Screening of Species-specific DNA Probes for Identification of Fallopia multiflora

To screen species-specific DNA probes for identification of Fallopia muhiflora, the genomic DNA （gDNA） suppression subtraction hybridization （SSH） between F. muhiflora and F. muhiflora var. ciliinervis was firstly performed. The obtained differential gDNA fragments by SSH were then hybridized with gDNA ar- rays consisting of multiple whole genomes of several species （adulterants and/or closely related species of F. muhiflora） and four differential fragments were screened uniquely representing F. muhiflora, which could be used as F. muhiflora species-specific probes. The screened DNA probes were tested by reverse dot blot hybridization and the results demonstrated that these probes could be used reliably to identify F, muhiflora. The species-specific DNA probes obtained in this study exhibited broad application prospects in the preparation of gene chips for identifying Chinese traditional medicines and the authentication of germplasm re- sources and crude drugs of F. muhiflora.

Evaluation of a sulfonated DNA probe (sulfoprobe) for diagnosis of falciparum malaria

In this study,the DNA probe pPF14 was nonradioactively labelled by sulfo-modifica-tion,and used in a dot blot hybridization assay for detection of P.falciparum.The resultsshowed that the sulfoprobe successfully detected 25pg purified DNA and 0.001% parasitemia ofcultured P.falciparum,and did not react with human leukocyte DNA.In the study of 179clinical blood samples of malaria patients from Yunnan province,the DNA probe results corre-lated well with blood smear ones.Of 107 P.falciparum samples determined by microscope ex-amination,99 were positive by sulfoprobe (92.5%);Of 72 P.vivax samples,1 was crosslyreacted;no cross reactions were found with any of 48 normal blood samples.It is indicated thatsulfoprobe may be useful in specific diagnosis and epidemiological investigation of P.falciparuminfection.

QUALITY CONTROL FOR CHINESE HERBAL DRUGS USING DNA PROBE TECHNOLOGY

ＩＮＴＲＯＤＵＣＴＩＯＮＴｈｅｔｙｐｅａｎｄｓｐｅｃｔｒｕｍｏｆｄｉｓｅａｓｅｓａｒｅｃｈａｎｇｉｎｇｓｉｇｎｉｆｉｃａｎｔｌｙａｓｔｈｅｓｏｃｉｅｔｙａｇｉｎｇｔｏｄａｙ .Ｔｈｅａｕｔｏｉｍｍｕｎｅｄｉｓｅａｓｅｓｓｕｃｈａｓｓｅｎｉｌｅｄｅｍｅｎｔｉａ ,ＡＩＤＳ ,ａｓｗｅｌｌａｓｃａｒｄｉｏ ｃｅｒｅｂｒａｌｖａｓｃｕｌａｒｄｉｓ ｅａｓｅｓｓｕｃｈａｓｈｙｐｅｒｔｅｎｓｉｏｎ ,ａｒｒｈｙｔｈｍｉａ ,ｍｙｏｃａｒｄｉａｃｉｎｆｒａｃｔｉｏｎｅａｒｅｂｅｃｏｍｉｎｇａｎｉｎｔｒａｃｔａｂｌｅａｎｄｇｌｏｂｕｌａｒｐｒｏｂｌｅｍ .Ｒｅｃｅｎｔｌｙ ,ｐｅｏｐｌｅａｒｅｖｅｒｙｍｕｃｈｃｏｎｃｅｒｎｅｄｗｉｔｈｓｉｄｅｅｆｆｅｃｔｓｏｆｓｙｎｔｈｅｔｉｃｐｈａｒ ｍａｃｅｕｔｉｃａｌｓａｎｄａｒｅａｎｘｉｏｕｓｔｏｒｅｔｕｒｎｔｏｔｈｅｕｓｅｏｆｎａｔｕｒａｌｍｅｄｉｃｉｎｅ .Ｂａｃｋｔｏｎａｔｕｒｅ ,ｔｈｅｎｅｅｄｆｏｒＣｈｉｎｅｓｅｈｅｒｂａｌｄｒｕｇｓｉｓｉｎｃｒｅａｓｉｎｇｇｒａｄｕａｌｌｙｆｏｒｐｒｅｖｅｎｔｉｏｎａｎｄｔｈｅｒａｐｙｏｆｄｉｓｅａｓｅｓｉｎｔｈｅｗｏｒｌｄ .Ａｒｅｃ...

