RAPD分析用白花丹参叶片DNA的提取方法

本研究采用改良CTAB法和SDS法提取新鲜白花丹参叶片的基因组DNA,初步筛选RAPD扩增引物。结果表明改良CTAB法提取的DNA较SDS法质量好,随机引物p2扩增条带相对较为清晰。再利用p2随机引物对2种方法提取的DNA进行RAPD检测比较,结果显示仅改良CTAB法能扩增出有效条带,说明改良CTAB法更适合用于白花丹参基因组DNA的RAPD检测分析。本文将为白花丹参叶片DNA的提取提供方法学参考。     

缢蛏基因组DNA的提取及其效果分析

应用酚/氯仿抽提法提取缢蛏基因组DNA,并以所提取的基因组DNA为材料进行酶切反应及RAPD分析。结果显示,用酚/仿提取法提取的缢蛏基因组DNA质量高、稳定性好,酶切效果明显,RAPD扩增条带清晰,能满足DNA的酶切、PCR和RAPD等分子生物学实验对于模板DNA质量高的要求。     

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。     

番茄灰霉病生防融合子基因组DNA提取方法的研究

以番茄灰霉病生防菌株木霉T-23和链霉菌A的融合子为实验材料,在SDS-CrAB法、改进CTAB法和氯化苄法的基础上加以改进,比较和研究了真菌融合子基因组DNA的提取,找到了一种快速、高效的基因组DNA提取方法,为进一步对融合子进行生防机制和分子生物学水平的研究提供基础。结果表明SDS-CTAB法提取的基因组DNA OD_(260)/OD_(280)为1．909,DNA浓度约为42．0ng/μL,可以满足分子生物学实验的需要。并将提取的基因组DNA直接用于PCR扩增,得到了多态性的RAPD图谱。     

太白红杉3种不同材料总DNA的提取

高质量DNA的获得是进行各项遗传操作的基础,提取DNA的方法、材料因不同的植物而异。我们用改良的CTAB法,对太白红杉的3种不同材料的基因组总DNA进行了提取,均成功获得了适于RAPD分析的总DNA。结果表明,提取太白红杉DNA并对其进行研究,幼苗是较佳材料,愈伤组织也是较好的可试材料,而针叶则相对较差。     

四种提取芸芥基因组DNA方法的比较

以芸芥为材料 ,分别用CTAB法、SDS法、尿素法、NaOH法四种方法对芸芥基因组DNA进行了提取 ;并用紫外光分光光度计法、琼脂糖电泳法和RAPD分析法对所提取的DNA进行检测 ,将它们在DNA的产量、质量和耗时、耗费等方面的优缺点进行比较 ,以便在实际工作中根据不同的试验条件选取最合适的提取方法。通过四种方法的比较 ,研究认为尿素法是芸芥基因组DNA的最佳提取方法。     

香蕉枯萎菌基因组DNA提取方法的研究

以香蕉枯萎菌菌株为试验材料,在SDS～CTAB法和高盐沉淀法等基础上加以改进,对两种提纯香蕉枯萎菌基因组DNA的方法进行了比较研究。结果表明：高盐沉淀法是适合于香蕉枯萎菌基因组DNA提取的方法。该方法提取的DNA OD260／OD280的比值为1．841,DNA产量为0．81mgDNA／g菌丝体。基因组DNA经琼脂糖凝胶电泳得到一条带型较宽且清晰的DNA谱带,基本无DNA碎带;将提取的DNA直接用于PCR扩增,得到带多而且清晰、整齐、基本无拖尾的RAPD图谱。     

石斛干品基因组DNA的提取与RAPD分析

市场中药干品的药性差异一直是影响中药标准化的瓶颈,而检测技术相对落后是导致这一现象的主要原因。DNA分子水平检测的困难是药材干品的基因组DNA难以提取。本文以铁皮石斛(Dendrobium candidum)干茎为材料,采用了四种方法从干品石斛中提取基因组DNA。结果表明,采用改良的CTAB法可从石斛干品尤其是干茎皮中提取质量较高的基因组DNA,其分子量大于23kb,以此DNA为模板进行不同引物的PCR扩增可获得清晰的RAPD条带。该研究初步建立了石斛干品合适的RAPD技术体系。     

