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1.
鲮鱼DAF和RAPD的比较研究   总被引:6,自引:0,他引:6       下载免费PDF全文
鲮鱼(Cirrhina molitorella)为南方“四大家鱼”之一,目前其产量约占两广地区池塘鱼总产量的20%左右,具有产量高、抗病力强、肉质鲜美等特点,也是驰名中外的加工淡水鱼类产品。在利用RAPD开展鲮鱼的遗传多样性研究中,发现珠江水系不同江段鲮RAPD遗传多样性不甚丰富。Caetano-Anoll啨s等学者在1991年利用DNA扩增指纹(DNA Amplifica-tion Fingerprinting,DAF)开展了基因组分析研究,他是利用非常短的随机引物(7—8bp,甚至5bp)对不同模板DNA的酶链式扩增,采用高分辨率的聚丙烯酰胺疑胶电泳和银染显色检测DNA的多态性,引物比RAPD更…  相似文献   

2.
用变性PAGE-银染法鉴定小麦抗条锈基因Yr5的RAPD标记   总被引:14,自引:0,他引:14  
以小麦抗条锈病基因Yr5的供体亲本Triticumspeltaalbum作为对照 ,对近等基因系Yr5 6×AvocetS和感病亲本AvocetS进行RAPD分析。扩增产物用 4%变性PAGE分离 ,银染显色。在变性PAGE上可以检测到 50~ 10 0条带 ,是琼脂糖凝胶电泳的 5倍以上。筛选了 2 40个随机引物 ,发现 2 3条稳定的多态性DNA片段 ,初步检测表明其中6条与Yr5基因具有连锁性。用 12 1株AvocetS和Yr5 6×AvocetS杂交制备的F2 代分离群体进一步进行的遗传连锁性检测表明 ,多态性DNA片段S13 2 0 2 0 7和S13 4 83 6 3 分别与Yr5基因完全连锁和紧密连锁。结果表明 ,用变性PAGE分离PCR产物并结合银染显色 ,提高了小麦RAPD分析的多态性水平 ,改善了实验的重复性  相似文献   

3.
九、碱性琼脂糖凝胶 科学家们认为,琼脂糖凝胶电泳是分离、鉴定和纯化DNA片段的一种极好的方法。这种技术操作简单,快速,并且还能解决在密度梯度离心中所存在的DNA片段不能充分分离的难题。此外,还能直接确定凝胶中DNA的位置:其方法是先用低浓度的能发荧光的具有插入本领的染料(溴乙锭),对胶中DNA条带进行染色;然后,把电泳胶置于紫外光下进行直接检测,其灵敏度可达1毫微克(ng)。  相似文献   

4.
目的使用随机扩增多态DNA标记建立标准化的布氏田鼠封闭群遗传质量控制分子标记库。方法使用高盐沉淀法从鼠尾中提取布氏田鼠基因组DNA。采用40条PRAD引物对布氏田鼠封闭群进行PCR扩增,琼脂糖电泳分离条带,参考标准分子量标记计算条带大小,并使用多态位点数、单态位点数以及多态位点比率评价种群的遗传多样性。结果筛选出8个能获得清晰稳定扩增条带的RAPD标记。这8个RAPD标记检测到的多态位点数存在明显差异。8个引物得到的遗传多态位点的数据之和能揭示种群的遗传结构。结论本实验建立了检测布氏田鼠封闭群遗传结构的RAPD标记。  相似文献   

5.
基于RAPD分析用百合DNA的提取   总被引:2,自引:0,他引:2  
对百合DNA的提取过程进行改良和优化,筛选出较理想的DNA提取方法。结果表明,该方法提取的百合DNA在1%的琼脂糖胶电泳上为清晰的一条带,RNA去除干净,无降解现象,分子量大于23kb,OD200nm/OD280nm值为1.80-2.00。DNA的质量符合百合RAPD(随机扩增多态DNA)分析要求,在百合遗传多态性分析和品种分子鉴定中具有良好的应用前景.  相似文献   

6.
用随机扩增多态性DNA产物做探针产生鸡的DNA指纹图   总被引:2,自引:0,他引:2  
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.  相似文献   

7.
双梯度超薄胶PAGE分离DNA及其银染与回收   总被引:2,自引:0,他引:2  
双浓度梯度超薄胶PAGE分离DDRT-PCR扩增产物表明,在30cm长的凝胶板上能清晰分辨长度仅差1bp的DNA,清楚的显示出100多条分离谱带,并可直接从银染PAGE胶中回收DNA谱带。  相似文献   