一种新型Al^(3+)荧光探针的合成及与DNA的相互作用

以2-羟基-1-萘甲醛、邻氨基苯甲醛为原料,设计合成了一种新型Schiff碱Al^(3+)荧光探针S,并通过1HNMR、^(13)CNMR、ESI-MS、IR对探针S进行结构表征。光谱实验结果表明,探针S在DMSO∶H_(2)O=4∶1(V/V)体系中,对Al^(3+)有良好的荧光和裸眼识别。Al^(3+)浓度在2.0~50μmol/L范围内,体系荧光强度呈现出良好的线性关系,检出限为0.033μmol/L,探针S与Al^(3+)形成1∶1的配合物。通过荧光光谱研究了荧光探针S-Al^(3+)配合物与DNA的相互作用,实验结果表明,探针S-Al^(3+)配合物对DNA的猝灭过程为动态猝灭,同时对不同温度下相互作用的结合点常数、结合位点数及热力学参数进行了计算,推断二者作用力的类型为疏水作用,作用方式为嵌插作用。

Evaluation of ECL Labeled DNA Probe for the Diagnosis of Falciparum Malaria

A commercially available non—radioactive DNA labelling kit "enhanced chemiluminescence"(ECL) was evaluated for the diagnosis of falciparum malaria. The results showed that ECL labeled probe successfully detected as little as 25 pg. Purified DNA of 0.001％ parasitemia of cultured Plasmodium falciparum and did not react with human

New DNA probe for studying cell membrane interactions

Subject Code:B05 With the support by the National Natural Science Foundation of China and the US National Institutes of Health,a team led by Dr.Tan Weihong(谭蔚泓)at Hunan University and the University of Florida reported a new DNA probe for studying cell membrane interactions,which was recently published

Use of (Dig)-DNA Probe for the Epidemiological Survey of Plasmodium falciparum Malaria

The Plasmodtum falctparum DNA fragment was isolated from a cloned recombinant plasmid pPF14. labelled with Digoxigenin (Dig) and used as a probe (pPF14-F-Dig)to detect 274 blood samples from the eqdemic area population in Hainan Province by dot hybridization.The results showed that out of 274 blood specimens one was positive when using microscopic examination and the positivity rate was 0.36 percent. Fifteen samples showed to be positive (including one positive specimen examined by microscopy) when this probe was applied to detect the 274 samples and its positivity rate was 5.47 percent.The positive coincidence rate between pPF14-F-Dig and microscopic examination was 1/1 and the negative coincidence rate was 94.87 percent. Since a piece of nitrocellulose membrane with the size of 9 by 12 square centimetres can accommodate blots of 96 samples,this probe is fit for large-scale epidemiological surveys.

异核双金属探针在活细胞细胞核和线粒体中选择性定位DNA

用于实时成像活细胞DNA的细胞核探针是非常罕见的。通过Ru(N^N)3、Ir(C^N)2(N^N)、Re(CO)3Cl(N^N)和Pt(C^N)(N^N)的结合,生成了4种异核双金属配合物:2种不同的功能金属位点被锚定在线性双齿螯和配体(N^N)-(N^N)的尾端,形成不对称哑铃状的双金属分子M1-M2,即Ru-Re、Ru-Pt、Ir-Re和Ir-Pt。Ru-Re和Ru-Pt的红色发射探针具有较大的斯托克斯位移、优异的核膜穿透能力、良好光稳定性的DNA结合能力。此外,Ru-Re和Ru-Pt探针的DNA成像可与专门用于活细胞细胞核DNA染色的Hoechst 33342商用染料相媲美,而Ir-Re和Ir-Pt探针直接靶向线粒体。不同探针的发光特性和选择性胞内定位高度依赖于在异核双金属配合物中的金属物种。

