酶标HBV DNA探针的研制与应用

本实验采用一种非放射性物质——碱性磷酸酶标记乙肝病毒HBV DNA制备分子探针。碱性磷酸酶在苯醌作用下与单链DNA联结,形成DNA和酶的共价复合物,即酶标探针。此探针通过分子杂交与待测DNA结合,与酶的底物作用显色,几小时内可观察结果,其最低检测量约为10pg。用此探针检测乙肝病人血清中的HBV DNA,与~(32)P标记的探针比较,酶标探针可检测出~(32)P标记探针检出率的95.7％。结果表明,所合成的酶标探针具有准确、简便、快速、安全而经济的优点,具有应用前景。     

水稻叶绿体DNA的提取和纯化

以籼稻品种珍讪97B为材料,采用溶液捣碎和不连续蔗糖梯度离心的方法提取了籼稻的叶绿体DNA,DNA经限制性内切酶酶解和琼脂糖胶电泳可以得到清晰的条带,来自蚕豆的核酮糖—1,5—二磷酸羧化氧合酶大亚基基因探针和23SrRNA基因探针可以与酶切条带杂交,由此确定了含这二种基因的BamHI酶切片段。     

水稻线粒体DNA酶切带型研究

水稻IR36线粒体DNA经6种限制酶酶切,用脉冲电泳和长距离琼脂糖凝胶电泳分离酶切片段,获得高分辨率的清晰带型。每组酶切片段加和测得水稻IR36线粒体基因组大小分别为227kb(HindⅢ)、253kb(EcoRⅠ)、253kb(XhoⅠ)、294kb(BamHⅠ)、239kb(SalⅠ)和283kb(xbal)采用9个来自水稻和玉米线粒体基因组的基因探针与酶切条带杂交发现,水稻线粒体基因组含有包括编码基因在内的重复顺序。     

酶标探针在转基因植物检测中的应用

采用碱性磷酸酶标记DNA制备分子探针, 并首次在植物中应用。酶在苯醌作用下与单链DNA联结, 形成DNA和酶的共价复合物即酶标探针。此探针通过分子杂交与待测DNA结合, 再与酶的底物作用显色, 3～6h 内可观察结果。用此探针检测转基因植物中的UidA基因, 点杂交和Southern杂交结果表明, 所合成的酶标探针具有快速、准确、安全而经济的优点。点杂交证明外源UidA基因被成功转化到受体植物中, Southern 杂交对转基因的材料检测的结果证明, 该材料包含多个外源UidA基因拷贝, 初步确定其外源UidA基因拷贝数在5个以上。     

玉米黑粉菌mt DNA的研究 Ⅰ.DNA克隆和基因图谱

本文对玉米黑粉菌mtDNA进行了如下研究:(1)将mt DNA的Bam HⅠ和Pss Ⅰ两套酶切片段分别克隆到pBR322的相应位点上,共克隆到占其基因组总长度89.3%的序列。(2)以植物或真菌来源的线粒体基因作探针,用低严紧度DNA分子杂交法鉴定出了玉米黑粉菌线粒体中的7个基因,并对照另文发表的限制性内切酶图谱,初步得出了这些基因在mt DNA上排列分布的基因图谱。它们排列的次序为:—COB—OⅫ—S—rRNA—OⅩⅢ—L—rRNA—ATPase6—OⅪ—。(3)对已鉴定出的3个含线粒体基因的克隆质粒,用E.coli极大细胞系统作了表达研究,但没见到线粒体基因的编码产物。     

用透析膜从琼脂糖胶中回收DNA片段

分离与回收DNA片段是基因操作的重要环节之一。本文介绍了一个用透析膜从琼脂糖胶中回收 DNA 片段的改进方法。利用本法回收DNA片段洗脱容易、节约时间、回收率在80％ 左右。回收的 DNA片段可用于酶切反应、连接反应和用缺口位移反应制备32p标记DNA探针。     

直接酶标——化学发光分子杂交体系的建立

本文通过调整发光剂、氧化剂、发光增强剂的浓度,并加入一组辅助剂,改进了依赖过氧化物酶化学发光液的配方。并通过比较实验证明:该发光液的检测灵敏度高于其它原有的发光液,同时还发现合成后的酶标复合物可经CM-32离子交换柱阶段洗脱纯化,以提高杂交灵敏度。从而建立了一套直接酶标DNA探针——化学发光自显影分子杂交体系。其狭缝杂交灵敏度可达0.03pg。并利用该体系进行了单拷贝基因分析,Western印迹杂交及检测血清HBV DNA等的应用研究。     

用随机扩增多态性DNA产物做探针产生鸡的DNA指纹图

我们用12个随机扩增多态性DNA(RAPD)引物对来自不同品系的4只鸡进行了RAPD分析,在扩增出的共99条带中,表现多态性的带为38条,占总带数的38％.回收了4个表现个体特异性的RAPD产物,当用鸡的基因组总DNA探针与它们杂交时,其中3个表现阳性,说明RAPD方法扩增出的高变异产物含有重复序列.用含重复序列的个体特异性RAPD产物作探针,与无关个体鸡基因组DNA的HaeⅢ酶切产物进行DNA印迹,获得了变异性较高的DNA指纹图谱.因此,高变异的RAPD产物可以有效地用作DNA指纹探针.     

