不断完善,近年来MinION在DNA测序中被广泛应用。MinION 测序一次需要的DNA量约1μg,其标准识别速度为一秒钟识别250个碱基,平均读长可至13kb~20kb,测序准确率可以达到98%。纳米孔测序的高识别速度和高准确率,完全满足快速检测的要求,将其应用于食品中微生物检测是完全可行的。     

Direct imaging of DNA motif sequences with encoded nanoparticles

We present a method for encoded tagging and imaging of short nucleic acid motif chains (oligomotifs) using selective hybridization of heterogeneous Au nanoparticles (Au-NP). The resulting encoded NP string is thus representative of the underlying motif sequence. As the NPs are much more massive than the motifs, the motif chain order can be directly observed using scanning electron microscopy. Using this technique we demonstrate direct sequencing of oligomotifs in single DNA molecules consisting of four 100-nt motif chains tagged with four different types of NPs. The method outlined is a precursor for a high density direct sequencing technology.     

Non-gel based techniques for plant pathogen genotyping

The introduction of real-time PCR technology has significantly improved and simplified the quantification of nucleic acids, and this technology has become an invaluable tool for many scientists working in different disciplines. Particularly in the field of molecular diagnostics and genotyping, real-time PCR-based assays have gained favour in the recent past. Rapid real-time PCR diagnosis can result in appropriate control measures and eradication procedures in a faster and more accurate way than traditional methods based on pathogen isolation. Real-time quantitative PCR represents a highly sensitive and powerful technique for the gel-free detection of nucleic acids. In this review, the main chemistries used for the detection of PCR product during real-time PCR, as well as advantages and limitations of real-time PCR will be depicted. Furthermore, the existing literature as it applies to plant pathogens detection in the routine and research laboratory will be reviewed in order to focus on one of the many areas in which the application of real-time PCR has provided significant methodological benefits.