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根据PCR反应的要求,用改良的CTAB法,以番茄植株为材料,实现了微量番茄叶片基因组DNA的快速提取。提取基因组DNA所用的组织量少,所得的DNA经过电泳检测虽有降解,但足以用于PCR检测,以其作模板扩增中国番茄黄化曲叶病毒诱导的硫黄素酶(Su)基因沉默植株中病毒组分中的DNAmβ和1.7 A,片段大小分别为1 300、500 bp。测序结果证明是相应基因的部分片段。该方法的材料不需要使用液氮,可以单人大批量提取,并在基因沉默的番茄植株中能稳定而准确的规模化PCR检测。 相似文献
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春小麦体细胞无性系变异的研究 总被引:3,自引:0,他引:3
利用RAPD技术检测春小麦愈伤组织和再生植株在离体培养过程中产生的变异,对培养不同时期的愈伤组织、再生植株检测结果表明,在小麦离体培养愈伤组织和再生植株中,RAPD谱带发生变化,表明发生了体细胞无性系分子水平变异.且具有明显的规律性和变异特点:杂交B代幼穗培养获得的愈伤组织发生变异的频率高于遗传稳定品种幼穗培养获得的愈伤组织。在愈伤组织培养75d时,在RAPD电泳图谱反映出高频率的亲本谱带缺失和非亲本谱带增加。不同基因型或外植体诱导的愈伤组织和再生植株中出现了相同的变异。与愈伤组织相比.再生植株中检测到的变异频率更高。不同外植体离体培养获得的再生植株,即使表型上没有观察到变异,但从RAPD图谱上却反映出变异的发生。表明RAPD技术可以快建方便地检测组织培养每个阶段出现的DNA水平变异。 相似文献
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苜蓿红豆草属间体细胞杂种的分子生物学鉴定 总被引:12,自引:0,他引:12
通过原生质体融合和培养获得苜蓿红豆草属间体细胞杂种植株。采用一种简便方法从杂种组织再生植株叶片、红豆草羟脯氨酸抗性系再生植株叶片和苜蓿根癌农杆菌702转化系愈伤组织提取DNA用于RAPD和Southern杂交分析。随机引物扩增结果显示两种亲本的RAPD多态具有明显差异。在所用20种随机引物中,6种产生较多的DNA片段。杂种组织具有两种亲本特有的DNA片段,但倾向于排除红豆草亲本的染色体,表明该杂种为非对称杂种,两种亲本染色体之间可能发生了重组。由于红豆草DNA的介入,杂种组织表现出较强的分化能力。分别利用RAPD扩增得到的OPA141000bp红豆草羟脯氨酸抗性系特异产物和OPA141600bp苜蓿根癌农杆菌702转化系特异产物为探针进行Southern分子杂交,证明杂种组织同时具有这两种DNA片段的同源序列。 相似文献
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抗虫转基因欧洲黑杨的培育* 总被引:6,自引:0,他引:6
用带有35 S-Ω-Bt-NOS嵌合基因的双元载体的农杆菌LBA 4404转化欧洲黑杨的叶片外植体,共获得54株转化再生植株。用这些再生植株对杨尺蠖(Apcchimia cinerarius)进行毒力测定,昆虫校正死亡率在80一96%的再生植株占总测定植株的15%。部分再生植株对舞毒娥进行测定,表明在5~9天内校正死亡率高达100%,存活昆虫的生长和发育也明显地受到抑制。根据苗木在苗圃中高生长和昆虫校正死亡率采用重心聚类法进行分析,初步选出高生长良好和杀虫率居中的3株再生植株。以选出的植株为主进行了PCR及PCR产物的South—ern blot和再生植株DNA Southern blot测定,结果证明Bt基因已插入到这些植株的DNA上,并表达出苏云金杆菌杀虫蛋白的杀虫活性。 相似文献
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A simple method for DNA extraction from marine bacteria that produce extracellular materials 总被引:9,自引:0,他引:9
We present a simple method for extracting DNA from the marine bacteria Hahella chejuensis, a Streptomyces sp., and a Cytophaga sp. Previously, DNA purification from these strains was hindered by the presence of extracellular materials. In our extraction method, the marine bacteria are lysed by freezing and grinding in liquid nitrogen, and treated with SDS. The extracted DNA is purified using a phenol/chloroform mixture, and precipitated in isopropanol. The extracted DNA is of high quality and suitable for molecular analyses, such as PCR, restriction enzyme digestion, genomic DNA blot hybridization, and genomic DNA library construction. We used this method to extract genomic DNA from several other marine bacteria. Our method is a reproducible, simple, and rapid technique for routine DNA extractions from marine bacteria. Furthermore, the low cost of this method makes it attractive for large-scale studies. 相似文献
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Wilson R 《Nucleic acid therapeutics》2011,21(6):437-440
The preparation of single-stranded DNA from double-stranded PCR products is an essential step in the identification of aptamers by Systematic Evolution of Ligands by EXponential enrichment (SELEX). The most widely used method for producing single-stranded DNA is alkaline denaturation of biotinylated PCR products attached to streptavidin-coated magnetic beads. Recently, it has been suggested that this method may be unsuitable due to the release of interfering amounts of streptavidin and biotinylated DNA. In this article, the alkaline method is compared with a thermal method that is known to release significant amounts of streptavidin and biotinylated DNA. Results show that trace amounts of streptavidin and biotinylated DNA are released in the alkaline method, but this can be curtailed by preconditioning the beads in aqueous sodium hydroxide. The main product in the alkaline method is single-stranded DNA, which is produced in high yield. 相似文献
