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1.
RAPD应用于蕈菌研究中的条件优化探讨   总被引:4,自引:0,他引:4  
在进行鹅膏菌属蕈菌遗传多样性的RAPD分析时,对RAPD分析过程中的DNA提取方法、DNA纯度、模板DNA和dNTP浓度、循环次数、DNA不同来源等影响因素进行了大量的实验探索,结果表明:DNA不同提取方法具有相同的扩增产物,DNA模板中的RNA对扩增产物无影响,模板浓度在一个相当大的范围内(50~400μg)不影响扩增结果.dNTP浓度达0.75mmol/L时无带谱出现,引物浓度达1μmol/L时出现非特异性带谱,从菌丝体和子实体中提取的DNA可获得一致的扩增产物.扩增循环40~45周期条件下扩增效果较好,本实验证明了RAPD产物具有很好的重复性,建立了适合蕈菌RAPD分析的PCR程序及条件,为RAPD应用于蕈菌的遗传研究打下了良好的基础.  相似文献   

2.
1RAPD的原理随机扩增多态性DNA(RandomAmplifiedPolymorphicDNA,RAPD)系1990年由美国科学家采用PCR技术发展起来的。在发现RAPD多态性的同时,也证明了RAPD标记的分离符合孟德尔独立分配规律,从而确立了RA...  相似文献   

3.
RAPD技术及其在微生物学方面的应用   总被引:4,自引:0,他引:4  
198 0年 ,Botsein提出DNA限制性片段长度多态性 (RFLP)可以作为遗传标记 ,从此开创了直接应用DNA多态的新阶段。 80年代后 ,DNA多聚酶链式反应 (PCR)的发展 ,使直接扩增DNA的多态性成为可能 ,并在此基础上产生了许多种新型分子标记 ,诸如扩增片段多态性 (ALFR)、串联重复序列(VNTR)、单链构型多态性 (PCR SSCP)、序列特异扩增区域 (SCAR)、随机扩增多态性DNA(RAPD)等。而RAPD是较为突出的一种。RAPD是由Williams和Welsh在 1 990年各自独立发现的一种DNA多态检…  相似文献   

4.
普通野生稻( Oryzarufipogon Griff.)的基因资源对水稻的育种起着至关重要的作用。报道了从其硅胶干燥的小量叶片中制备DNA的方法。用此方法制备的DNA分子量大(40~45 kb) ,产率也较高(50 ~200 μg/g) ,且成功地进行了RAPD扩增。用制备的44 个居群,1168 个个体的总DNA 建立了中国普通野生稻的总DNA 库作长期冷冻保存,可用于基于PCR 的DNA水平上的各种目的的研究。根据实验结果,从在室温下贮存1 周、3 个月、6 个月、1 年的硅胶干燥的叶片中提取的DNA 用于RAPD扩增所得的扩增产物没有差异;模板DNA浓度在3 .1 ~50 ng 的范围内均得到很好的RAPD扩增结果。这说明了从硅胶干燥的叶片中提取的普通野生稻的DNA 用于RAPD扩增的产物很稳定,将其用于群体遗传分析具有很好的可比性和可靠性。同时也讨论了模板DNA的纯度和浓度对RAPD扩增的影响  相似文献   

5.
RAPD分析快速鉴定双歧杆菌   总被引:8,自引:0,他引:8  
本文应用RAPD技术,选用11种引物,以嗜酸乳杆菌为对照,对6种13珠双歧杆菌菌株基因组DNA进行PCR扩增,分析其DNA指纹图谱,并计算其相似性指数。结果表明,双歧杆菌和非双歧杆菌之间,其相似性指数有显著差异;选择合适的引物进行扩增,双歧杆菌不同种间和同种不同株间可表现不同的DNA指纹图谱。本文还对RAPD技术应用于双歧杆菌分类鉴定的可能性进行讨论。  相似文献   

6.
RAPD在植物育种上的应用及其技术校正   总被引:1,自引:0,他引:1  
RAPD(RandomAmplifiedPolymorphicDNA) ,是在PCR的基础上发展起来的一种DNA多态性检测技术 ,由Willams[1] 和Welsh[2 ] 于 1990年建立 ,原理是以一系列不同的 ,少数碱基组成的随机核甘酸序列为引物 ,对样本基因组DNA进行PCR扩增 ,每个RAPD片段的产生要求在可增范围内存在与引物匹配的反向互补序列。引物结合位点DNA序列的改变以及两扩增位点之间碱基的缺失插入或转换都能导致扩增片段的数目和长度的差异 ,经PAGE或琼脂糖凝胶电泳分离和EB染色来检测DNA片段的多态…  相似文献   

