Primer Design and Optimization for RAPD Analysis of Nepenthes

Primers with higher G+C content produced better random amplified polymorphic DNA (RAPD) profiles in Nepenthes. The occurrence of clustered G's and C's in the center of the primer seemed also to influence the banding patterns. It was also observed that for certain polymerases, the use of different buffers other than that recommended by the manufacturer provided a better amplification profile for Nepenthes.     

烟草RAPD双引物与单引物PCR扩增多态性效率的比较分析

以两个亲缘关系较远的烟草（Nicotiana tabacum）品种台烟7号和白肋21为材料（GS=0.58）,研究了单引物扩增和双引物聚合扩增对RAPD-PCR分子标记多态性的影响。结果显示：双引物反应能够比单引物反应扩增出更多的多态性片段,双引物在白肋21和台烟7号中扩增出的多态性片段总数是单引物反应扩增出的多态性片段总数的6.6倍。因此,双引物RAPD的运用有助于提高烟草多态性分子标记的有效性。     

采用随机引物组合对绞股蓝作RAPD分析及SCAR鉴定

为寻找简单、可重复的分子鉴定方法,对绞股蓝（Gynostemma pentaphyllum（Thunb．）Makino）新鲜品、药材干品及其混淆品乌蔹莓（Cayratia japonica（Thunb．）Gagnep）进行了鉴定。首先从2000余条10碱基的随机引物中筛选出20条,并以5条为1组随机分成4组,然后采用RAPD方法对7份不同来源的绞股蓝新鲜品进行扩增,对扩增得到的绞股蓝特征条带进行基因克隆、测序,分析序列特征,再设计特异性引物,最后．用绞股蓝新鲜品、药材干品及其混淆品乌蔹莓等样品进行PCR验证。结果显示：RAPD扩增能够得到7份绞股蓝新鲜品重复的共有特征条带;经基因克隆、测序并设计特异性引物后进行PCR验证,获得了绞股蓝特异序列扩增区域标志（Sequence—characterized amplified region maker,SCAR）。初步建立了区分绞股蓝及其混淆品乌蔹莓的分子鉴定标准,并首次将SCAR应用于绞股蓝分子鉴定中。     

Primer R108 performs best in the RAPD strain typing of threeAspergillus species frequently isolated from patients

We evaluated the suitability of various primers for the RAPD (random amplified polymorphic DNA) accurate species identification and strain typing of Aspergillus clinical isolates. Five primers described previously were tested for their discriminatory power in three Aspergillus species (A. fumigatus, A. niger agg. and A. flavus - 23 clinical isolates and 2 reference strains). Clustering of RAPD fingerprints corresponded well with the identification based on morphological features. All isolates were resolved as different strains using the primer R108 and the RAPD protocol optimized for a Robocycler thermal cycler. RAPD with the primer R108 thus can be considered to be a valuable, simple and powerful tool for identification and strain delineation of Aspergillus spp.     

悬钩子属植物的RAPD分析

用RAPD标记技术对13个黑莓（blackberry）、树莓（raspberry）品种和5个野生悬钩子种类（Rubus spp．）的26个居群的遗传多样性进行了分析。用15个寡聚核苷酸随机引物共扩增出条带131条,其中多态性条带118条,占扩增条带总数的90％,表明悬钩子属植物种间和品种间存在丰富的遗传多样性。通过聚类分析可将供试的26个居群分为4组,其中A组有粗叶悬钩子（R．alceaefolius Poir．）、山莓（R．corchorifolius L．）、插田泡（R．coreanus Miq．）、掌叶覆盆子（R．chingii Hu）、‘威廉姆特’、‘托拉咪’、‘泰勒’、‘金克维’、‘布里斯托’、‘宝森’7号、‘布莱兹’及其实生苗、‘基奥瓦’、‘萨尼’、‘黑布特’等15个居群;B组包括‘宝森’1号～‘宝森’6号和‘马林’、‘乔克多’等8个居群;C组包括‘赫尔’和‘切斯特’的2个居群;D组仅有蓬藁（R．hirsutus Thunb．）1种。这一聚类结果与传统形态学分类结果基本吻合。     

利用RAPD分析鉴定花椰菜杂种纯度

应用随机扩增多态性DNA(RAPD)分析方法,鉴定花椰菜F1代杂种纯度。筛选出20个10bp随机引物对杂种F1代和父母本基因组DNA进行RAPD分析,共获得扩增片段124条,分子量在0.3-3kb之间,其中2个引物S120和S174可用来鉴定杂种纯度。RAPD分析结果与田间形态鉴定结果基本一致,表明RAPD分析方法适用于花椰菜杂种的纯度鉴定。     

