甘蓝品种''''争春''''和''''寒光2号''''的DNA指纹图谱构建

用SDS法提取甘蓝（Brassfca oleraceavat．capitata）品种‘争春’、‘寒光2号’及其各自亲本的基因组DNA,通过SRAP、RAPD两种分子标记方法,构建其DNA指纹图谱,用于种子纯度鉴定。利用30对SRAP引物组合和200个RAPD随机引物,以各品种及其亲本的基因组DNA为模板组进行筛选,结果显示：多数SRAP引物组合对模板组的扩增带型一致,少数组合扩增出差异,但未能找到具有互补差异的引物组合;通过RAPD标记方法筛选出能鉴定2个品种纯度的引物分别为S42、S103、S193和S42、S89、S151,其中引物S42对2组材料均能扩增出特异的RAPD指纹图谱,并将RAPD指纹图谱转变为相应的数字指纹。     

DNA指纹图谱在乳酸菌分类鉴定中的应用

目的 探讨DNA指纹图谱在乳酸菌分类鉴定中的应用。方法 选用S23随机引物,对乳酸菌基因组DNA进行RAPD随机扩增,获得能够区分不同菌株的DNA指纹图谱,依据图谱DNA条带的多态性,对10株乳酸菌菌株进行分类与鉴定。结果 实验室保藏菌株LAP2、LAT、LAM、LAC和LAO之间的基因组DNA相似性达80%,亲缘关系最为相近,而LAB菌株与所有菌株的亲缘关系最远。结论 DNA指纹图谱技术与常规方法结合使用,将使乳酸菌的分类、鉴定更为准确、便捷。     

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.     

DNA指纹图谱鉴别双歧杆菌的研究

采用RAPD技术选用10条引物对7种9株双歧杆菌基因组DNA进行PCR扩增,根据在优化条件下所得DNA指纹图谱分析了双歧杆菌菌株的遗传多样性,并构建了相似性指数矩阵和树状图.结果表明,不同序列的随机引物可扩增不同形式的RAPD图谱,但并非所有图谱都具有分类学意义,其中引物S256对双歧杆菌种及同种不同菌株均具有良好的区分能力,由该引物扩增的RAPD图谱计算出的相对性指数矩阵以及由此构建的聚类树状图均能正确地反映出双歧杆菌的系统发育关系,同时对RAPD图谱作为工业双歧杆菌分子标记的可能性进行了探讨.     

应用DNA指纹图谱技术鉴定整肠生菌株BL63516的研究

目的建立应用DNA指纹图谱技术鉴定微生态制剂——整肠生菌株BL63516的方法,提高菌种鉴别水平。方法应用RAPD（随机扩增多态性）方法,采用50条随机引物对7株地衣芽胞杆菌进行基因组DNA指纹图谱分析,选择多态性好、重复性好、稳定性强的随机引物,对BL63516与其他地衣芽胞杆菌进行区分。结果发现选用引物$87或$88分别对7株地衣芽胞杆菌进行基因组DNA指纹图谱分析,BL63516菌株扩增的DNA片段的大小、数量均与其他地衣芽胞杆菌有明显差异。结论此方法具有可重复性,方便、快速和准确的优势,可用于微生态制剂整肠生菌株的鉴别。     

直接从人工老化的菜心干种子中提取基因组DNA用于RAPD分析

利用 4 0℃、1 0 0 %RH对菜心种子进行人工加速老化处理获得了不同活力的种子批 ,利用平衡酚_氯仿法直接从人工老化的菜心干种子中提取基因组DNA ,并对提取的基因组DNA进行了RAPD扩增。结果表明 ,所提取的基因组DNA量多 ,而且比较整齐一致。引物S2 0 8扩增所获得的基因组DNA指纹图谱上的DNA带清晰、明亮 ,从而表明利用本方法从人工老化菜心干种子中直接提取的基因组DNA完全可以用于RAPD分析。     

莲藕品种DNA指纹图谱的绘制

采用RAPD技术对14个莲藕品种进行遗传多态性分析,用5个Operon引物和80个SBS的RAPD引物进行筛选,从中选出来自SBS的RAPD-C13和RAPD-D15扩增出的8条多态性条带,绘制了14个品种的DNA指纹图谱,在该图谱中每个品种均有各自特异的DNA指纹。     

直接从人工老化的菜心干种子中提取基因组DNA用于RAPD分析

利用40℃、100％朋对菜心种子进行人工加速老化处理获得了不同活力的种子批,利用平衡酚-氯仿法直接从人工老化的菜心干种子中提取基因组DNA,并对提取的基因组DNA进行了趾PD扩增。结果表明,所提取的基因组DNA量多,而且比较整齐一致。引物S208扩增所获得的基因组DNA指纹图谱上的DNA带清晰、明亮,从而表明利用本方法从人工老化菜心干种子中直接提取的基因组DNA完全可以用于RAPD分析。     

