基因芯片技术检测肾综合征出血热病毒核酸的研究

To establish a rapid, sensitive and specific diagnostic assay for Hantavirus with microarray techniques, specific primers and probes were designed according to the conservative and specific DNA sequence of 76-118 strain and R22 strain. The probes were spotted on glass slides to form microarrays.The Cy3-1abled single stranded DNA fragments prepared by dissymmetical PCR were hybridized with the probes on the glass slides. The microarrays were scanned and analyzed with a scanner. The results showed that the DNA microarray could detect the different typed DNA of HTN and SEO with adequate specificity and sensitivity. The developed DNA microarray and techniques might be a very useful method for diagnosis and prevention, and could be widely applied in specific pathogens detection ofinfectious diseases such as hemorrhagic fever with renal syndrome.     

Construction of a DNA library from chromosome 4 of rice （Oryza sativa） by microdissection

A simple method to create a chromosome-specific DNA librqary of rice,including microdissection,amplification,charterization and cloning,is described.Rice chromosome 4 from a metaphase cell has been isolated and amplified by the Linker Adapter PCR (LA-PCR).The PCR products were labeled as probes with DIG-11-dUTP using the random priming method.Southern blot analysis with rice genomic DNA and specific RFLP markers demonstrated that the PCR products were derived from rice chromosome 4.A large library comprising over 100,000 recombinant plasmid microclones from rice chromosome 4 was constructed.Colony hybridization showed that 58% of the clones contained single or low-copy sequences and 42% contained repetitive sequences.The size of inserts generated by PCR ranged from 140bp to 500bp.This method will facilitate cloning of the specific chromosome DNA markers and important genes of rice.     

小麦及其近缘种中基因组特异性DNA重复序列的研究进展

本文对小麦族植物中基因组特异性DNA重复序列的分类、基本特征、分离和鉴定方法、在小麦遗传改良中的应用以及未来研究的发展趋势进行了简述。综合已有的研究结果可以看出基因组特异性DNA重复序列是小麦族植物基因组特异性形成的重要构成部分。对基因组特异性DNA重复序列的研究是认识小麦族植物基因组的有效途径之一,基因组特异性DNA重复序列的应用将进一步促进小麦族植物分子细胞遗传学和普通小麦遗传改良研究的进展。 Advances in Studies of Genome-Specific Repetitive DNA Sequences in Wheat and Related Species BAI Jian-rong1,2,JIA Xu1,WANG Dao-wen1 1.The State Key Laboratory of Plant Cell and Chromosome Engineering,Institute of Genetics and Developmental Biology,The Chinese Academy of Sciences,Beijing 100101,China; 2.Crop Genetics Institute,Shanxi Academy of Agricultural Sciences,Taiyuan 030031,China Abstract:In this paper we review recent advances in studies of several aspects of genome specific repetitive DNA sequences in wheat and related species.The available results demonstrate that genome specific repetitive DNA sequences are important components of genome specificity in wheat and related species.Research on genome specific repetitive DNA sequences is essential to the elucidation of genome function.The application of genome specific repetitive DNA sequences will aid molecular cytogenetic studies in wheat and related species and contributes to genetic improvement of common wheat. Key words：wheat;genome specific repetitive DNA sequence;chromosome     

枫泾猪、香猪和长白猪的DNA指纹图分析

本文用M13和3个人源小卫星探针(33.6、 33.15、α-珠蛋白-3’HVR)获得了枫泾猪、香猪及长白猪的DNA指纹图,分析了各品种内和品种间的遗传变异性。结果表明,各品种的平均DNA指纹图相似系数在0.440-0.532的范围内,极显著地高于品种间的相似系数(0.223-0.307)。各品种的DNA指纹图还存在着一些品种特异性图带,这些图带在以后的基因定位中值得重视。 Abstract:This paper demonstrated DNA fingerprints of Fengjing,Xiang and Large White breeds of swine generated by M13 and three human minisatellite probes(33.6,33.15 andα-globin-3’HVR),and determined the intrabreed and interbreed variabilities of swine DNA fingerprints.It was shown that the mean band sharing probability within breeds was 0.440-0.532,significantly higher than that between breeds(0.223-0.307).Some breed-specific bands were noted in each breed,which should draw our attention in the course of gene mapping.     