8.
本实验以16个石榴品种为实验材料,筛选出10个重复性及多态性均较好的引物进行RAPD分析。分别采用琼脂糖凝胶以及聚丙烯酰胺凝胶(PAGE)电泳检测方法对PCR扩增结果进行检测并对其结果进行比较,结果显示,两种电泳方式均能得到较为清晰的扩增条带,且两种电泳方式获得的条带总数及多态性条带数均有所不同,琼脂糖凝胶电泳方法共检测出76条带,其中有43条为多态性谱带,多态性比率为56.4%;而在PAGE电泳方法共检测出123条谱带,多态性谱带数为87条,多态性比率为70.95%。PAGE电泳方法检测出的条带数约为琼脂糖凝胶电泳方法检测出条带数的1.5倍。基于两种电泳方法所得RAPD标记的多态性位点,利用NYSYS软件计算遗传相似系数,并构建遗传关系聚类图,分析结果显示,石榴遗传多样性丰富,两种电泳方法所得聚类结果大致相同,可以利用RAPD分子标记及两种电泳检测方法对不同数量的石榴进行分子水平的品种鉴定和遗传多样性的分析。同时通过对来自几个引物随机挑选的17个片段进行克隆,测序结果显示17个片段都是对应引物的RAPD扩增产物,其中有3条是编码蛋白的基因片段,表明了RAPD不仅扩增基因组上的非编码蛋白序列,同时也可以扩增编码蛋白的基因片段,这为更好地认识RAPD技术的实质以及促进石榴产业的发展提供了理论依据。  相似文献   

9.
用引物G1和L1扩增的16S-23SrDNA间隔区作为模板DNA,再用引物AP50进行随机扩增多态性DNA(Random Amplified PolymorphicDNA,RAPD)分棉线AP50扩增出的各菌株DNA片段,用1.2%的琼脂糖凝胶电泳EB染色,可获得1或2个大小不同的DNA片段(390-1031bp)。各株间差异明显,结果表明,不同菌株有不同的DNA多态性,药物敏感株与耐药株之间多态性差异明显,耐药株中耐药谱相似或相同的菌株间其多态性也相似,RAPD有助于简便,快速,有效地了解菌株间的克隆相关性及耐药性传播。  相似文献   

10.
王银东  熊邦喜 《昆虫知识》2006,43(3):355-360
为了研究摇蚊科昆虫种群遗传的多样性,以促进对其资源的合理保护,以萨摩亚摇蚊Chironomus samoensisEdwards基因组DNA为模板,对摇蚊幼虫的RAPD扩增条件进行优化,建立了摇蚊幼虫RAPD扩增反应的最佳体系:按照利用优化的RAPD扩增条件进行研究,实验有着良好的重现性。用16个随机引物对3种摇蚊幼虫类群各10个个体进行RAPD扩增,其中萨摩亚摇蚊共扩增出78个条带,多态座位率为41.03%,Shannon遗传多样性指数为0.2570,群体内相似度为0.8730;红裸须摇蚊Propsilocerus akamusi(Tokunaga)共75个条带,多态座位率为44.0%,Shannon遗传多样性指数为0.2472,群体内相似度为0.8731;刺铗长足摇蚊Tanypus punctipennis(Fabricius)共67个条带,多态座位率为41.79%,Shannon遗传多样性指数为0.1943,群体内相似度为0.9066。聚类分析结果表明,刺铗长足摇蚊与红裸须摇蚊的亲缘关系较近。  相似文献   

11.
Fast and sensitive silver staining of DNA in polyacrylamide gels.   总被引:288,自引:0,他引:288  
The photochemically derived silver stain of nucleic acids in polyacrylamide gels originally described by Merril et al. (1981, Science 211, 1437-1438) was modified to reduce unspecific background staining and increase sensitivity (down to 1 pg/mm2 band cross-section). Detection limits for double-stranded DNA fragments from HaeIII endonuclease digests of phage phi X174 were maintained despite eliminating oxidation pretreatment of fixed gels and reducing silver nitrate concentration. Preexposure to formaldehyde during silver impregnation enhanced sensitivity and the inclusion of the silver-complexing agent sodium thiosulphate in the image developer decreased background staining. Higher formaldehyde concentration during image development resulted in darker bands with good contrast. The procedure almost halves the number of steps, solutions and experimental time required and can be used for the staining of DNA fragments in polyacrylamide gels bound to a polyester backing film by controlling temperature during image development. We have applied this improved staining procedure for the routine analysis of complex DNA profiles generated by DNA amplification fingerprinting (DAF).  相似文献   