利用RAPD分子标记快速鉴定3个奇楠种质

利用随机扩增多态性DNA(random amplified polymorphic DNA,RAPD)分子标记技术,分析3个奇楠种质(ChiNan germplasm)亲缘关系,并快速鉴定。通过提取基因组DNA,筛选适用于奇楠种质分析的RAPD引物,PCR扩增获得扩增条带,分析奇楠种质的遗传多样性,并利用人工绘制品种鉴别示意图方法(manual cultivar identification diagram,MCID)将奇楠种质区分开来。从120条RAPD分子标记引物中筛选到10条适合于奇楠种质分析的引物,利用这10条引物发现奇楠种质在物种水平上遗传多样性高,3个奇楠种质不同居群间的遗传一致性高、遗传距离小,在聚类图上各自聚为一支;根据引物S63、S18和S100扩增的多态性谱带构建奇楠种质的MCID,很好地区分了3个奇楠种质。3个奇楠种质均具有特异性强、一致性好、稳定性高的特点,RAPD分子标记技术的MCID可以有效快速地鉴定区分3个奇楠种质。     

沙梨8个栽培品种的DNA指纹鉴定

利用RAPD标记技术,对8个沙梨(Pyrus pyrifolia)主栽品种进行RAPD分析。结果显示,从33个10碱基随机引物中筛选出了2个多态性高的引物S2075和S1296,扩增位点分别为11个和10个,以此为基础建立了8个品种的DNA指纹图谱;根据这2个引物中任何一个的扩增图谱均可以将这8个品种区分。研究结果为沙梨品种的区别与鉴定提供了一种有效方法。     

天麻特异DNA序列的克隆及其在天麻鉴定中的应用

应用改进的RAPD方法测定了名贵中药材天麻基因组DNA指纹图谱;通过选择和回收各种天麻种群共有和优良种群特有的DNA片段,加以克隆、测序和生物信息学分析,证明其中5个DNA序列是未报道的,已被美国基因数据库收录,并运用高效液相色谱技术测定了天麻样本的有效成分天麻素含量。运用PCR技术研究了这些DNA序列在9个天麻种群中的分布及其与天麻素含量的关系。结果表明这5个DNA序列在这些天麻种群中的分布各不相同,其中DNA序列1是所研究的全部天麻种群共有、而其伪品没有的特异DNA分子标记;DNA序列2可能与天麻的天麻素含量高有关。这些DNA标记序列可用于天麻的真伪鉴别、品种鉴定和优选优育等。     

粘类小麦细胞质雄性不育系的RAPD分析

利用250条10-聚寡核苷酸随机引物对具粘果山羊草(Aegilops kotschyi)、易变山羊草(Ae.variabilis)、偏凸山羊草(Ae.ventricosa)和二角山羊草(Ae．bicornis)细胞质不育系及其保持系5-1的总DNA进行了RAPD多态性分析,其中31条引物对4种不育系及其保持系总DNA均无扩增,217条引物扩增条带完全相同。有2条随机引物在2种不育系之间有特异的扩增片段,其中引物S22在偏凸山羊草细胞质雄性不育系基因组DNA中扩增出分子量约为1600bp的特异带,引物S202在粘果山羊草细胞质雄性不育系基因组DNA中扩增出约1300bp特异带。线粒体基因组DNA的RAPD分析表明,4种不育系及其保持系mtDNA存在明显的差异。证明了S22—1600为偏凸山羊草细胞质不育系及其mtDNA基因组DNA的RAPD特异片段．S202—1300可能为粘果山羊草细胞质不育系及其ctDNA基因组DNA的RAPD特异片段。     

Development of RAPD protocol for typing of strains of lactic acid bacteria and enterococci

P.S. COCCONCELLI, D. PORRO, S. GALANDINI AND L. SENINI. 1995. A protocol for typing strains of lactic acid bacteria and enterococci based on randomly amplified polymorphic DNA (RAPD) fragments has been developed. Using a single 10-mer primer, fingerprints were achieved without the need to isolate genomic DNA. Different conditions of DNA release and amplification were investigated in order to obtain reproducible results and high discrimination among strains. This RAPD protocol was successfully applied for the typing of strains belonging to the species Lactobacillus acidophilus, Lact. helveticus, Lact. casei, Lact. reuteri, Lact. plantarum, Enterococcus faecalis, Ent. faecium and Streptococcus thermophilus.     

Optimization of RAPD for fingerprinting Salmonella

Random amplification of polymorphic DNA (RAPD) is proving to be a useful technique in studying the epidemiology of micro-organisms. The technique can be troublesome and time consuming to establish due to the essentially empirical approach to optimization. By standardization of certain parameters and use of a commercially available PCR buffer optimization kit, a particularly promising primer was identified and RAPD conditions for a highly discriminatory and reproducible characterization of Salmonella isolates was achieved. In addition, a technique to obtain reproducible RAPD fingerprints of Salmonella isolates without the need to purify genomic DNA is described.     