A Strategy to Optimize the Oligo-Probes for Microarray-based Detection of Viruses

DNA microarrays have been acknowledged to represent a promising approach for the detection of viral pathogens. However, the probes designed for current arrays could cover only part of the given viral variants, that could result in false-negative or ambiguous data. If all the variants are to be covered, the requirement for more probes would render much higher spot density and thus higher cost of the arrays. Here we have developed a new strategy for oligonucleotide probe design. Using type I human immunodeficiency virus (HIV-1) tat gene as an example, we designed the array probes and validated the optimized parameters in silico. Results show that the oligo number is significantly reduced comparing with the existing methods, while specificity and hybridization efficiency remain intact. The adoption of this method in reducing the oligo numbers could increase the detection capacity for DNA microarrays, and would significantly lower the manufacturing cost for making array chips.     

PCR方法制备地高辛标记DNA探针检测中国对虾非包涵体型杆状病毒

A pair of primers created from information of PmNOBⅢ genome DNA Sal I fragment produced a 355bp band by using Penaeus chinensis non occluded baculovirus (PcNOBV),the WSBV isolate from P.hinensis in mainland China,as the DNA template.The specific PCR product was cloned,sequenced and labeled with digoxigenin (DIG)DNA labeling kit(Boehringer Mannheim).The DIG labeled fragment was tested by dot blot hybridization for sensitivity and specificity with purified PcNOBV nucleocapsid,PcNOBV infected shrimp tissues and healthy shrimp tissues.The detection limit of the DNA probe is 6.8pg of purified PcNOBV DNA.No hybridization signals were observed using DNA from healthy shrimp as template.Healthy P.chinensis,artificially infected P.chinensis and pond reared adult P.chinensis were screened for PcNOBV infection by both PCR and the hybridization assay.The results showed a good relationship between PCR and the hybridization assay.These findings demonstrate that the DIG labeled probe can be used as a sensitive,specific and cost effective reagent for detection of PcNOBV.     

两种泥鳅中PdSox8和PdSox9的RFLP分析

以α-32P-dCTP标记的大鳞副泥鳅的PdSox8和PdSox9基因片段为探针,研究了二者在泥鳅和大鳞副泥鳅中的限制性片段长度多态性。结果表明两种探针在6尾大鳞副泥鳅（3♀3 ♂）和6尾泥鳅（3♀3 ♂）的经EcoRI和HindIII完全酶切的基因组DNA中检测到了较丰富的阳性杂交带,并讨论了其产生的可能原因。未发现性特异杂交带。 Abstract：With the α-32 P-dCTP-labeled PdSox8 and PdSox9 fragments of Paramisgurnus dabryanus as probes, the Restricted Fragment Length Polymorphism(RFLP) were analyzed in two species of loaches P. dabryanus and Misgurnus anguillicaudatus. The results showed that the plentiful positive bands were observed in the genomic DNA of 6 individuals(3♀3♂) of P. dabryanus and 6 individuals(3♀3♂) M.anguillicaudatus, which were digested thoroughly by EcoRI and HindIII. The possible reasons were discussed. No sex-specific band was observed.     