7.
分别利用5’RACE和3’RACE确定了CMV-SDRNA2的5’和3’未端序列,在此基础上,利用RT-PCR得到了RNA2的5’端一半的cDNADQ BTG Pc25和3’端一半的cDNA克隆PC23,并通过拼接构建了RNA2全长cDNA克隆PC2F。  相似文献   

8.
烟草黑胫病菌株亲缘关系的RAPD分析   总被引:15,自引:1,他引:14  
从220个RAPD(Random Amplified Polymorphic DNAs)随机引物中所选出的多态扩增性强的21个引物对来源不同的33个烟草黑胫病菌株进行全基因组DNA遗传多样性分析和指纹构建。选用引物对受试菌株进行RAPD-PCR扩增,共产生243条DNA标记图带,其中191条为多态性图带,多态检测率为78.6%。利用UPGMA(Unweigthted Pair-group Meth  相似文献   

9.
几种因素对山茶属植物RAPD分析的DNA扩增的影响   总被引:10,自引:0,他引:10       下载免费PDF全文
唐绍清  施苏华  林海波   《广西植物》1998,18(2):185-188
多种因素会影响RAPD扩增,本研究试验了引物、Mg2+和dNTP的浓度以及Taq酶来源对山茶属植物进行RAPD分析的DNA扩增的影响。结果表明这些因素对扩增结果都会产生影响,通过比较分析,得到了一个对于山茶属植物进行RAPD分析较理想的扩增条件。  相似文献   

10.
RAPD分析氮离子注入甜菊种子后的幼苗基因组DNA变异   总被引:19,自引:2,他引:17  
应用RAPD 技术检测经低能氮离子注入甜菊纯系种子引起的幼苗基因组DNA 变异。筛选出OPJ系列中的15 种引物对实验及对照基因组DNA 进行了PCR 扩增,共获扩增片段103 条,分子量在0.3 - 3kb 之间,其中5 种引物OPJ- 1 ,7 ,9,11 ,12 扩增出差异片段12 条。结果表明,低能氮离子注入甜菊种子可引起体内基因组DNA 发生突变;RAPD 技术是检测基因组DNA 发生诱变的一种简便、有效方法。本文同时探讨了离子强度和Tag DNA 聚合酶用量对甜菊RAPD 分析结果的影响,以及氮离子注入诱变效应的可能机制。  相似文献   

11.
本文对三峡水库大坝至香溪河段所设A、B、C、D、E、F和G等7个站点浮游生物群落DNA进行了RAPD分子生物学研究,并分析了其与水体理化因子的关系。各站点间RAPD研究表明:D和E首先聚到一组, 然后与A聚到一起, 最后与C聚成一大类;B和F聚成一大类;而站点G独自归于一类。而理化因子聚类结果显示:B首先与C聚为一小类,再与D聚到一起,然后与G、F聚成的小类聚为一类,而E与A分别单独归为一类。比较发现,RAPD聚类结果中相距较近的站点在理化因子聚类中显示为相距较远的站点(如站点A、C、D、E之间),而在RAPD聚类中相距较远的站点在理化因子聚类中显示为相距较近的站点(如站点B、F分别与G之间),这可能因为它们之间存在负相关性,也可能部分因为试验条件本身所造成的误差。为确定浮游生物DNA指纹结构与理化因子的关系提供了新的信息,进而为建立一种新的水质评价体系积累了理化因子的一些背景资料。  相似文献   

12.
RAPD (Random Amplified Polymorphic DNA) technique has been widely used in animal, plant, human and microorganism research since it was first established by Williams in 1990[1-3]. But, because of low annealed temperature and short 10-nt primers, the resolution and repetition is low in RAPD. The stability of RAPD is influenced by many factors such as the concentration of template, primers, dNTP, Mg++,and Taq DNA polymerase[4-6]. The influence on amplified products of different commercial Taq DNA polymerase in RAPD was studied in this paper.  相似文献   

