利什曼原虫DNA压电晶体传感器的初步研究

目的：研制利什曼原虫DNA压电晶体传感器。方法:对压电石英晶体表面进行处理并结合上探针DNA制成DNA传感器，在不同的杂交时间和杂交浓度条件下测定其频率响应。结果：在5h内随着杂交时间的增加，压电晶体振荡频率的改变值△f也呈现性增加，在相同的杂交时间内，低浓度范围△f浓度的增加而增加。结论：在一定条件下，利什曼原虫DNA压电晶体传感器有望用于检测利什曼虫。     

核酸杂交方法：测定与研究药物耐药基因扩散的应用

DNA-DNA 杂交法在生物学和医学方面应用的前景核酸杂交法是当代生物学最革命和公认的方法之一。它是建立在分子的单链互补生成双链杂交特性的基础上,杂交可以在溶液中或固体底垫如硝基纤维或尼龙膜上进行,在后一种情况下,需将 DNA 或消化及游离出核酸的细胞悬乳液固定在膜上,将双链的DNA 变性,生成的单链分子与膜共价结合。当加上 DNA 探针即标记的变性核酸分子时,则在有一定同源性的探针与固定在膜上的DNA 之间产生杂交。探针可以应用研究者感兴趣的基因或任何其它的 DNA 片段,以及基于已知核苷酸序列的 DNA 片段而合成的寡核苷酸。采用大小为10～40个核苷酸组成的合成寡核苷酸可以绝对准确地判断存在于生物材料中的必需基因或核苷酸都份。DNA 探针在不同医学和生物学领域得     

压电基因芯片技术及其在医药学研究中的应用

压电基因芯片是将基因芯片技术、传感器技术、微电子技术结合起来的新的基因分析技术。它是基于石英晶体的质量效应，即石英晶体表面质量改变引起晶体振荡频率变化，从而将杂交信号转变成电信号。其优点是不需要对探针进行标记，可以进行杂交动力学的实时分析，操作简单，容易实现分析的自动化、微型化、集成化。     

miR-196a2单核苷酸多态与乳腺癌易感性关系

miRNA是一类长约21-24bp、内源性、非编码、单链RNAs,进化上高度保守,广泛存在于动、植物细胞中。可以调节基因表达[1]。miRNAs可以与靶mRNAs互补序列配对结合,有效并且稳定地降低mRNAs的翻译活性。哺乳动物成熟miRNAs大多通过不完全互补配对与靶mRNA 3′-UTRs结合,调节靶基因的表达,有时也可以直接将靶mRNAs切断。生物信息学数据表明一条单链miRNA可以[2]     

核酸原位杂交技术在细胞病理学的应用

<正>杂交的技术原理是核酸碱基互补,指DNA互补链,DNA与RNA或RNA互补链间发生杂交。原位杂交是互补碱基序列形成氢键的过程。构成稳定的复合体或杂交体。主要工具是标记的特定序列探针。目前应用的探针标记物多是非放射性核位素。探针种类主要有四类:双链DNA,单链DNA,反义RNA和自动化合成的寡核苷酸探针。它们各有其本身特点。     

二氯乙酸对ras基因的DNA损伤作用

目的研究二氯乙酸(dichloroacetic acid,DCA)对ras基因的DNA损伤作用,探讨其致癌作用分子机制。方法制备各ras基因外显子单链探针;DCA染毒TK6细胞,提取基因组DNA,再经RDPCR扩增,扩增产物与探针杂交显色。结果成功制备各ras基因外显子单链探针,Southern杂交检测到DCA对N-ras基因外显子2和H-ras基因外显子2的DNA损伤片段杂交条带,未检测出其对N-ras基因外显子1和H-ras基因外显子1的DNA损伤作用。结论DCA可造成ras基因DNA损伤,可能与其致癌作用机制密切相关,属于遗传毒性物质。     