用FISH技术对人、恒河猴、食蟹猴染色体的研究

用人类5号、 9号、13号、15号、17号、20号整条染色体探针分别对人、恒河猴和食蟹猴的中期细胞进行荧光原位杂交,结果表明:人的5号、13号、17号探针分别杂交到恒河猴的5号、16号、17号染色体上;9号探针杂交到恒河猴14号染色体的长臂及部分短臂上; 15号探针杂交到恒河猴7号染色体短臂及部分长臂上;20号探针杂交到恒河猴的13号染色体长臂上。食蟹猴的杂交结果与恒河猴完全一致。结合G带带型分析,对人与猕猴的染色体同源性及其进化进行了讨论。 Abstract:Fluorescent in situ hybridizaiton(FISH)was used on the metaphase of Macaca mulatta and Macaca fasicularis with human chromosome specific DNA libraries for chromosome 5、9、13、15、17 and 20.In Macaca mulatta,the result showed that chromosome 5、16 and 17 was entirely painted by human chromosome 5、13 and 17 specific libraries respectively.The long arm and the partial short arm of chromosome 14 and the short arm and the partial long arm of chromosome 7 were painted by human chromosome 9 and 15 specific libraries respectively.And the long arm of chromosome 13 was painted by human chromosome 20 library.The result was the same in Macaca fasicularis.Combinded with the comparative analysis of G-banding,the evolutional relationship of these chromosomes between human and macaques was discussed.     

Identification and analysis of genes present in Leptospira interrogans serovar lai but absent in L. biflexa serovar monvalerio

Genes present in virulent bacterial strains but absent in avirulent close relatives can be of great biologic and clinical interest. This project aimed to identify strain specific DNA sequences of Leptospira interrogens serovar lai, which is absent in the saprophytic L. biflexa serovar monvalerio, via suppression subtractive hybridization with the former as the tester while the latter as the driver. The mixture of PCR amplified DNA fragments from two subtractive hybridization experiments were cloned into pMD 18-T vector and the positive clones were identified by dot blotting against the chromosome DNA of the two strains individually. After DNA sequencing and analysis, the distribution of these genomic fragment sequences in a panel of pathogenic and nonpathogenic leptospires was investigated employing dot blot analysis. Among the 188 positive clones randomly chosen, 24 contained the tester strain specific genomic regions, of which, 5 were non-coding fragments while the others contained 23 distinct protein coding sequences. Besides 9 genes encoding functional proteins, 12 genes encode unknown proteins and the rest two genes encode proteins with recognizable domain structures, one for a putative leucine-rich repeats (LRR) family protein while the other as an outer-membrane protein. Our experiment results indicated that suppression subtractive hybridization is effective for screening specific DNA sequences between two leptospiral strains, and some of these sequences might be responsible for virulence determination. Further analysis of these DNA sequences will provide important information on the pathogenesis of Leptospira.     

两种DNA探针杂交检测结核分支杆菌方法的研究

为改进结核杆菌DNA探针的特异性与实用性,研制了以生物素标记的两种对结核分支杆菌特异的DNA探针:一个5’端标记的20bp的寡核苷酸探针和一个采用PCR方法合成的188bp长链探针。两种探针分别与结核分支杆菌的全染色体DNA,以及基因组上IS6110序列的一段317bp的PCR扩增产物进行斑点杂交,以碱性磷酸酶(AP)催化的染色反应检测,测试了两个探针的敏感性和特异性。系统地比较研究了两种探针杂交检测条件:探针的浓度选择,杂交温度与洗膜温度的选择,以及杂交与洗膜温度对检测的敏感性与特异性的影响。寡核苷酸探针和188bp探针杂交检测纯化结核分支杆菌基因组DNA的敏感性分别为100ng与6ng,杂交检测PCR产物的敏感性分别是400pg与50pg。两探针的最佳杂交浓度均为40～160ng/ml,最佳杂交温度分别是42℃与68℃,最佳洗膜温度分别是60℃与60～68℃之间。两种探针均仅与结核分支杆菌及BCG有杂交信号,而与其它受试分支杆菌及非分支杆菌杂交结果都呈阴性。它们的特异性都很强,但188bp探针的敏感性约是寡核苷酸探针的7～16倍,而且188bp探针检测本底较低,是检测结核分支杆菌的较佳选择     