13.
A standardized-reagents commercial kit for random amplified polymorphic DNA (RAPD) analysis was used for typing 58 Escherichia coli strains that were recovered from the milk of sows, having coliform mastitis, within a single swineherd in Sweden. Previously, the 58 E. coli strains were characterized serologically and profiled biochemically. They were also evaluated for their serum resistance and their ability to adhere to fibronectin and bovine fetal fibroblasts. The RAPD analysis was fast, easily performed, and required only a nanogram of DNA. The indistinguishable banding patterns obtained with repeated analyses of 2 isolates from each strain demonstrated that RAPD analysis using standardized beads is a technique that provides reproducible results for typing E. coli strains that cause mastitis in sows. The results of the RAPD analyses demonstrated that E. coli sow mastitis strains are highly variable in serotype, biochemical profiles, virulence factors, and RAPD type, and that all 58 strains can be differentiated by means of the RAPD technique. The strains grouped into 24 RAPD types by combining the results of 2 primers, and into 38 groups by combining the results of serotype and RAPD type. No relationship between serotypes, virulence factors and RAPD types was found.  相似文献   

14.
Phenotypic approaches based on metabolic and biological characteristics of Corynebacterium pseudotuberculosis have been limited due to insufficient discrimination between closely related isolates. In this paper we present performance and convenience of three molecular typing methods: BOX-PCR, random amplification of polymorphic DNA (RAPD) and amplification of DNA fragments surrounding rare restriction site (ADSRRS-fingerprinting) in genome analysis of these bacteria. Among examined 61 strains there were distinguished four, eight and 10 different genotypes by BOX-PCR, RAPD and ADSRRS-fingerprinting, respectively. The value of discrimination index was the lowest for BOX-PCR (D = 0.265), much bigger for RAPD (D = 0.539) and the highest for ADSRRS-fingerprinting (D = 0.604). The good discriminatory ability and reproducibility of RAPD and ADSRRS-fingerprinting indicates that those techniques may be particularly applied for epidemiological studies of C. pseudotuberculosis isolates. We found that ADSRRS-fingerprinting is a rapid method offering good discrimination power, excellent reproducibility and may be applied for epidemiological studies of intraspecific genetic relatedness of C. pseudotuberculosis strains.  相似文献   

15.
Restriction fragment length polymorphism (RFLP) and random amplified polymorphic DNA (RAPD) markers are being used widely for evaluating genetic relationships of crop germplasm. Differences in the properties of these two markers could result in different estimates of genetic relationships among some accessions. Nuclear RFLP markers detected by genomic DNA and cDNA clones and RAPD markers were compared for evaluating genetic relationships among 18 accessions from six cultivated Brassica species and one accession from Raphanus sativus. Based on comparisons of genetic-similarity matrices and cophenetic values, RAPD markers were very similar to RFLP markers for estimating intraspecific genetic relationships; however, the two marker types gave different results for interspecific genetic relationships. The presence of amplified mitochondrial and chloroplast DNA fragments in the RAPD data set did not appear to account for differences in RAPD- and RFLP-based dendrograms. However, hybridization tests of RAPD fragments with similar molecular weights demonstrated that some fragments, scored as identical, were not homologous. In all these cases, the differences occurred at the interspecific level. Our results suggest that RAPD data may be less reliable than RFLP data when estimating genetic relationships of accessions from more than one species.  相似文献   

16.
Random amplified polymorphic DNA (RAPD) analysis is a valuable tool in studying inter- and intra-specific genetic variations, patterns of gene expression, and for the identification of specific genes using nearly isogenic variants. Here we used RAPD analysis to study the genetic variation in Ginkgo biloba grown in the eastern United States. Our results support the evidence that Southern blot hybridization of RAPD using probes made from cloned DNA fragments allows a more accurate analysis of the RAPD pattern than dye-stained gels or Southern blot hybridization of RAPD blots using probes made from purified PCR products. Using these techniques, we observed a high degree of relatedness among plants grown in certain localities although significant genetic variation may exist in the species, and could be a possible explanation for the observed variations in the efficacy of medications derived from G. biloba extract.  相似文献   