Identification of cultivars and validation of genetic relationships in Mangifera indica L. using RAPD markers

Twenty-five accessions of mango were examined for random amplified polymorphic DNA (RAPD) genetic markers with 80 10-mer random primers. Of the 80 primers screened, 33 did not amplify, 19 were monomorphic, and 28 gave reproducible, polymorphic DNA amplification patterns. Eleven primers were selected from the 28 for the study. The number of bands generated was primer- and genotype-dependent, and ranged from 1 to 10. No primer gave unique banding patterns for each of the 25 accessions; however, ten different combinations of 2 primer banding patterns produced unique fingerprints for each accession. A maternal half-sib (MHS) family was included among the 25 accessions to see if genetic relationships could be detected. RAPD data were used to generate simple matching coefficients, which were analyzed phenetically and by means of principal coordinate analysis (PCA). The MHS clustered together in both the phenetic and the PCA while the randomly selected accessions were scattered with no apparent pattern. The uses of RAPD analysis for Mangifera germ plasm classification and clonal identification are discussed.     

人体蠕形螨的DNA提取与随机引物PCR检测

【目的】探索人体毛囊蠕形螨和皮脂蠕形螨DNA的提取方法。【方法】采用液氮反复冻融研磨法破碎螨体细胞, 选用改良小昆虫DNA提取法、碱裂解法和试剂盒提取法, 分别提取冻存时间在5个月内和8~10个月的毛囊蠕形螨和皮脂蠕形螨基因组DNA, 并用随机引物PCR方法进行检测。【结果】蛋白核酸测定仪检测结果显示, 试剂盒法提取的DNA纯度较高、量较多, 明显优于改良小昆虫法和碱裂解法。随机引物扩增结果显示清晰的DNA指纹图谱, 两种人体蠕形螨DNA指纹具有明显差异。蠕形螨冻存时间影响DNA提取的量, 但对DNA提取的纯度和RAPD指纹图谱影响较小。不同DNA提取方法提取的同一种蠕形螨DNA指纹图谱基本相似, 试剂盒法和改良小昆虫法提取的DNA样本条带多而清晰, 碱裂解法提取的样本条带少而模糊。【结论】液氮反复冻融研磨法破碎蠕形螨细胞是有效的, 蠕形螨冻存时间不宜超过6个月, 试剂盒提取法是提取蠕形螨DNA的好方法。RAPD技术可以用于这两种人体蠕形螨DNA分子水平上的检测和分类。     

RAPD analysis of Campylobacter isolates: DNA fingerprinting without the need to purify DNA

A method was developed to obtain reproducible DNA fingerprints from Campylobacter by PCR-based amplification, without the need to isolate total DNA. Randomly amplified polymorphic DNA (RAPD) profiles were generated with three randomly designed 10-mers, using each separately as an amplification primer. A range of C. jejuni serotypes could be typed by RAPD analysis. Depending on the primer, the analysis of RAPD profiles resulted in different levels of discrimination between the strains. Clear correlations were observed between results of RAPD analysis and serotyping. Two of the primers tested generated RAPD profiles which allowed discrimination of strains within given Penner and Lior serotypes.     

Optimization of primer screening for evaluation of genetic relationship in rose cultivars

Optimization of primer screening for evaluation of genetic relationship in 34 cultivars of rose through random amplified polymorphic DNA (RAPD) markers was investigated. Four series of decamer primers were used for screening and optimization of RAPD analysis between which A and N series performed good amplification of fragments as compared with other series. The primers OPN-07 and OPN-15 produced maximum number of DNA fragments in Rosa hybrida cv. Anuraag. Some primer either did not produce amplification or produced very poor amplification. Further, ten selected primers were used for genetic analysis of 34 rose cultivars. The primer OPN-15 amplified 21 fragments in all cultivars tested. A total of 162 distinct DNA fragments (bands) ranging from 100 to 3400 base pairs were amplified by using 10 selected random primers. The cluster analysis indicated that these rose cultivars formed nine clusters.     

大血藤DNA提取及RAPD研究初探

以浙江天台山大血藤为材料,对其DNA提取和RAPD分子标记方法进行了研究。结果表明:所采用的经改进的SDS提取法可获得高质量的大血藤DNA,分子量大于23kb,可满足RPAD扩增;用15个不同的随机引物对所提取的12个个体的大血藤DNA进行了RAPD分子标记分析,其中14个引物扩增产物具有多态性。建立了大血藤DNA提取和RAPD标记的分析程序,为RAPD分析应用于大血藤遗传多样性研究打下了良好的基础。