叶绿体DNA的限制性内切酶谱分析在植物系统分类学中的应用

业已证明,叶绿体DNA为一共轭闭合环状分子。绝大多数植物叶绿体DNA均含有一对反向重复序列。对于所有已检测过的植物,叶绿体基因组所编码的基因数量、基因组成和基因排列是基本一致的。这表明叶绿体DNA具有进化上的保守性。由于其进化上的保守性,叶绿体DNA常为研究层次较高的分类单位间(属间、科间、目间、纲间等)进化关系和进化历史提供较准确的信息。1976年,Vedel等首先建立了叶绿体DNA的内切酶谱分析方法,并将其应用于植物系统分类学中。Vedel等用限制酶ECoRI酶切从豌豆     

辣根过氧化物酶标记DNA探针及光增强检测DNA指纹图在法医应用的研究

本文介绍了用辣根过氧化物酶直接标记DNA探针,光增强检测DNA指纹图的技术,所得图谱清晰易读,分辨率高,灵敏度为0.8μg,接近~(32)P同位素标记的水平。就150名无关个体的DNA指纹图分析表明两个个体图谱完全相同的机率是3.7×10~(-14);方法稳定性测定表明,足以取代同位素标记,用作准确的个人识别和亲子鉴定,为侦破强奸、凶杀等案件提供重要科学依据,该法简便,快速,适用于各实验室,易于推广应用。     

Pi30 DNA probe may be useful for the identification of Prevotella intermedia at the species or strain level

Recently, we introduced a new method for the rapid screening of bacterial species-or subspecies-specific DNA probes, named the "inverted dot blot hybridization screening method." This method has subsequently been then applied to develop species-or strain-specific DNA probes for Prevotella intermedia and Prevotella nigrescens. In a previous study, the inverted dot blot hybridization data showed that a probe, Pi30, was specific for P. intermedia. In this study, the DNA probe Pi30 was evaluated by Southern blot analysis to determine if it could distinguish P. intermedia from P. nigrescens. The data showed that the probe Pi30 reacted with the genomic DNAs from the reference strains and clinical isolates of both P. intermedia and P. nigrescens, but the size of the signal bands was different. In addition, the probe Pi30 reacted with a 1.4 kbp fragment from the genomic DNAs digested with Pst I of the P. intermedia strains but not with any fragments of P. nigrescens strains. The result indicates that the probe Pi30 could be useful for the identification of P. intermedia by restriction fragment length polymorphism (RFLP) at the species or strain level.     

Southern and dot blot analysis of DNA from formalin-fixed,paraffin-embedded tissue samples from colonic carcinomas

Two extraction methods for the isolation of DNA from formalin-fixed, paraffin-embedded tissue samples from colonic carcinomas were compared. The processed DNAs were compared with DNAs from fresh specimens of the same tumors. The two extraction methods gave similar results. Formalin-fixation and paraffinembedding irreversibly denatured DNA and consequently decreased the extraction yield and interfered with the quantitative measurement of DNA. Southern blot and dot blot analysis of processed and native DNA was performed using a c-myc and an actin probe. The results show that for Southern analysis processed DNA can be used but, due to the generation of random breaks, the restriction fragments have to be small. Furthermore, the fixation-induced crosslinking of DNA appears to hamper hybridization. For these reasons processed DNA can be analyzed better by dot blot rather than Southern blot hybridization.     

光生物素标记DNA探针检测感染细胞的BHV—1 DNA

将七株从不同地区、不同牛种或不同部位分离出的牛Ⅰ型疱疹病毒(BHV-1)培养于MDBK细胞中,从感染的细胞中提取病毒并抽提其DNA将克隆的BHV-1LA株DNA的HindⅢ—Ⅰ片段(11.7Kb)用光生物素标记作为探针,检测上述七株感染细胞的BHV-1 DNA,其中有六株(均属IBR临床型)呈阳性反应,另一株(属IPP临床型)呈阴性反应,这一探针将作为一种有效的检疫工具在本病的防制中起重要作用.     