Detection of hepatitis B virus DNA by real-time PCR using TaqMan-MGB probe technology

AIM: To develop a real-time PCR for detecting hepatitis B virus-(HBV) DNA based on TaqMan technology using a new MGB probe.METHODS: Plasmid containing the sequence of X gene (1414-1744 nt) was constructed as HBV-DNA standard for quantitative analysis. A TaqMan-MGB probe between primers for amplification was designed to detect PCR products. The interested sequence contained in the plasmid and in clinical specimens was quantitatively measured.RESULTS: The detection limit of the assay for HBV DNA was 1 genome equivalent per reaction. A linear standard curve was obtained between 100 and 109 DNA copies/reaction (r＞0.990). None of the negative control samples showed false-positive reactions in duplicate. HBV DNA was detected in 100% (50/50) of HBV patients with HbeAg, and in 72.0% (36/50) with HBsAg, HBeAb and HBcAb. The coefficient of variation for both intra- and inter-experimental variability demonstrated high reproducibility and accuracy.CONCLUSION: Real-time PCR based on TaqMan-MGB probe technology is an excellent method for detection of HBV DNA.

Hierarchically spacing DNA probes on bio-based nanocrystal for spatial detection requirements

Sterically spacing and locating functional matters at the nanoscale exert critical effects on their application,especially for the fluorescence probes whose aggregation causes emission quenching.Here we achieved a hierarchical spacing strategy of DNA fluorescence probes for ion detection via locating them separately on rod-like cellulose nanocrystals(CNCs)and further isolating CNCs by pre-grafting long molecular chains.Controlling chemical structure of CNC and location degree could adjust the interspace of DNA probes(with a molecular length of ca.3.6 nm)in a range of 3.5-6.5 nm with a gradient about 0.2 nm.A length up to micrometer scale of the CNC nanorods was necessary to provide DNA probes with well-separated grafting locations and enough freedom,which brought a vast linear detection range from 10 nmol/L to 5 μmol/L of Hg^2+ concentration.The abundant reactive sites on CNC allowed a grafting pre-location of poly(tert-butyl acrylate)(PtBA)to promote the isolation of DNA probes.Controlled radical polymerization was employed to adj ust the length of PtBA molecular chains,which increased the linear sensitivity coefficient of Hg^2+ detection by ca.2.5 times.This hierarchical nanoscale spacing concept based on chemical design can hopefully cond uce to the development of biosensor and medical diagnosis.A hierarchical spacing strategy was applied to separate DNA fluorescent probes on CNCs and detect ion concentration linearly.The first-level spacing was to locate probes uniformly on CNCs,obtaining a wide linear range;and the second-level spacing was to isolate CNCs with polymer,obtaining an increased linear coefficient.

Carbazole tricationic salt:A novel potential two-photon fluorescent DNA probe for nucleic imaging of cells

A new carbazole tricationic salt,4,4'-(1E,1'E)-2,2'-(9-(2-(1-(2-hydroxyethyl)pyridinium-4-yl)ethyl)-9H-carbazole-3,6-diyl) bis(ethane-2,1-diyl) bis(1-(2-hydroxyethyl)pyridinium) iodide (THEPC) was synthesized. Photophysical experiments have shown that THEPC has large two-photon excited fluorescence action cross-sections (33 GM in the presence of DNA),which ranks THEPC as a good biological fluorophore. The results from electronic absorption,circle dichroism and single-/two-photon fluorescence emission spectra suggest that THEPC can strongly bind to DNA,with an intrinsic binding constant of 5.79 × 106 L mol-1. THEPC has better photostability under one-or two-photon excitation conditions. Finally,the staining photos from two-photon fluorescence microscopy (TPM) show that THEPC can exclusively label the nucleus with high contrast and without image distortion. These remarkable properties and optimized imaging ability make THEPC an attractive DNA probe in TPM.