17.
RAPD在双歧杆菌菌种鉴定及分型研究中的应用   总被引:3,自引:0,他引:3  
采用近年来兴起的一种新的分子生物学分型技术RAPD即AP-PCR对20株4种不不双歧杆菌进行了基因指纹图谱的构建,其结果不同双歧杆菌种间存在同源性和多态性。本研究结果表明RAPD技术可用于双歧杆菌菌种鉴定及分型。  相似文献   

18.
Randomly amplified polymorphic DNA (RAPD) analysis was performed for the molecular genetic typing of 30 Salmonella enterica subsp. enterica strains isolated from chickens and ducks in Thailand. Six different primers were tested for their discriminatory ability. While some of the primers could only differentiate between the different serovars, the use of multiple primers showed that the RAPD method could also subdivide within a given serovar. The Ready-To-Go RAPD analysis beads used, resulted in reproducible and stable banding patterns. As the RAPD technique is simple, rapid and rather cheap, we suggest that it may be a valuable new tool for studying the molecular genetic epidemiology of S. enterica ssp. enterica, both inter- and intra-serovars.  相似文献   

19.
飞蝗总DNA的抽提及其RAPD分析条件的摸索   总被引:39,自引:0,他引:39       下载免费PDF全文
通过试验寻求得到一种快速、简便抽提飞蝗(Locusta sp.)总DNA方法,使每头雄性和雌性成虫分别可以得以50和100μg的总DNA。所得到的总DNA OD260/OD280为1.5-2.2,分子量45kb。为了获得高分子量的DNA产品,使RPAD结果具重复性,酚氯仿抽提后的DNA沉淀用灭菌Tip头挑出,而不用离心收集。对各种分析条件如摸板、Taq酶、dNTP及引物的浓度、不同的PCR仪、反应管进行了比较试验,发现在一定的范围内,它们对RAPD结果影响。用优化的试验条件对我国3个飞蝗亚种5个地理种群进行RAPD分析。结果在3个亚种UPGMA聚类图中,东亚飞蝗和西藏飞蝗珠2个种群以100%Bootstrap分别聚类在一起,亚洲飞蝗与东亚飞蝗的2个种群以66%的Bootstrap聚类在一起,在3个亚种所有个体的UPGMA聚类图中,亚种内的所有个体都聚类在一起,各自形成独立分支,说明3个飞蝗亚种有明显的区别。西藏飞蝗的2个种群之间,群居型与散居型东亚飞蝗之间在聚类图中混合聚类,说明它们之间存在基因交流。  相似文献   

20.
 Random amplified polymorphic DNA (RAPD) was used to determine whether such markers can be employed for detecting genomic modification during plant development or under certain stress environments. Pairwise comparisons in RAPD patterns of leaf and root DNA amplifications were studied for 11 soybean accessions representing different origins. Hydroponic culture was used for the ease of harvesting roots. From a total of 40 primers screened, it was found that 16 can detect leaf DNA polymorphism, 19 for root DNA polymorphism, while 10 show a greater consistency for detecting polymorphism between leaf and root (L/R) DNAs. Nevertheless, problems were encountered when the newly synthesized oligo-primers and different thermal cyclers were used to check the data. Several factors were then tested for their reproducibility. The results indicated that the amplified differences between root and leaf DNAs are mostly not affected by template DNA concentrations. The addition of DMSO (dimethyl sulphoxide) or TMAC (tetramethyl-ammonium chloride) also did not mask the L/R differences. However, DNA polymerase and oligo-primers synthesized from different manufacturers, as well as the thermal cyclers, reacted differently sometimes. Regardless of the general problems of reproducibility in RAPD patterns, some amplified differences remain between the L/R DNAs. The most distinct patterns involve differences in the relative intensity of amplified bands. Differential amplification might have occurred during plant leaf and root development. Southern hybridization of the eluted polymorphic bands against restriction digestion of total genomic DNA confirms their being homologous to soybean DNA fragments. Polymorphism of these specific L/R differences also exists among varieties. RAPD should be a useful tool in detecting genomic alterations during plant development or under certain stress environments, as long as the factors affecting the reproducibility of RAPD patterns can be properly controlled. An additional cycle of selection would be possible if such a type of polymorphism is proved to be correlated with certain developmental characters. Received: 7 October 1996 / Accepted: 20 May 1997  相似文献   

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