卡瓦胡椒RAPD反应体系的建立

卡瓦胡椒RAPD分子标记的研究,目前国内外尚未见有报道。该试验通过CTAB法提取卡瓦胡椒基因组DNA,通过对模板DNA用量、Mg^2＋浓度、退火温度、电泳上样量等几个单因子试验来建立RAPD稳定扩增体系和反应条件,RAPD扩增结果重复性好,稳定可靠,为卡瓦胡椒RAPD分子标记的研究打下基础。     

RAPD条件优化及天麻基因组DNA多态性分析

建立了RAPD扩增条件快速优化程序与方法．并应用于天麻基因组DNA扩增条件的优化及多态性的测定：获得了天麻基因组DNA的RAPD扩增优化条件和DNA指纹图谱;分析了模板DNA、引物、dNTP、Taq DNA聚合酶等的浓度和退火温度对RAPD扩增的影响．结果表明：天麻基因组DNA用引物S1扩增的片段具有更明显的多态性,这种指纹图谱更适合于天麻遗传分化研究;而用引物S12扩增的DNA指纹图谱具有更大的相似性,这种指纹图谱更适合于天麻真伪鉴别．该方法使RAPD扩增条件优化过程实现了程序化和数量化,是获得RAPD优化条件的简便快速、经济实用方法．应用该方法进行RAPD扩增,可获得图谱清晰、稳定可靠的实验结果．     

两个地区东方田鼠基因组RAPD分析比较研究

目的　从DNA的水平分析比较两个地区东方田鼠的分子遗传特征,探讨以RAPD标记鉴别两个地区的东方田鼠。方法　筛选6条10bp的随机引物对洞庭湖和青铜峡地区的东方田鼠基因组进行了随机扩增多态DNA(RAPD)分析,并对这两个地区的东方田鼠的基因组DNA进行了比较。结果　①两个地区东方田鼠的所有受试个体中共有的片段数为20条,这是两个地区东方田鼠的共性所在;②两个地区东方田鼠各有其特异性扩增片段;③引物S17和S80可作为鉴别两个地区东方田鼠的特异性引物;④不同地区的东方田鼠其不同个体之间的共享度较低,且存在较大差异;两个地区东方田鼠的遗传背景均呈非均一性。结论　运用RAPD方法可以作为鉴别不同地区东方田鼠的基因多态性的标记。     

Rapid estimation of genetic relatedness among heterogeneous populations of alfalfa by random amplification of bulked genomic DNA samples

A procedure which involves the use of RAPD markers, obtained from bulked genomic DNA samples, to estimate genetic relatedness among heterogeneous populations is demonstrated in this study. Bulked samples of genomic DNA from several alfalfa plants per population were used as templates in polymerase chain reactions with different random primers to produce RAPD patterns. The results show that the RAPD patterns can be used to determine genetic distances among heterogeneous populations and cultivars which correspond to their known relatedness. The results also indicate that, by using ten primers with bulked DNA samples from ten individuals, 18–72 populations or cultivars can be distinguished from each other on the basis of at least one unique RAPD marker. We anticipate that DNA bulking and methods for comparing RAPD patterns will be very useful for identifying cultivars, for studying phylogenetic relationships among heterogeneous populations and for selecting parents to maximize heterosis in crosses.     

DNA fragments of organellar origin in random amplified polymorphic DNA (RAPD) patterns of sugar beet (Beta vulgaris L.)

The technique of random amplified polymorphic DNA (RAPD) offers a broad range of applications in the investigation of plant genomes. A promising prospect is the use of RAPD products as genetic markers. We have investigated a possible organellar source of fragments in RAPD patterns of total DNA. Two nearly-isogenic lines of cytoplasmic male-sterile and male-fertile sugar beet (Beta vulgaris L.) were subjected to RAPD analysis with six different primers. Total, nuclear, mitochondrial (mt), and chloroplast (cp), DNA from each line were investigated. Reproducible DNA fingerprints could be obtained from both organellar DNAs. Differences in band patterns of mtDNA between cytoplasmic male-sterile and -fertile lines were observed with five out of six primers, whereas different cpDNA patterns were generated by one of the primers. Consequently, the RAPD technique can be used to discriminate between different cytoplasms. Clear evidence is provided for the organellar origin of fragments in genomic (total DNA) RAPD patterns. The consequences of these results for the interpretation of RAPD analyses are discussed.     