12.
银杏DNA提取及RAPD分析   总被引:2,自引:0,他引:2  
采用SDS裂解液和苯酚/氯仿/异戊醇提取液从银杏叶中提取银杏总DNA,并进行DNA样品分光光度测定和琼脂糖凝胶电泳分析。通过引物筛选和反应参数优化,选用3个随机引物对DNA样品进行RAPD扩增,获得较为清晰并有一定多态性差异的扩增谱带,初步摸索出适合于以银杏叶为材料的DNA提取方法和RAPD扩增程序,为研究银杏遗传多态性及种质资源研究提供一种实用的分析方法。  相似文献   

13.
RAPD (random amplification of polymorphic DNA) molecular markers can be utilized for analyzing genetic variability in populations for which only a few or no molecular markers are available. They were used in a study of an endangered species, Peripatus acacioi, found in the Tripuí Ecological Station, in Ouro Preto, MG, Brazil. The ecological station was specifically created to protect this velvet worm species, the first of this group found in Brazil. For an initial evaluation of the genetic diversity of this species, DNA samples from the lobopods of four individuals, collected at random, were analyzed using RAPD. Each reaction was run with a different primer (Operon RAPD 10-mer Kits), totaling 13 primers (OPC2, OPC3, OPC4, OPC6, OPC8, OPC10, OPC11, OPL2, OPL7, OPL11, OPL13, OPL18, and OPL19). Due to the low amplification yield, RAPD fragments were separated in polyacrylamide gels and stained with silver nitrate. Numerous bands were observed. Fifty-five of the amplified bands proved to be reproducible, both in terms of presence and intensity. Among these, 27 were variable and 28 were constant. The average number of bands per gel was 4.2. Nine of the 13 primers tested allowed the identification of constant and variable bands among these four individuals. RAPD analysis of genetic variation using silver-stained polyacrylamide gel electrophoresis provided measures of band sharing among the individuals, and therefore could be used in population genetics studies of P. acacioi.  相似文献   

14.
DNA amplification fingerprinting: A strategy for genome analysis   总被引:4,自引:1,他引:3  
A novel strategy to detect genetic differences among organisms, DNA amplification fingerprinting (DAF), uses a thermostable DNA polymerase directed by usually one short (≥5 bp) oligonucleotide primer of arbitrary sequence to amplify short segments of genomic DNA and generate a range of DNA extension products. These products can be analyzed by polyacrylamide gel electrophoresis and silver staining. DAF is rapid and sensitive and is independent of cloning and prior genetic characterization. Here we describe this new methodology, its application to plant genotyping, and its perspectives in DNA fingerprinting and genome mapping.  相似文献   

15.
The Random Amplified Polymorphic DNA (RAPD) assay was used tostudy genetic variation within and between 9 species of thegenus Bulinus and to determine whether RAPD profiles could beused as markers for identification purposes. RAPDs were generatedwith 8 primers of two different sizes (l0mers & 15mers)and were visualised using both polyacrylamide gel electrophoresis(PAGE) with silver staining and agarose gel electrophoresiswith ethidium bromide staining. The species groups of Bulinushad few similarities in their RAPD profiles and there was interspecificvariation within groups. Intrapopulation variation was observed,with all primers, for B globosus collected from a single sitein Zimbabwe PAGE/silver staining methods visualised a greaternumber of RAPDs in comparison with agarose/ethidium bromidemethods. Phenetic analysis indicated that distance estimatesbetween taxa were sometimes non-additive and the phylo-geneticanalysis of such non-metnc data is discussed. The resultantphenograms, constructed using a least squares method, were constrainedalmost into a polytomy with topologies often differing betweendata sets. It was concluded that this phenomenon was most likelyattributable to large nucleotide divergences between the speciesgroups which go beyond the phylogenetic scope of RAPD analysis.RAPD profiles, when used in conjunction with other taxonomicmethods, may contribute to the identification of species ofBulnus on a regional basis, but the observed variability ina natural population suggests that a diagnostic RAPD profilefor each species throughout its geographic range is unlikely. (Received 19 April 1995; accepted 1 September 1995)  相似文献   

16.
一种用于PCR模板制备的电泳产物简易回收方法   总被引:1,自引:0,他引:1  
为了探索一种简便、有效而且能从琼脂糖凝胶中大量回收用于第2次PCR扩增的DNA电泳条带的方法,采用刀片切胶法和牙签插胶法从琼脂糖中回收DNA,并进行了两种方法的比较.结果显示牙签插胶法回收的DNA用作第2次PCR的模板,获得了清晰、稳定的PCR产物电泳条带,用该法成功地制备了一批DNA微阵列探针.由此可见牙签插胶法是一种简便、快速、有效的用于PCR模板的DNA琼脂糖凝胶回收法.  相似文献   