RAPD analysis of Yersinia enterocolitica

A total of 87 isolates of Yersinia enterocolitica were examined with randomly amplified polymorphic DNA (RAPD) by use of three different primers. Based on the RAPD profiles, the strains could be divided into three major groups: (1) the pathogenic American serotypes, O: 8, O: 13ab, O: 20 and O: 21; (2) the pathogenic European serotypes, O: 3, O: 5,27 and O: 9; and (3) the nonpathogenic serotypes. Five tested strains of the American serotype O: 4 gave unique profiles with YCPEL, but did not give reproducible profiles with the other primers. The European serotypes could be further subdivided into a group consisting of strains of O: 3 and O: 5,27 and a group of strains of O: 9. RAPD profiling provides an easy approachable method to divide isolates of Y. enterocolitica into pathogenic and nonpathogenic strains and further to differentiate between the pathogenic isolates.     

泡桐属七种植物的RAPD分析

利用 1 3个 1 0 mer的随机引物对中国泡桐属的 7个种进行了 RAPD分析 ,对扩增形成的谱带进行了对比 ,发现 7个种间存在较丰富的多态性带纹。经用 UPGMA法聚类结果说明可把研究的 7个种分成 2个类群。其中白花泡桐与川泡桐亲缘关系最近 ,二者与揪叶泡桐、南方泡桐、山明泡桐归为 1个类群。毛泡桐与兰考泡桐亲缘关系也较近 ,二者在系统演化上比较原始 ,归为 1个类群。     

葱属12种植物的RAPD分析

采用随机引物扩增多态DNA技术对葱属部分植物进行了种间亲缘关系的研究。结果说明12种材料之间存在丰富的多态性,遗传距离变幅在0.2500-0.7887之间,聚类分析说明蒙古韭,山韭,野韭,韭菜（栽培韭）,野生韭菜,矮韭亲缘关系较近,聚为一支,其中韭菜与野生韭菜亲缘关系最近。天蒜,薤白,蒜聚为一支,葱,洋葱,红葱聚为一支,其中葱与洋葱,红葱的遗传分化较大。     

Primer design for PCR and sequencing in high-throughput analysis of SNPs

To achieve high-throughput analysis of allele frequencies in human SNPs, we have developed automated methodsfor designing PCR and DNA sequencing primers. We found we could run the PCR assays at quite stringent, uniform conditions. The design process used freely available databases, including dbSNP, SNPper, and TSC, and publicly available software including RepeatMasker and Primer3. We describe parameters for the software and other considerations that increase experimental success. As anticipated. some assays filed at the design stage due primarily to the genomic locations of repetitive sequences, extreme GC content regions, or lack of sufficient sequence. However, over 23,000 assays, including 96% of those recently analyzed, have been experimentally successfuL Similar design methods could be usedfor PCR assays in any organism with substantial available sequence.     

RAPD analysis of genome polymorphism in the family Lemnaceae

The multilocus RAPD analysis of intergeneric, inter- and intraspecific nuclear genome polymorphism was used for the first time to assess intergeneric, interspecific, and intraspecific polymorphism in Lemnaceae growing on the territory of Russia. The origin of the chosen accessions overlapped with the natural range of duckweeds in Russia. Seventy-five Lemnaceae accessions representing eight species (L. minor, L. gibba, L. turionifera, L. japonica. L. trisulca, L. aequinoctialis, S. polyrhiza, and L. punctata) from three genera (Lemna, Spirodela, and Landoltia), were analyzed. The highest variability levels were revealed in L. minor accessions (0.03-0.20). Species L. trisulca and S. polyrhiza were characterized by values of genetic distance 0.01-0.18 and 0.03-0.16, respectively. The lowest polymorphism levels were detected for L. turionifera (0.01-0.11). The dendrogram based on RAPD data showed that L. aequinoctialis was the most genetically distant species of the genus Lemna. Accessions of species L. turionifera and L. japonica, as well as L. minor and L. gibba, did not form separate species-specific subclusters; rather, they fell into clusters with L. japonica/L. turionifera and L. minor/L. gibba. Accessions of the genera Spirodela and Landoltia formed two separate clusters combined into one group.