Use of random amplified polymorphic DNA (RAPD) for generating specific DNA probes for microorganisms

We report the rapid generation of DNA probes for several Azospirillum strains. This method does not require any knowledge of the genetics and/or the molecular biology of the organism (genome) to be investigated. The procedure is based on the generation of random amplified polymorphic DNA (RAPD) fingerprints using primers with an embedded restriction site. The amplification product(s) peculiar to one strain or common to two or more strains can be purified, cloned, sequenced and used as molecular probes in hybridization experiments for the detection and identification of microorganisms. We have tested this methodology in the nitrogen-fixing bacterium Azospirillum by amplyfing the total DNA extracted from several Azospirillum strains. We have used amplification bands with different specificity as molecular probes in hybridization experiments performed on amplified DNA. Results obtained have demonstrated the usefulness of this methodology for Azospirillum. Its use in microbial ecology studies as a general strategy to generate specific DNA probes is also discussed.     

生物素标记寡核苷酸探针探测扩增的病理性突变珠蛋白基因

用末端转移酶催化生物素核苷酸底物(Biotin-ll-dUTP)共价连接在合成的寡核苷酸3’羟基末端,从而合成了两种寡核苷酸探针(β~T_(41-42)及β~A_(41-42))。用它们分别与克隆化扩增的正常和突变的β—珠蛋白基因片段杂变。结果表明该探针都具有与~(32)P探针相似的特异性,其杂交的灵敏度为2—3pg(特异序列)。进而将探测HbS基因的正常和异常两种寡核苷酸19聚体(β~A_6和β~S_6)用~(32)P和生物素分别标记;将HbS杂合子病人的白细胞DNA经聚合酶链反应(PCR)法扩增,并以含正常β—珠蛋白基因的DNA片段作对照,与两种探针分别进行斑点杂交。所得结果完全一致;Hbs杂合子DNA对正常和异常探针都显出杂交信号,而正常DNA只与β~A探针显杂交信号。     

细小病毒B19 Oligo探针设计

利用BLAST软件对细小病毒B19的序列进行序列比对,获得特异序列;利用生物学软件Oligo6.40设计特异性高、Tm值接近、长度均一的Oligo探针。结果获得了13条70bp的Oligo探针,用于芯片打印及细小病毒B19的检测。表明利用BLAST系统和生物学软件Oligo6.40设计细小病毒B19诊断芯片的探针是一种简便而有效的方法。     

蛋白质检测新技术——邻位连接技术及初步应用

邻位连接技术（proximity ligation assay,PLA）,是新研发的一项高灵敏度的蛋白质体外分析技术。该方法利用一对邻位探针（proximity probes）对靶分子进行双识别,通过连接反应产生可扩增的检测信号,以实时 PCR进行放大和检测,将对蛋白质的检测转变成为对DNA的检测,实现痕量蛋白的分析,具有极高的检测灵敏度和特异性。综述了邻位连接技术的原理、研究进展以及该技术在蛋白质分析及疾病诊断领域的初步应用。     

制备炭疽芽胞杆菌检测基因芯片的初步研究

建立制备炭疽芽胞杆菌检测基因芯片的技术,并探讨研制检测炭疽芽胞杆菌基因芯片的方法。酶切炭疽芽胞杆菌的毒素质粒和荚膜质粒,通过建立质粒DNA文库的方法获取探针,并打印在经过氨基化修饰的玻片上,制成用于炭疽芽胞杆菌检测的基因芯片。收集了290个阳性克隆探针,制备了检测炭疽芽胞杆菌的基因芯片。提取炭疽芽胞杆菌质粒DNA与基因芯片杂交,经ScanArray Lite芯片阅读仪扫描得到初步的杂交荧光图像。通过分析探针的杂交信号初步筛选出273个基因片段作为芯片下一步研究的探针。     