生物素标记贾第虫全基因组DNA探针的制备及其特异性敏感性测定

用生物素标记的贾第虫全基因组ＤＮＡ探针,在斑点杂交试验中显示高度的敏感性和特异性。用它可检出１０ｎｇ贾第虫ＤＡ,１０＾３个贾第虫滋养体或包囊,且不与阴道毛滴虫、溶组织内阿米巴、弓形虫和ＢＡＢＬ／ｃ小鼠肝细胞ＤＮＡ,以及贾第虫患者粪便上清液发生交叉反应。本探针可用于贾第虫病病原体检测和虫株鉴定研究。     

DNA hybridization probe for the Pseudomonas fluorescens group.

Plasmid pHF360 was constructed from cloned rRNA genes (rDNA) of Pseudomonas aeruginosa and used as hybridization probe for the Pseudomonas fluorescens group. The probe was tested by dot and in situ colony hybridizations to chromosomal DNAs from a wide variety of organisms. pHF360 DNA hybridized exclusively to chromosomal DNAs from bacteria representing the P. fluorescens group and separated them clearly from all other bacteria tested in the present study. Determination of the nucleotide sequence of the cloned DNA showed that it is a fragment from a 23S rRNA gene of P. aeruginosa. It was compared with the published 23S RNA sequence from Escherichia coli.     

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.     

DNA hybridization probe for the Pseudomonas fluorescens group

Plasmid pHF360 was constructed from cloned rRNA genes (rDNA) of Pseudomonas aeruginosa and used as hybridization probe for the Pseudomonas fluorescens group. The probe was tested by dot and in situ colony hybridizations to chromosomal DNAs from a wide variety of organisms. pHF360 DNA hybridized exclusively to chromosomal DNAs from bacteria representing the P. fluorescens group and separated them clearly from all other bacteria tested in the present study. Determination of the nucleotide sequence of the cloned DNA showed that it is a fragment from a 23S rRNA gene of P. aeruginosa. It was compared with the published 23S RNA sequence from Escherichia coli.     

Southern and dot blot analysis of DNA from formalin-fixed, paraffin-embedded tissue samples from colonic carcinomas

Two extraction methods for the isolation of DNA from formalin-fixed, paraffin-embedded tissue samples from colonic carcinomas were compared. The processed DNAs were compared with DNAs from fresh specimens of the same tumors. The two extraction methods gave similar results. Formalin-fixation and paraffin-embedding irreversibly denatured DNA and consequently decreased the extraction yield and interfered with the quantitative measurement of DNA. Southern blot and dot blot analysis of processed and native DNA was performed using a c-myc and an actin probe. The results show that for Southern analysis processed DNA can be used but, due to the generation of random breaks, the restriction fragments have to be small. Furthermore, the fixation-induced crosslinking of DNA appears to hamper hybridization. For these reasons processed DNA can be analyzed better by dot blot rather than Southern blot hybridization.     

地高辛标记核酸探针斑点杂交法检测轮状病毒疫苗中残余MA－104细胞DNA含量的研究

本文报导用地高辛标记核酸探针和分子斑点杂交技术检测轮状病毒基因重配株Ｌ－３株活疫苗中残余ＭＡ－１０４细胞ＤＮＡ含量的方法。提取和纯化ＭＡ－１０４细胞ＤＮＡ,将其ＡｌｕＩ酶切片段用随机引物法引导ＤＮＡ标记为探针。待检样品抽提核酸后点膜进行斑点杂交。此法灵敏度高,可检出０．１４ｐｇ的ＤＮＡ,特异性强,与非同源性ＤＮＡ无杂交。用此法检测轮状病毒基因重配株Ｌ－３株制备的疫苗,ＭＡ－１０４细胞残余ＤＮＡ含量低于１４ｐｇ低于ＷＨＯ限量标准,结果表明此重配株用于研制疫苗是安全的。     

Development of Prevotella intermedia-specific PCR primers based on the nucleotide sequences of a DNA probe Pig27

The aim of this study was to develop Prevotella intermedia-specific PCR primers based on the P. intermedia-specific DNA probe. The P. intermedia-specific DNA probe was screened by inverted dot blot hybridization and confirmed by Southern blot hybridization. The nucleotide sequences of the species-specific DNA probes were determined using a chain termination method. Southern blot analysis showed that the DNA probe, Pig27, detected only the genomic DNA of P. intermedia strains. PCR showed that the PCR primers, Pin-F1/Pin-R1, had species-specificity for P. intermedia. The detection limits of the PCR primer sets were 0.4 pg of the purified genomic DNA of P. intermedia ATCC 49046. These results suggest that the PCR primers, Pin-F1/Pin-R1, could be useful in the detection of P. intermedia as well as in the development of a PCR kit in epidemiological studies related to periodontal diseases.