Microarray of DNA probes on carboxylate functional beads surface

The microarray of DNA probes with 5’ -NH2 and 5’ -Tex/3’ -NH2 modified terminus on 10 um carboxylate functional beads surface in the presence of 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) is characterized in the preseni paper. it was found that the microarray capacity of DNA probes on the beads surface depends on the pH of the aqueous solution, the concentra-tion of DNA probe and the total surface area of the beads. On optimal conditions, the minimum distance of 20 mer single-stranded DNA probe microarrayed on beads surface is about 14 nm, while that of 20 mer double-stranded DNA probes is about 27 nm. If the probe length increases from 20 mer to 35 mer, its microarray density decreases correspondingly. Mechanism study shows that the binding mode of DNA probes on the beads surface is nearly parallel to the beads surface.

实时PCR和cycling probe技术检测母血浆游离胎儿DNA筛选重型β地中海贫血胎儿

目的通过检测孕妇外周血中的游离胎儿DNA来筛选重型β-地中海贫血胎儿。方法选择行产前基因诊断的夫妇6对,孕妇孕周23-26周。血液学检查:胎儿的父亲均为β-地中海贫血17M/N型,孕妇本人为携带除17M/N型之外的另一β-地中海贫血突变类型。针对CD17(A→T)无义突变,设计β-珠蛋白肽链上该等位基因的一对特异性引物和通过cycling probe法分别设计检测正常基因序列和基因突变位点的两条荧光探针,分别用FAM和HEX荧光标记。结合RT-PCR技术检测孕妇外周血中游离胎儿DNA,诊断胎儿是否遗传了其父亲的β地中海贫血17M/N碱基突变位点。同时与脐血血液学检查所诊断的胎儿地贫基因型对照。结果提取的6例孕妇血浆DNA模板中有3例同时显示FAM和HEX荧光信号值阳性结果,即这3例孕妇的胎儿遗传了父亲β-珠蛋白肽链上CD17位点的突变碱基(A→T)。另外3例孕妇血浆DNA模板的FAM信号值阳性,HEX信号值阴性,即所孕胎儿没有遗传父亲的CD17位点的突变碱基。结论利用RT-PCR和cycling probe技术检测孕妇外周血中的游离胎儿DNA可用来筛选患重型地中海贫血的胎儿。

TB AccuProbe、INNO-LIPA^(TM)RIF.TB探针和rpoβDNA测序三种方法鉴定结核分枝杆菌的研究

目的用 3种先进的技术 -TBAccuProbeDNA杂交法、INNO LIPATMRIF TB(LiPA)探针试验和rpoβDNA基因序列测定的前瞻性研究 ,以鉴定结核分枝杆菌复合物 (MTBC)和非结核分枝杆菌(NTM)及其成本效益。方法在BactecMGIT 96 0培养系统中将获得的 10 5例阳性培养物筛选出 70例MTBC和 35例NTM ,用 3种技术鉴定并检测其rpoβ基因序列突变位点。 结果TBAccuProbe杂交阳性率是 95 7% (6 7/70 ) ,LiPADNA探针阳性率是 98 6 % (6 9/70 ) ,rpoβDNA序列测定阳性率是 97 1% (6 8/70 )。 3种方法两两比较差异在统计学上均无显著意义 (P >0 0 5 )。每份阳性培养物花费时间 :TBAc cuProbe杂交法和LiPADNA探针法在 2 4小时内完成 ,全部完成需 6～ 37天 ,平均为 15天 ;rpoβ基因序列测定需 4～ 5天完成 ,全部完成需 8～ 4 0天 ,平均 17天。比传统的 4～ 8周鉴定时间减少了很多。每份标本所需费用 3种方法各为 12 0、194和 96元。在 35例NTM中 ,3种方法均为MTBC阴性 ,同时rpoβDNA序列测定鉴定了NTM的种类。结论TBAccuProbe杂交和LiPADNA探针操作简便 ,所需时间短 ,只能鉴定MTBC和NTM。但LiPADNA探针还能鉴定rpoβ基因位点突变 ;rpoβ基因序列测定所需时间稍长 ,要有特定仪器和专门人员 ,但所需费用较低 ,除能鉴定MTBC外 。