Broad range DNA probes for detecting and amplifying eubacterial nucleic acids

In this report we describe and characterize two oligomer probes that are broadly homologous to conserved eubacterial 16S ribosomal RNA (rRNA) sequences not present in human 18 rRNA or human mitochondrial 12S rRNA. One or both of the probes can detect all of 23 phylogenetically diverse eubacterial nucleic acids against which they were tested by dot blot hybridization. A sensitivity of about 1 bacterium per 10 eukaryotic cells was achieved. By using these oligomer sequences or their complements as primers in the polymerase chain reaction (PCR), the equivalent of 1 pg of E. coli DNA was detected in the presence of a large excess of eukaryotic DNA. Information useful for partial phylogenetic classification of detected organisms may be obtained by direct sequence analysis of the amplified DNA and comparison with known sequences or catalogs. Such broadly homologous probes offer advantages over more narrowly specific probes for detecting organisms whose identity is unknown. They could thus be employed for recognizing infection by organisms that cannot be cultured as may occur, for example, in tissue culture or in plant or animal diseases of unknown cause, provided the probes fail to hybridize with host nucleic acids.     

Isolation of a specific DNA fragment and development of a PCR-based method for the detection of Mycobacterium genavense

The rise of Mycobacterium genavense infections is making identification ever more important for diagnosis and treatment. Moreover, isolation and identification of M. genavense are made difficult by the lack of growth on solid media and by its low generation rate in BACTEC liquid media. Thus, amplification by PCR or similar techniques represents the only possibility of detecting and identifying M. genavense from tissue samples. In order to set up a simple and species-specific method based on the use of PCR and non-radioactive hybridization technique, we decided to search for and clone a specific DNA fragment of this bacterial species. In the present study, a 1734-bp fragment was isolated. This fragment was found to be highly specific for M. genavense strains. A species-specific pair of primers (MG22 and MG23) and two oligonucleotide probes (MG18 and MG19) were selected. They were successfully used to amplify and detect a 155-bp DNA fragment from the 13 available strains of M. genavense which were isolated from clinical specimens or from birds. Conversely, the primers and probes did not hybridize with DNA from any of the 20 other mycobacterial species tested. It is worth noting that the chosen primers and probes did not hybridize with DNA of M. simiae, although it is closely related to M. genavense. The present PCR technique uses species-specific primers for M. genavense. Followed by a non-radioactive hybridization technique on microplates it is able to distinguish M. genavense from other mycobacteria in one step, without sequencing or restriction analysis. On the basis of the Southern blot hybridization, PCR and sandwich hybridization results, we concluded that the isolated 1.7-kb sequence was specific for the M. genavense chromosome. The method developed here for M. genavense identification uses a simple methodology and commonly available reagents. Furthermore it can be easily automated.     

DIG标记DNA探针检测noggin在爪蟾胚胎中的表达

整体原位杂交（Whole-mount in situ hybridization)用于基因表达定位和表达分布模式的研究,已经成为一种非常重要的手段。PCR方法进行探针标记,可以获得特异性高,片断大小可变的探针。采用DIG标记,检测已知基因noggin在爪蟾胚胎时空分布,所得结果与文献报道一致,表明PCR方法获得的DNA探针在一定的条件下可以用于爪蟾胚胎整体原位杂交。     

A new method for rapid screening of bacterial species- or subspecies-specific DNA probes

A simple assay for the rapid screening of bacterial species- or subspecies-specific DNA probes for the random cloning method is presented, involving the use of genomic DNAs as probes and recombinant plasmid DNAs containing genomic DNA digested with HindIII as targets. The optimal amount of target DNAs and the concentration of digoxigenin-labeled genomic DNA probes were 20 ng and 100 ng ml(-1) (or 10 ng and 200 ng ml(-1)), respectively. The method was applied to the development of Fusobacterium nucleatum subspecies-specific probes. Our results showed that four out of 96 probes were F. nucleatum subspecies-specific, which was confirmed by Southern blot analysis. Our results indicate that the new method can be used for the rapid screening of species- or subspecies-specific probes.