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.

Construction of Agropyrum intermedium 2Ai-2 Chromosome DNA Library and Cloning of Species-Specific DNA Sequences

The univalent from the meiosis-metaphase spreads of F1 (Z2× wheat variety Wan7107) wasidentified to be Agropyrum intermedium 2Ai-2 chromosome by GISH. The 2Ai-2 chromosomes weremicroisolated and collected. After two rounds of PCR amplification, the PCR products wereranged from 150-3 000 bp,with predominant fragments at about 200-2 000 bp. Using Ag.intermedium genomic DNA as a probe, Southern blotting analysis confirmed the products originatedfrom Ag. intermedium genome. The products were purified, ligated to pUC18 and then transformedinto competence E.coli DH5αto produce a 2Ai-2 chromosome DNA library. The microcloningexperiments produced approximately 5 ×105 clones, the size range of the cloned inserts was 200-1 500 bp, with an average of 580 bp. Using Ag.intermedium genomic DNA as a probe, dot blottingresults showed that 56% clones are unique/low copy sequences, 44% are repetitive sequences inthe library. Four Ag. intermedium clones were screened from the library by RFLP, and threeclones(Mag065, Mag088, Mag139)belong to low/single sequences, one clone(Mag104)was repetitivesequence, and GISH results indicated that Mag104 was Ag.intermedium species-specific repetitiveDNA sequence.

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。

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。

Development of oligonucleotide probes for FISH karyotyping in Haynaldia villosa,a wild relative of common wheat

Haynaldia villosa is a wild relative of wheat and a valuable gene resource for wheat improvement.Owing to the limited number of probes available for fluorescence in situ hybridization(FISH),the resolution at which the karyotype of H.villosa can be characterized is poor,hampering accurate characterization of small segmental alien introgressions.We designed ten oligonucleotide probes using tandem repeats in DNA sequences derived from the short arm of H.villosa chromosome 6 V(6 VS).FISH with seven of them resulted in clear signals on H.villosa chromosomes.Using these,we constructed FISH karyotypes for H.villosa using oligo-6 VS-1 and oligo-6 VS-35 oligonucleotides and characterized the distribution of the two probes in five different H.villosa accessions.The new FISH probes can efficiently characterize H.villosa introgressions into wheat.

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.

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。

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.

Detection of HBV and HCV Coinfection by TEM with Au Nanoparticle Gene Probes

Gold（Au） nanoparticle HBV DNA or HCV cDNA gene probes were prepared and were used to detect HBV DNA and HCV RNA extracted from positive serum of patients with HBV and HCV coinfection directly by transmission electron microscopy （TEM）. PCR identifying HBV and HCV in serum of patients with HBV and HCV coinfection was established. Alkanethiol-modified oligonucleotide was bound with self-made Au nanoparticles to form nanoparticle HBV DNA or HCV cDNA gene probes through covalent binding of Au-S. HBV DNA and HCV RNA extracted from positive serum of patients with HBV and HCV coinfection was added to the detection system composed of nanoparticle HBV DNA and（or） HCV cDNA gene probes, The results showed that HBV DNA and HCV RNA could be specifically amplified by PCR. The zones of DNA amplification appeared in 431 bp and 323 bp respectively. When HBV DNA and HCV RNA extracted from positive serum of patients with HBV and HCV coinfection were added to the detection system, TEM displayed the nanoparticles self-assembled into large network aggregates. It was concluded that the detection of HBV and HCV coinfection by TEM was convenient and efficient with high specificity and sensitivity.

A Rapid Identification Method of Bactrocera cilifera (Hendel) with Species-Specific Primers(SS-COI)

[Objective]The paper was to establish a rapid identification method of Bactrocera cilifera(Hendel)with species-specific primers(SS-COI).[Method]Using B.cilifera(Hendel)as the positive control,and 19 species of fruit flies such as B.diaphora(Coquillett)and B.dorsalis(Hendel)as the negative controls,a pair of species-specific primers,YF290 and YR511,were designed and screened for accurate identification of B.cilifera,based on mitochondrial DNA COI sequence.[Result]The PCR products were amplified and detected by electrophoresis.Only a clear and single band was observed at about 222 bp in the positive control,while no bands were found in the other negative controls.[Conclusion]The established rapid identification method with species-specific primers(SS-COI)is of great practical significance for rapid identification of fruit flies intercepted from import and export fruits and vegetables at ports,and for rapid clearance and early warning of import fruits and vegetables at ports.

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.

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 。

QUALITY CONTROL FOR CHINESE HERBAL DRUGS USING DNA PROBE TECHNOLOGY

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

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。

虫草素与DNA作用的光谱研究

利用紫外光谱及以溴化乙锭 (EthidiumBromide ,EB)为荧光探针 ,研究了虫草素与小牛胸腺DNA的作用机理。讨论了不同浓度虫草素与DNA作用的紫外光谱 ,荧光光谱及磷酸盐对荧光的猝灭作用。从紫外光谱图上可看出DNA发生了明显的增减色效应并有轻微的红移现象 ,说明虫草素可能以其腺嘌呤碱基嵌入到DNA双螺旋碱基对间。DNA的荧光光谱先是增强然后减弱 ,最终伴随轻微的蓝移进一步表明了虫草素可能插入DNA双螺旋碱基对间。同时磷酸盐的猝灭作用说明虫草素与DNA的磷酸集团也能发生作用。最后通过Scatchard方程作图证明 :虫草素与DNA可能存在两种作用方式即插入方式和与DNA的磷酸基团结合。

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 .

DNA指纹图在法医学鉴定中应用的研究(Ⅱ)——应用‘Myo’小卫星探针进行血斑、精斑、个体组织的个体认定

应用‘Myo’小卫星 DNA 探针,Southern 印迹杂交技术,对血斑、精斑、同一个体不同组织进行 DNA 指纹图分析,均获得清晰的图谱。同一个体的血斑与血液、精斑与精液以及不同的组织其 DNA 指纹图谱完全相同。可以根据斑痕或组织与嫌疑个体的血液或某一组织 DNA 的指纹图谱比对以做出同一认定。50μl 血液量的血斑、5μl 精液量的精斑可以获得清晰易辨的指纹图谱。五年的精斑、两年的血斑亦可做出与同源个体新鲜精液、血液完全一致的 DNA 指纹图谱。对杀人、强奸杀人、碎尸等不同案件的血痕、精斑、不同组织碎块进行了 DNA 指纹图检验,均做出了正确的个体认定。本方法的应用为我国法医物证检验提供了新的分析手段,使个体认定得以实现。