双链探针实时荧光PCR核酸检测新技术研究*

目的:采用一种“双链探针”实时荧光PCR技术,提高HBV核酸检测灵敏度,并在同一反应管中实现代谢酶CYP2C19^*2基因型检测。方法:采用双链探针与TaqMan探针同时检测不同浓度HBV血清样本,使用上海宏石SLAN 96实时荧光PCR仪进行核酸Ct值检测和结果统计分析;采用双链探针检测代谢酶CYP2C19^*2不同基因型样本,使用上海宏石SLAN 96实时荧光PCR仪进行核酸Ct值检测和基因型确定。结果:不同浓度HBV血清样本检测,双链探针荧光本底低,检测灵敏度更高,与TaqMan探针检测结果相比,两者核酸检测Ct值存在显著性差异(P<0.05);双链探针检测36份样本的代谢酶CYP2C19^*2基因型,检测结果与Sanger测序结果完全一致。结论:双链探针实时荧光PCR检测技术可完成目的基因的高灵敏核酸检测,也可实现基因型分析。     

地高辛标记Ucp2基因RNA探针的制备和应用

目的:制备用于检测小鼠胚胎早期Ucp2基因表达的地高辛标记的特异性RNA探针。方法:提取小鼠胚胎脑组织总RNA,设计引物,通过RT-PCR方法获取Ucp2基因片段,将其克隆到pGEM-T载体。分别利用Sp6、T7和Ucp2特异性引物,PCR扩增获得转录模板,通过Sp6及T7 RNA聚合酶,获得地高辛标记的正义、反义Ucp2 RNA原位杂交探针。检测标记探针的效价后,通过全胚胎原位杂交分析制备探针的特异性和杂交效果。结果:成功获得Ucp2基因正义、反义探针,反义探针能高效灵敏检测到Ucp2基因在小鼠胚胎Ed9.5、Ed10.5神经系统呈现高表达,而正义探针未能检测到表达信号。结论:成功制备了特异高效的地高辛标记Ucp2 RNA原位杂交探针,为进一步研究Ucp2基因在小鼠胚胎组织中的表达,尤其在神经组织的定位奠定基础。     

应用小鼠整体原位杂交技术检测MDM2在鼠胚发育不同阶段的表达

整体原位杂交(whole-mountinsituhybridization,WMH)已经成为基因表达定位和表达分布模式研究的一种重要手段.该技术能在整体水平上精确地研究胚胎发育过程中基因表达的三维信息,而且为大规模筛选区域及组织特异性候选克隆提供了有利的技术手段.采用体外转录地高辛标记的RNA探针,检测已知基因MDM2在鼠胚胎发育不同阶段的表达模式.     

基于H2DCFDA荧光探针的植物活性氧检测方法

活性氧(reactive oxygen species, ROS)是植物体内的一把“双刃剑”。ROS作为信号分子在植物生命活动中发挥关键作用, 但ROS过量积累会对生物大分子造成氧化损伤。准确测定ROS含量对于评估植物细胞内的氧化还原状态至关重要。由于植物体内ROS各组分半衰期短且反应活性强, 定性定量检测较为困难。因此, 选择合适的检测方法以提高检测的时空准确性非常重要。目前, 荧光分析法因其具有灵敏度高、选择性好、检出限低和直观性强等优点, 受到研究人员的广泛关注。该文详细描述基于流式细胞仪和激光共聚焦显微镜, 利用2′,7′-二氯二氢荧光素二乙酸酯(H2DCFDA)荧光探针检测水稻(Oryza sativa)体内ROS水平和时空分布的操作流程及注意事项。该技术也可用于直接检测拟南芥(Arabidopsis thaliana)、玉米(Zea mays)和大豆(Glycine max)等模式植物组织中ROS的水平和分布。     

钙荧光探针Fura-2/AM对大肠杆菌细胞的负载行为

Fura-2/AM作为一类高效的钙离子荧光探针,在动物细胞中已得到较成功的应用,但在微生物细胞分析方面鲜见报道。该文系统研究了该探针的荧光光谱特征及其在大肠杆菌细胞内的负载行为,并考察了在大肠杆菌细胞中钙离子与Fura-2的结合情况。为利用其研究大肠杆菌等微生物细胞内钙离子浓度及钙离子跨膜行为奠定了基础。     