17.
AIMS: To develop a rapid, sensitive and reproducible screening test for the detection of nosocomial spreading of Pseudomonas aeruginosa. METHODS AND RESULTS: Ps. aeruginosa genomic DNA extraction, RAPD-PCR, electrophoresis on acrylamide gel and silver staining were performed by using standardized reagents and conditions. The results were compared with the agarose gel electrophoresis followed by ethidium bromide staining. CONCLUSIONS: The coupling of acrylamide gel electrophoresis and silver staining gave about 80% more DNA bands than the traditional method, allowing a finer discrimination among different Ps. aeruginosa strains. SIGNIFICANCE AND IMPACT OF THE STUDY: By enhancing the resolution of the electrophoretic separation and the sensitivity of the staining, random amplification could be easily applied to the surveillance and prevention of nosocomial infections by clinical microbiology laboratories.  相似文献   

18.
The use of random amplified polymorphic DNA from the polymerase chain reaction (RAPD-PCR) allows efficient construction of saturated linkage maps. However, when analyzed by agarose gel electrophoresis, most RAPD-PCR markers segregate as dominant alleles, reducing the amount of linkage information obtained. We describe the use of single strand conformation polymorphism (SSCP) analysis of RAPD markers to generate linkage maps in a haplodiploid parasitic wasp Bracon (Habrobracon) hebetor and a diploid mosquito, Aedes aegypti. RAPD-SSCP analysis revealed segregation of codominant alleles at markers that appeared to segregate as dominant (band presence/band absence) markers or appeared invariant on agarose gels. Our SSCP protocol uses silver staining to detect DNA fractionated on large thin polyacrylamide gels and reveals more polymorphic markers than agarose gel electrophoresis. In B. hebetor, 79 markers were mapped with 12 RAPD primers in six weeks; in A. aegypti, 94 markers were mapped with 10 RAPD primers in five weeks. Forty-five percent of markers segregated as codominant loci in B. hebetor, while 11% segregated as codominant loci in A. aegypti. SSCP analysis of RAPD-PCR markers offers a rapid and inexpensive means of constructing intensive linkage maps of many species.  相似文献   

19.
Species of Ganoderma are used in traditional medicines. An improved random amplified polymorphic DNA (RAPD) analysis, where the RAMP time is prolonged, has been used to characterize the genetic variation in some well known species of Ganoderma. The DNA materials were collected from ten Ganoderma strains, amplified with randomly selected 24 RAPD primers and evaluated by agarose gel electrophoresis. A cluster dendrogram was constructed for genetic analysis on the basis of amplification results. The improved RAPD amplified DNA with consistent and clear banding patterns. A total of 316 bands were found with 93% polymorphism. There was a significant genetic distance between the different strains of Ganoderma, with an index of similarity coefficient in the range of 0.52–0.74. The inter-simple sequence repeat (ISSR) analysis of the Ganoderma DNA samples showed similar trend results to the RAPD analysis with 0.49–0.81 similarity coefficients. This study reports the high level of genetic differences between different species or strains of a single species of Ganoderma and confirms the significance of the improved RAPD method in genetic characterization of organisms. Therefore, the improved RAPD combined with ISSR techniques might be used for the genetic characterization of organisms.  相似文献   

20.
Similarity among a number of aquatic microbial communities was examined using randomly amplified polymorphic DNA (RAPD), a common polymerase chain reaction (PCR)-based DNA fingerprinting technique. After amplification of whole-community DNA extracts, the PCR products were resolved by agarose gel electrophoresis and the band patterns compared to determine percent similarity. Twelve different primers were used to amplify approximately 100 fragments (total) from each DNA sample; the bands were scored as present or absent and the similarity between each sample was determined using Jaccard's coefficient. From this information. dendrograms were constructed and a bootstrapping procedure was used to assess how well supported the tree topologies were. Principal component analyses were also conducted as a means of visualizing the relationships among samples. Results obtained for two different experimental systems (a pair of tidal creeks and several wells in a shallow groundwater aquifer) correlated well with the temporal and spatial variations in environmental regime at the sites confirming that arbitrarily primed PCR-based DNA fingerprinting techniques such as RAPD are useful means of discriminating among microbial communities and estimating community relatedness. Moreover, this approach has several advantages over other DNA-based procedures for whole-community analysis; it is less laborious and uses smaller quantities of DNA, making it amenable to sample-intensive monitoring, and it does not depend on culturing or the use of selective PCR primers.  相似文献   

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