DNA Fingerprints of Animals Generated With Total Genomic DNA Probes

DNA fingerprinting, developed in 1985, has now become a powerful tool forindividual identification and paternity testing in forensic casework and medicine. It hasalso found wide application to population genetics, evolution, systematics and geneticlinkage analysis in animals, plants and even microorganisms. Conventional DNAfingerprinting requires specific multilocus minisatellite probes-or microsatellite

用DNA探针检测我国栽培的无核葡萄及辅助育种初探

用葡萄无核基因的特异探针 18bp (5’CCAGTTCGCCCGTAAATG3’)检测了我国栽培的 2 1个无核葡萄品种和 9个有核对照品种的无核性 ,证实了该探针可以对葡萄无核基因进行准确的检测和鉴定 ;通过该探针对有核×无核 (红地球×红光无核 )杂交后代无核性状的鉴定 ,并对照大田结果 ,证明了 18bp检测葡萄无核基因探针具有预测葡萄无核性状的作用。

荧光光谱法研究抗癌药物与DNA的相互作用

荧光探针技术可以用来测定药物核酸相互作用的强度。通过药物与核酸相互作用,使DNA与探针键合的程度减小,反映在探针荧光光谱的改变,从而可以了解药物和核酸的作用机理。相互作用常数D为DNA+探针中加入药物后探针的荧光强度相对于DNA+探针荧光强度减去纯探针荧光强度的下降百分数。文章利用盐酸小檗碱(Berberine)作为探针,测定几种抗癌药物加入DNA与荧光探针(盐酸小檗碱)混合溶液的荧光谱,探讨它们与DNA的相互作用。并根据相同浓度的不同药物与核酸相互作用的常数D确定作用强弱。

二茂铁标记DNA电化学探针的研制及性质研究

以乙基 -( 3-二甲基丙基 )碳化二亚胺盐酸盐 ( EDC)为偶联活化剂 ,利用缩合反应分别将电化学活性物质氨基二茂铁 ( Aminoferrocene,AFC)和醛基二茂铁 ( Ferrocenecarboxaldehyde,FCA)成功地标记在变性小牛胸腺 DNA片段上 ,制备成二茂铁标记 DNA探针 .分别采用电化学、紫外、红外光谱等方法研究了二茂铁标记 DNA探针的性质 ,计算了二茂铁的标记效率 ,变性小牛胸腺 DNA的反应效率以及二茂铁化合物与变性小牛胸腺 DNA的反应比率 .实验表明 ,氨基二茂铁和醛基二茂铁与 DNA上的磷酸基是以 1∶ 1比率进行的反应 ,该标记反应不影响 DNA链碱基的紫外吸收 ,二茂铁标记 DNA探针在石墨电极上有良好的电化学响应 .将制备好的二茂铁标记 DNA电化学探针置于冰箱中冷冻 (温度低于 -1 5℃ )保存 3个月后用于测定 ,峰电流仅下降 1 .

基于硫化镉纳米团簇标记DNA电化学传感的研究

合成了表面具有自由羧基的硫化镉 (CdS)纳米团簇 ,以乙基 (3 二甲基丙基 )碳二亚胺盐酸盐 (EDAC)为偶联活化剂 ,将其标记于人工合成的 5′端氨基修饰的寡聚核苷酸 (ODN)片段上 ,制备成CdS纳米团簇标记DNA探针 ,该寡聚核苷酸片段与大肠杆菌肠毒素基因相关 .在一定的条件下 ,使其与固定在玻碳电极表面的待测DNA序列进行杂交反应 ,利用阳极溶出示差脉冲伏安法 (ASDPV)间接测定Cd(II)的量 ,实现对互补、非互补DNA片段的识别和电化学检测 。