Quantitative Properties of Random Amplified Polymorphic DNA (RAPD)

Random Amplified Polymorphic DNA analysis (RAPD) is a methodology that has been used as a tool for monitoring microbial communities. To be useful in this application RAPD, and any other methodology, must show properties that allows for the detection of quantitative changes in composition of the microbiota. Therefore, the objective of this study was to establish whether RAPD possesses such properties. The strategy was to use genomic DNA, extracted from a set of tertiary bacterial mixtures defined according to an experimental mixture design, and containing varying proportions of Escherichia coli, Bacillus subtilis, and Pseudomonas CF600. RAPD-PCR was performed on the mixed DNA extracts and the amplified DNA fragments were separated on sequencing gels to produce genomic fingerprints that were digitized and modeled by Partial Least Squares regression (PLS). Significant predictions were obtained using an external test set for validation, with Root Mean Square Error of Predictions (RMSEP) of 0.21, 0.19 and 0.20 for the proportion of E. coli, B. subtilis and Pseudomonas CF600 respectively. Taken together, the results showed that RAPD patterns quantitatively represented the initial mixture proportions. Therefore, the view that RAPD could be useful for whole microbial community monitoring was strengthened.     

梨不同DNA提取方法的效果研究

以7个梨品种为实验材料,比较分析了SDS法、CTAB法、SDSCTAB法、改良的CTAB法、高盐低pH值法、分步离心法对梨总DNA提取的效果。结果表明:利用以上6种方法提取的梨总DNA在纯度和量上有很大的差别。所得到的平均DNA量从大到小依次为:分步离心法、SDS法、SDSCTAB法、改良的CTAB法、CTAB法、高盐低pH值法。DNA提取纯度依次为分步离心法、SDSCTAB法、改良的CTAB法、高盐低pH值法、CTAB法、SDS法。RAPD和自交不亲和基因(S基因)特异性引物扩增实验结果都比较理想,但分步离心法和SDSCTAB法提取的DNA双酶切效果较好。分步离心法提取的梨总DNA更适用于后续的分子生物学实验操作。     

Identification of structural DNA variations in human cell cultures after long-term passage

Random amplified polymorphic DNA (RAPD) analysis was adapted for genomic identification of cell cultures and evaluation of DNA stability in cells of different origin at different culture passages. DNA stability was observed in cultures after no more than 5 passages. Adipose-derived stromal cells demonstrated increased DNA instability. RAPD fragments from different cell lines after different number of passages were cloned and sequenced. The chromosomal localization of these fragments was identified and single-nucleotide variations in RAPD fragments isolated from cell lines after 8–12 passages were revealed. Some of them had permanent localization, while most variations demonstrated random distribution and can be considered as de novo mutations.     

洞庭湖四种黄颡鱼基因组DNA遗传多样性的RAPD分析

以洞庭湖瓦氏黄颡鱼、光泽黄颡鱼、长须黄颡鱼、普通黄颡鱼的基因组DNA为材料对 4种黄颡鱼进行了遗传多样性随机扩增多态性DNA(RAPD)分析。通过筛选的 90个引物对 4种黄颡鱼基因组DNA的扩增 ,获得了 36个有效引物 ,并计算出了 4种黄颡鱼群体间的遗传相似系数和遗传距离 ,遗传距离最大的为普通黄颡鱼和瓦氏黄颡鱼 (D =0 9895 ) ,遗传距离最小的为普通黄颡鱼和光泽黄颡鱼 (D =0 672 0 )。同时运用聚类分析 (UPGMA)的方法建立了 4种黄颡鱼的聚类图。