对斑点杂交方法检测治疗性单克隆抗体制品中残留宿主细胞DNA含量的影响因素研究

目的:对斑点杂交方法检测重组生物制品中残留宿主细胞DNA含量试验中的6个影响因素进行评价。方法:对不同的样品稀释液,样品中是否含有蛋白,含蛋白样品是否采用蛋白酶K处理,采用不同方法预处理的模板DNA作为标记用模板,采用不同体积的离心管进行探针标记,分别采用不同的杂交温度,共计6个因素对检测结果的影响进行评价。结果:在使用随机标记的探针通过斑点杂交方法检测重组生物制品中残留宿主细胞DNA含量试验中:标准品和样品稀释中使用水进行稀释时结果可信度较高;样品中含有蛋白会降低检测的灵敏度;在含蛋白样品中加入蛋白酶K处理可以在一定程度上提高检测的灵敏度;使用超声处理的模板DNA作为探针标记的模板时,检测灵敏度较高,同时背景较低;探针标记过程中应采用0.2 mL薄壁PCR管作为容器以保证试验成功率;最适的杂交温度为42℃左右。结论:在斑点杂交方法检测治疗性单克隆抗体制品中残留宿主细胞DNA含量中,采用水作为稀释液,对含蛋白样品使用蛋白酶K消化,标记用模板DNA使用超声处理,使用0.2 mL薄壁PCR管作为标记反应管和在42℃杂交时,结果的稳定性和灵敏度有较大提高。     

miRNA与消化系统非恶性疾病关系的研究进展

microRNA是近年来在多种真核细胞及病毒中发现的一类来源于内源性染色体上的非编码单链RNA,在进化上具有高度的保守性,能够通过与靶mRNA特异性的碱基互补配对,引起靶mRNA降解或者抑制其翻译,从而对基因进行转录后的表达调控,在体内发挥重要的生物学功能.     

PCR方法在水蛭素基因克隆方面的新应用

目的应用PCR方法获得水蛭素基因。方法在传统PCR方法的基础上进行了改进 ,根据已知天然水蛭素基因序列 ,设计了一对 12 3nt且 3′端具有 15nt互补序列的单链DNA ,通过低温复性使其互补结合后 ,进行延伸反应 ,得到微量双链水蛭素基因。再以该基因作为模板设计一对引物 ,进行常规PCR扩增反应。结果获得水蛭素基因。结论此法对于获得象水蛭素基因这类序列已知、片段不太长 ,且不易得到的基因提供了一种可行方法     

高脂饮食胰岛素抵抗大鼠模型解偶联蛋白基因表达的变化

目的　研究高脂喂养致胰岛素抵抗 (IR)动物模型中胰岛素敏感性的变化及解偶联蛋白 (UCPs)基因表达的改变。方法　在高脂喂养大鼠的IR模型中 ,以高胰岛素 -正葡萄糖钳夹术评价胰岛素敏感性的变化 ,用半定量的逆转录 -聚合酶链反应 (RT -PCR)检测UCPs基因mRNA表达的改变。结果　高脂喂养可以显著降低葡萄糖输注率 (GIR2 8 0 3± 8 0 2VS 9 89± 3 2 8P <0 0 1) ,显著增加血清甘油三酯 (TG)、自由脂肪酸(FFA) (0 6 1± 0 17VS 1 2 4± 0 34,0 2 4± 0 10VS 0 4 3± 0 13) ,增加动物的体重 ,可使肝脏内UCP - 2基因、肌肉内UCP - 3基因mRNA表达降低 ,对肌肉组织内UCP - 2基因mRNA表达无影响。结论　高脂喂养可引起血脂增高、体重增加、降低胰岛素的敏感性 ,胰岛素敏感性降低可能与UCPs基因表达减弱有关     

Taqman real-time PCR assay based on ORFV024 gene for rapid detection of orf infection

Abstract

In this study, a TaqMan real-time polymerase chain reaction (PCR) assay was developed to detect and quantify orf virus (ORFV) DNA in infected cell culture and clinical samples. Primers and probes were designed to amplify an 87?bp fragment DNA based on the sequence of ORFV024 gene encoding an NF-κB inhibitor of orf virus. The assay was highly specific and sensitive for ORFV DNA and no cross-reactions were detected with any other poxviruses; the sensitivity was 5?fg or 15 copies of ORFV genomic DNA. Both intra- (1.490?±?1.261%) and inter-assay (1.958?±?0.568%) variabilities were within the acceptable range, indicating the high efficiency and reproducibility of the assay. Further, the assay has shown a relative diagnostic sensitivity and specificity of 100%, when compared to B2L gene-based semi-nested PCR. The assay is simple, rapid, specific and sensitive with a wide potential for rapid field diagnosis of orf in sheep and goats.     