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DNA is one of the most basic and essential genetic materials in the field of molecular biology.To date,isolation of sufficient and good-quality DNA is still a challenge for many plant species,though various DNA extraction methods have been published.In the present paper,a recycling DNA extraction method was proposed.The key step of this method was that a single plant tissue sample was recycled for DNA extraction for up to four times,and correspondingly four DNA precipitations(termed as the 1st,2nd,3rd and 4th DNA sample, respectively) were conducted.This recycling step was integrated into the conventional CTAB DNA extraction method to establish a recycling CTAB method.This modified CTAB method was tested in eight plant species,wheat,sorghum,barley,corn,rice,Brachypodium distachyon,Miscanthus sinensis and tung tree.The results showed that high-yield and good-quality DNA samples could be obtained by using this new method in all the eight plant species.The DNA samples were good templates for PCR amplification of both ISSR and SSR markers.The recycling method can be used in multiple plant species and can be integrated with multiple conventional DNA isolation methods,and thus is an effective and universal DNA isolation method. 相似文献
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This paper describes the effective determination of DNA scission site using a novel approach that is based on the Sanger method of nucleotide sequencing. The DNA scission site is determined by contrast with the nucleotide sequence of the DNA. Here, instead of the traditional Maxam-Gilbert method for the determination of the DNA sequence, we utilized the Sanger method and studied its effectiveness in the determination of DNA scission sites. Using this method, the determination of DNA scission site becomes more facile and exact. And the total time for the determination is reduced nearly by half in comparison to the Maxam-Gilbert method. Further advantages of this novel approach include the reduced risks of radiation exposure for researchers and contamination of the apparatus. 相似文献
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改进的SDS-CTAB法提取濒危植物连香树总DNA 总被引:17,自引:0,他引:17
对珍稀濒危植物连香树(Cercidiphyllum japonicum)的6种总DNA提取方法进行了对比试验,结果表明改进的SDS-CTAB法更适合于连香树总DNA提取。该方法提取的DNA经紫外消光值检测,其A260/A280为1.8532,优于CTAB法(1.4872)、SDS法(1.3552)、PVP法(1.5079)、尿素法(1.1858)和高盐低pH法(1.4534)。琼脂糖凝胶电泳和PCR扩增结果也得出同样的结论。 相似文献
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Helen E. O'connor David R. Stevens Stuart V. Ruffle Jonathan H. A. Nugent Saul Purton 《Plant Molecular Biology Reporter》1993,11(3):207-211
The isolation of chloroplast DNA fromChlamydomonas reinhardtii requires the efficient separation of this AT-rich genome from the GC-rich nuclear genome by density-gradient centrifugation.
We describe a simple and efficient method for separating these DNA fractions by using a sodium iodide gradient in combination
with the DNA-binding dye, bisbenzimide. The yield of chloroplast DNA is close to the theoretical maximum and the DNA is suitable
for restriction enzyme analysis and cloning. This method is applicable to the isolation of AT-rich plastid genomes from other
organisms and may be appropriate as a general method for separating species of DNA that differ in their AT/GC ratios.
An erratum to this article is available at . 相似文献
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水稻基因组DNA微量提取 总被引:2,自引:0,他引:2
随着植物学向分子水平的深入发展,研究中经常需要获得高质量的植物DNA样品,因此,建立植物DNA提取与纯化的常规实验方法对教学和科研都显得非常必要。介绍一种快速提取微量DNA的方法,该方法简单易行,无需任何特殊设备,所需样品量少,提取的DNA纯度高,可满足以PCR扩增为基础的实验需要。 相似文献