钙荧光探针Fura2/AM对大肠杆菌细胞的负载行为

Fura2/AM作为一类高效的钙离子荧光探针,在动物细胞中已得到较成功的应用,但在微生物细胞分析方面鲜见报道。该文系统研究了该探针的荧光光谱特征及其在大肠杆菌细胞内的负载行为,并考察了在大肠杆菌细胞中钙离子与Fura2的结合情况。为利用其研究大肠杆菌等微生物细胞内钙离子浓度及钙离子跨膜行为奠定了基础。     

分子生物学技术讲座（五）：放射性标记的DNA和RNA探针的制备

放射性标记的核酸探针（DNA和RNA探针）目前已被广泛地应用于分子生物学的各个领域,例如克隆的筛选,Southern杂交分析,基因表达水平的测定等等。放射性核酸分子探针是指特定的已知核酸分子片段,内含放射性核素（例：‘千、‘H和”S）,并能与被检测的核酸分子退火杂交（核酸序列互补）,因此可用于待测核酸样品中特定基因序列片段的探测。1探针的种类和选择根据核酸分子探针的来源及其性质可将其分成3大类,即：DNA探针、RNA探针和人工合成的寡聚核青酸探针。而DNA探针又可分为基因组DNA探针和。DNA探针。探针的选择是根据不…     

Alu探针及dT探针在原位杂交质控中的应用

随着现代生物技术的发展 ,核酸杂交技术已成为分子生物学和细胞生物学领域中研究基因及其表达的重要手段。其中 ,原位杂交技术能对生物体整个基因组中单一的基因拷贝或其转录物进行探测 ,能从染色体组数百万个核苷酸序列中识别某个基因的转录物以了解基因的表达状态及表达情况 ,从而在基因定位、细胞分化调控、肿瘤遗传学等研究中得到了极其广泛的应用[1 ] 。但是 ,生物组织标本极易被ＤＮＡ酶或ＲＮＡ酶 (尤其是ＲＮＡ酶 )污染 ,从而造成标本中的核酸降解 ,导致假阴性结果甚至误导某些实验研究的结论。近年来发展的ｄＴ探针及Ａｌｕ探针有…     

用DNA杂交技术检测绿脓杆菌氨基糖甙类钝化酶基因的研究

目前已知的氨基糖甙类抗菌素钝化酶基因有许多种,2″—0—腺苷转移酶[ANT(2″)]基因为其中之一。本文从一株含ANT(2″)基因的重组质粒pFCT3103的基因克隆株,分离出310bp的ANT(2″)基因片段,将它制成基因探针,用该探针检查了30株绿脓杆菌是否含有氨基糖甙类钝化酶基因的存在情况,结果表明ANT(2″)基因的检出率为43.3%。     

Selective activation of SHP2 activity by cisplatin revealed by a novel chemical probe-based assay

Src homology-2 (SH2) domain-containing phosphatase 2 (SHP2) is known to participate in several different signaling pathways to mediate cell growth, survival, migration, and differentiation. However, due to the lack of proper analytical tools, it is unclear whether the phosphatase activity of SHP2 is activated in most studies. We have previously developed an activity-based probe LCL2 that formed covalent linkage with catalytically active protein tyrosine phosphatases (PTPs). Here, by combining LCL2 with a SHP2 specific antibody, we established an assay system that enables the direct monitoring of SHP2 activity upon cisplatin treatment of cancer cells. The protocol is advantageous over conventional colorimetric or in-gel PTP assays as it is specific and does not require the use of radioisotope reagents. Using this assay, we found SHP2 activity was selectively activated by cisplatin. Moreover, the activation of SHP2 appeared to be specific for cisplatin as other DNA damage agents failed to activate the activity. Although the role of SHP2 activation by cisplatin treatments is still unclear to us, our results provide the first direct evidence for the activation of SHP2 during cisplatin treatments. More importantly, the concept of using activity-based probe in conjunction with target-specific antibodies could be extended to other enzyme classes.     

Design and evaluation of a novel fluorescent CB2 ligand as probe for receptor visualization in immune cells

Cannabinoid CB2 receptor has emerged as a very promising target over the last decades. We have synthesized and evaluated a new fluorescent probe designated NMP6 based on 6-methoxyisatin scaffold, which exhibited selectivity and K(i) value at hCB2 of 387 nM. We have demonstrated its ability to be an effective probe for visualization of CB2 receptor binding using confocal microscopy and a flow cytometry probe for the analysis of CB2 protein expression. Furthermore, NMP6 was easily obtained in two chemical steps from commercially available building blocks.     