46XY女性性反转综合征一例sry基因的错义突变分析

目的 探讨46XY女性性反转综合征患者发病的分子机制. 方法 应用荧光原位杂交技术(FISH)、聚合酶链反应(PCR)扩增、限制性内切酶分析、单链构象多态性分析及基因测序技术对1例46XY女性性反转综合征进行sry基因的错义突变分析. 结果 本例FISH证实为X、Y染色体结构,无嵌合体存在;PCR扩增出现609 bp特...     

Electrochemical detection of enzyme labeled DNA based on disposable pencil graphite electrode

Electrochemical biosensor for the detection of DNA hybridization using the reduction signal of alpha-naphthol is described. A pencil graphite electrode was used as a working electrode. Capture probes were covalently attached on to the pencil graphite electrode surface (PGE) at the 5' end amino group by using N-(dimethylamino)propyl-N'-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysulfosuccinimide (NHS) as a coupling agent on to PGE. After capture probe immobilization on to PGE surface; probe was hybridized with complementary biotinylated oligonucleotide. Alkaline phosphatase labeled with extravidin (Ex-AP) binds to biotinylated hybrid via biotin-avidin interaction. alpha-Naphthyl phosphate (alpha-NAP) was added and the reaction between alkaline phosphatase (AP) and alpha-NAP was occurred consequently as a substrate of AP, alpha-NAP reduction signal was obtained from this reaction, at -0.100 V by using differential pulse voltammetry (DPV). Other experimental parameters were studied such as; optimizations of hybridization time, and the concentrations of capture probe, biotinylated oligonucleotide and enzyme.     

Highly specific,quantitative polymerase chain reaction probe for the quantification of human cells in cynomolgus monkeys

Quantification of human cells may be performed using quantitative polymerase chain reaction (qPCR). In preclinical studies, the human Alu sequence is widely used as biomarker for human DNA. However, because the Alu gene is shared by primates, its use is limited to non-primate studies. The biodistribution of human cells in primates is also necessary for translational studies. Therefore, we aimed to design a novel, human-specific primer/probe that enables the quantification of human cells in primates and other animal models. A novel primer/probe set was successfully designed based on highly repetitive LINE1 sequences. qPCR efficiency (94.95–99.21%) and linearity of calibration curves (r² = 0.996–0.999) were confirmed in tissue homogenates of cynomolgus monkey. The lower limit of detection was 10 cells per 15-mg tissue sample, a sensitivity that is equivalent to existing Alu primers/probes. The set was also effective in other animal models such as mice, rabbits, pigs, and common marmosets.To our knowledge, this is the first study describing the successful design of a human-specific qPCR primer/probe for human cell quantification in various animals, including non-human primates, using LINE1 sequence. The excellent selectivity, sensitivity, and versatility of the LINE1 primers/probes make it a promising quantification tool in preclinical biodistribution studies.     

Large-scale SNP scoring from unamplified genomic DNA

Discoveries from the Human Genome Project (HGP) continue to spur changes in medical technology that will lead to new diagnostic procedures in the clinical lab. As more single nucleotide polymorphisms (SNPs) are discovered and correlated to human diseases, demands for genetic tests will increase. The enormity of the number of SNPs makes developing inexpensive and reliable high-throughput methods for SNP scoring imperative. High-throughput screening (HTS) means, at a minimum, a production rate of thousands of assays per day. Ideally, the technology will be easy, inexpensive and amenable to automation. The Invader assay offers a simple diagnostic platform to detect single nucleotide changes with high specificity and sensitivity from unamplified, genomic DNA. The Invader assay uses a structure-specific 5' nuclease (or flap endonuclease) to cleave sequence-specific structures in each of two cascading reactions. The cleavage structure forms when two synthetic oligonucleotide probes hybridise in tandem to a target. One of the probes cycles on and off the target and is cut by the nuclease only when the appropriate structure forms. These cleaved probes then participate in a second Invader reaction involving a dye-labelled fluorescence resonance energy transfer (FRET) probe. Cleavage of this FRET probe generates a signal, which can be readily analysed by fluorescence microtitre plate readers. The two cascading reactions amplify the signal significantly; each original target molecule can lead to more than 10(6) cleaved signal probes in one hour. This signal amplification permits identification of single base changes directly from genomic DNA without prior target amplification. The sequences of the oligonucleotide components of the secondary reaction are independent of the target of interest and permit the development of universal secondary reaction components useful to identify any target.     

Decline in Exogenous Gene Expression in Primate Brain Following Intravenous Administration Is Due to Plasmid Degradation

Purpose Nonviral gene transfer to the brain of adult Rhesus monkeys is possible with a single intravenous administration of plasmid DNA that is encapsulated in the interior of pegylated immunoliposomes, which are targeted across membrane barriers in vivo with a monoclonal antibody to the human insulin receptor. Methods The present studies measure the rate of decay of luciferase gene expression in the Rhesus monkey with luciferase enzyme assays, Southern blotting, and real-time polymerase chain reaction. Results Luciferase enzyme activity in frontal cortex, cerebellum, and liver decays with a t_1/2 of 2.1 ± 0.1, 2.6 ± 0.2, and 1.7 ± 0.01 days, respectively. Luciferase plasmid in brain and liver was detectable by Southern blotting at 2 days, but not at 7 or 14 days. The concentration of luciferase plasmid DNA in brain and liver was measured by real-time polymerase chain reaction, and decayed with t_1/2 of 1.3 ± 0.3 and 2.7 ± 0.5 days, respectively. Conclusions The maximal concentration of luciferase plasmid DNA in Rhesus monkey brain was 3–4 molecules/cell following an i.v. administration of 12 μg/kg pegylated immunoliposome encapsulated plasmid DNA. These results demonstrate that the rate of loss of exogenous gene expression in the primate in vivo correlates with the rate of DNA degradation of the exogenous plasmid DNA.     

Suppression of a DNA base excision repair gene, hOGG1, increases bleomycin sensitivity of human lung cancer cell line

Bleomycin (BLM) has been found to induce 8-oxoguanine and DNA strand breaks through producing oxidative free radicals, thereby leading to cell cycle arrest, apoptosis and cell death. Cellular DNA damage repair mechanisms such as single strand DNA break repair/base excision repair (BER) are responsible for removing bleomycin-induced DNA damage, therefore confer chemotherapeutic resistance to bleomycin. In this study, we have investigated if down-regulation of human 8-oxoguanine DNA glycosylase (hOGG1), an important BER enzyme, could alter cellular sensitivity to bleomycin, thereby reducing chemotherapeutic resistance in human tumor cell. A human lung cancer cell line with hOGG1 deficiency (A549-R) was created by ribozyme gene knockdown technique. Bleomycin cellular sensitivity and DNA/chromosomal damages were examined using MTT, colony forming assay, comet assay as well as micronucleus assay. We demonstrated that hOGG1 gene knockdown enhanced bleomycin cytotoxicity and reduced the ability of colony formation of the lung cancer cell lines. We further demonstrated that bleomycin-induced DNA strand breaks resulted in an increase of micronucleus rate. hOGG1 deficiency significantly reduced DNA damage repair capacity of the lung cancer cell lines. Our results indicated that hOGG1 deficiency allowed the accumulation of bleomycin-induced DNA damage and chromosomal breaks by compromising DNA damage repair capacity, thereby increasing cellular sensitivity to bleomycin.     