A near‐infrared fluorescent probe based on boric acid hydrolysis for hydrogen peroxide detection and imaging in HeLa cells

Using the characteristics of hydrogen peroxide that are able to cleave phenyl‐boric acid selectively and efficiently, we here report a dicyanoisophorone‐boric acid ( DCP‐BA )‐based near‐infrared (NIR) fluorescent probe for detection of hydrogen peroxide. This probe shows a rapid, highly selective, and sensitive detection process for hydrogen peroxide with a significant NIR fluorescent turn‐on response that has been successfully applied to detect exogenous hydrogen peroxide in HeLa cells.     

Hg2+‐selective ratiometric and colorimetric probe based on dansyl–rhodamine and its staining function in cell imaging

A new ratiometric probe composed of a dansyl–rhodamine dyad for the detection of Hg²⁺ via fluorescence resonance energy transfer was designed and synthesized. Rhodamine, dansyl chloride, and hydrazide were selected as the acceptor, donor, and reaction site, respectively. It displayed high selectivity and sensitivity to Hg²⁺ with obvious colour change and fluorescence change due to Hg²⁺‐assisted hydrolysis of rhodamine hydrazide. A good linear relationship ranging from 0 to 16 μM and 0–28 μM for the Hg²⁺ concentration was found based on absorbance and fluorescence assay, respectively. Detection limits of absorbance and fluorescence for Hg²⁺ were calculated to be 1.22 μM and 9.10 μM, respectively.     

A colorimetric probe for detection of Cu2+ by the naked eye and application in test paper

A novel colorimetric probe RP1 was synthesized using rhodamine derivatives and heterocyclic compounds for the purpose of detecting Cu²⁺. RP1 showed good selectivity, high sensitivity and affinity toward Cu²⁺ over other competing ions in CH₃OH–H₂O (1/1, v/v) solution. Absorbance intensity showed a good linear fit between probe R1 and Cu²⁺ over the concentration range 1–8 μM and the association constant was also calculated to be 1.145 × 10⁵ M^?1. The sensing mechanism was deduced using Job's plot, Fourier transform infrared spectroscopy, and density functional theory studies. In addition, the colorimetric experiment indicated that probe RP1 could be made into test paper to detect Cu²⁺ with a colour change from colourless to pink.     

A colorimetric fluorescent probe for rapid and specific detection of nitrite

The method of fluorescent probes has been an important technique for detection of nitrite (NO₂^?). As an important inorganic salt, excessive nitrite would threaten humans and the environment. In this paper, a colorimetric fluorescent probe P‐N (1,2‐diaminoanthraquinone) with rapid response and high selectivity, which could detect NO₂^? by visual colour changes and fluorescence spectroscopy is presented. The probe P‐N solution (pH 1) changed from pink to colourless with the addition of NO₂^? and fluorescence intensity at 639 nm clearly decreased. Good linear exists between fluorescence intensities and NO₂^? concentrations for the range 0–16 μM, and the detection limit was 54 nM (based on a 3σ/slope). Moreover, probe P‐N could also detect NO₂^? in real water samples, and results were all satisfactory. Probe P‐N shows great practical application value for detecting NO₂^? in the environment.     

植物特异性DNA探针的制备与应用研究进展

本文简述了植物特异性DNA探针的种类,介绍了特异性探针的制备方法,主要是特异性DNA序列的分离与筛选,总结了植物特异性DNA探针在种质鉴定、外源染色体识别、核型分析和基因组研究等方面的应用,提出了存在的问题与应用前景。 Abstract:In this paper,the types of specific DNA probes from plant were briefly described,and some methods of constructing specific probes were introduced,mainly were the isolation and screening of specific DNA sequences.The applications of the specific probes in the germplasm and foreign chromosome identifications,the karyotype and genome analyses were also summarized and discussed.     

New broad-host-range promoter probe vectors based on the plasmid RK2 replicon

Broad-host-range plasmid RK2-based promoter probe vectors with a known nucleotide sequence were constructed. In the absence of an upstream promoter, the expression of two tested reporter genes (luc and lacZ) in Escherichia coli was virtually zero, while insertion of the Ptrc promoter resulted in strong inducer-dependent expression. The lacZ-based vectors were mobilized into Pseudomonas fluorescens ST, Pseudomonas putida KT2442, Sphingomonas spp. and Burkholderia spp. LB400, and expression analyses indicated that the properties observed in E. coli are maintained across the species barriers. In addition, the previously established knowledge of RK2 molecular biology allows easy manipulations of features such as plasmid copy number, further extending the application potential of the vectors.