Nanoparticle based bio-bar code technology for trace analysis of aflatoxin B1 in Chinese herbs

A novel and sensitive assay for aflatoxin B1 (AFB1) detection has been developed by using bio-bar code assay (BCA). The method that relies on polyclonal antibodies encoded with DNA modified gold nanoparticle (NP) and monoclonal antibodies modified magnetic microparticle (MMP), and subsequent detection of amplified target in the form of bio-bar code using a fluorescent quantitative polymerase chain reaction (FQ-PCR) detection method. First, NP probes encoded with DNA that was unique to AFB1, MMP probes with monoclonal antibodies that bind AFB1 specifically were prepared. Then, the MMP-AFB1-NP sandwich compounds were acquired, dehybridization of the oligonucleotides on the nanoparticle surface allows the determination of the presence of AFB1 by identifying the oligonucleotide sequence released from the NP through FQ-PCR detection. The bio-bar code techniques system for detecting AFB1 was established, and the sensitivity limit was about 10^?8 ng/mL, comparable ELISA assays for detecting the same target, it showed that we can detect AFB1 at low attomolar levels with the bio-bar-code amplification approach. This is also the first demonstration of a bio-bar code type assay for the detection of AFB1 in Chinese herbs.     

氯化镍在体内诱导大鼠DNA-蛋白质交联

目的　了解NiCl2 在体内对大鼠细胞的DNA产生何种损伤以及与聚腺苷二磷酸核糖聚合酶(PARP)活性的关系。方法　以 10 ,2 0 ,30mg·kg- 1NiCl2 ip给大鼠进行染毒 ,2 4h后处死动物 ,分离外周血淋巴细胞和肺细胞 ,应用单细胞凝胶电泳 (彗星试验 ) ,检测DNA单链、双链断裂和DNA 蛋白质交联 ,同时用 [3H]NAD渗透法检测PARP活性的变化。结果　外周血淋巴细胞和肺细胞均没有出现明显的DNA单链和双链断裂。所有剂量组的外周血淋巴细胞都出现了不同程度的DNA 蛋白质交联 ,交联率为 14 %～ 36 %,但没有剂量反应关系。 2 0mg·kg- 1NiCl2 也能诱导肺细胞DNA 蛋白质交联 ,交联率达 30 %。所有剂量组的NiCl2 均能明显抑制大鼠外周血淋巴细胞PARP酶的活性 ,酶活性下降至对照组的 38%～ 5 7%,但没有表现出剂量反应关系。大鼠肺细胞中该酶活性不受NiCl2 染毒的影响。结论　NiCl2 在体内环境下能诱导大鼠外周血淋巴细胞和肺细胞的DNA损伤 ,主要是引起DNA 蛋白质交联 ,而不直接造成DNA链的断裂 ;NiCl2 还能明显抑制外周血淋巴细胞的PARP酶活性 ,进而可能影响DNA修复。     

Intravenous Microdialysis in the Mouse and the Rat: Development and Pharmacokinetic Application of a New Probe

Purpose. A flexible microdialysis probe was designed for intravenous sampling in small laboratory animals. Methods. Surgical techniques were developed to implant this probe via the femoral vein in the vena cava of the mouse and the rat. The in- and outlet of the probe were exteriorized above the tail of the animal and were directly connected to the microsyringe pump for perfusate delivery and to the injection valve for on-line HPLC analysis of the microdialysate samples. Results. The in vitro recoveries of flurbiprofen and naproxen for these probes were 68.2 ± 6.9% (mean ± S.D., n= 12) and 66.5 ± 7.3%, respectively. The relative loss by in vivo retrodialysis, measured the day after the implantation of the probes, was 66.1 ± 8.8% for flurbiprofen and 60.9 ± 9.9% for naproxen. The pharmacokinetics of unbound flurbiprofen were studied following i.v. bolus administration of flurbiprofen to the mouse (n = 4) and the rat (n = 6) with on-line HPLC analysis of microdialysates every 10 minutes during 6 to 8 hours. Flurbiprofen microdialysate concentrations were converted to unbound concentrations using the in vivo loss of flurbiprofen by retrodialysis carried out just before the start of the pharmacokinetic experiment. The integrity of the probe throughout the experiment was monitored by continuous retrodialysis of naproxen. Conclusions. The developed techniques can be used to carry out routine pharmacokinetic studies in the mouse and the rat as illustrated by our experiments with flurbiprofen, a compound with very high plasma protein binding.