基于AFLP分析用吴茱萸叶高质量DNA的提取

目的：研究吴茱萸叶基因组DNA的提取方法,以用于扩增片段长度多态性（AFLP）分析。方法：设计了一种改良CTAB法：以石英砂代替液氮研磨;抽提前用不溶性PVP结合酚形成络合物,然后用缓冲液除去;抽提中加入Vc。将改良CTAB法所提吴茱萸叶DNA与传统的SDS法、CTAB法所提DNA进行比较。利用植物的核糖体DNA（rDNA）保守序列设计引物行PCR扩增鉴定吴茱萸DNA及其质量。并确定提取方法中最佳样本含量和β-巯基乙醇浓度。结果：改良CTAB法提取石虎、疏毛吴茱萸总DNA呈白色,A260/A280为1.721~1.886,DNA分子完整,约20kb左右,PCR扩增条带清晰、明亮,无杂带和脱尾。并确定0.10g为最佳样本量,2.0%为最佳β-ME浓度。结论：石英砂研磨简便、迅速、均匀,该实验所建立的改良CTAB法可有效避免次生代谢物的氧化褐变,是一种小量、快速提取吴茱萸叶DNA优化方法。     

A Rapid DNA Extraction Method for RFLP and PCR Analysis from a Single Dry Seed

A single-seed DNA extraction method was developed for rapid identification of plant genotype. The method was applied to 12 plant species, including the oil seeds sesame and soybean. The results were comparable to those obtained for oil-less seeds such as rice. This method will be useful for genotypic selection which requires rapid screening of large populations. It can also be used to identify varietal purity of seed stocks by PCR and RFLP analysis. The method includes two major steps, (i) treatment by proteinase K in an SDS extraction buffer, and (ii) grinding of a single half seed in the buffer after incubation. About 1.5–2 µg of DNA per half seed (the endosperm part) of rice was obtained and more than 200 half seed samples could be handled by one person in a day. The DNA could be used for fingerprinting and detection of target genes in a transgenic plant by PCR. The amplified PCR products from the half seed DNA exhibited the same banding patterns as those from leaf DNA. Yield and quality of DNA extracted from half seeds of rice was also sufficient for RFLP analysis. The remnant half seeds containing the embryo can be maintained for later germination of selected genotypes.     

A Simple, Reliable, and Fast Protocol for Thraustochytrid DNA Extraction

DNA extraction of thraustochytrids, common marine unicellular organisms, is usually accomplished by either the cetyltrimethylammonium bromide (CTAB) or proteinase K protocols. A novel lysis buffer protocol for thraustochytrid total DNA extraction is described. The average isolated total DNA is 20 to 40 kb, and DNA samples are suitable for a variety of uses including 18S–ribosomal DNA polymerase chain reaction, restriction enzyme digestions, and amplified fragment length polymorphism analyses. The new protocol is also faster than the other protocols. Received July 31, 2000; accepted November 2, 2000.     

Repetitive DNA of grapevine: classes present and sequences suitable for cultivar identification

Summary Repetitive DNA sequences present in the grapevine genome were investigated as probes for distinguishing species and cultivars. Microsatellite sequences, minisatellite sequences, tandemly arrayed genes and highly repetitive grapevine sequences were studied. The relative abundance of microsatellite and minisatellite DNA in the genome varied with the repeat sequence and determined their usefulness in detecting RFLPs. Cloned Vitis ribosomal repeat units were characterised and showed length heterogeneity (9.14–12.15 kb) between and within species. A highly repetitive DNA sequence isolated from V. vinifera was found to be specific only to those species classified as Euvitis. DNA polymorphisms were found between Vitis species and between cultivars of V. vinifera with all classes of repeat DNA sequences studied. DNA sequences suitable for DNA fingerprinting gave genotype-specific patterns for all of the cultivars and species examined. The DNA polymorphisms detected indicates a moderate to high level of heterozygosity in grapevine cultivars.On leave from the Biochemical Research Institute, Nippon Menard Cosmetic Co, Ltd, Ogaki Gifuken, 503 Japan     

A Simple and Rapid Procedure for Isolation of Total DNA Suitable for Fingerprint Analysis from shape Amaranthus

A simple, efficient and reliable method is described for isolation of total DNA from young leaves of Amaranthus species. This procedure yields a high amount (600–800 µg DNA/g fresh leaf tissue) of good quality DNA free from contaminating proteins, polysaccharides, and coloured pigments. The DNA is suitable for digestion with several restriction endonucleases, preparation of Southern blots, and PCR amplification. The DNA has been successfully used for generating DNA fingerprint profiles and RAPD banding patterns in two species of Amaranthus. The procedure is suitable for processing of a large number of samples simultaneously.     

快速、高效的羊绒羊毛织品DNA提取方法的建立

目的：建立一种快速、高效的羊绒羊毛纺织品DNA提取的方法。方法：采用chelex-100法的3种处理、试剂盒法分别提取羊绒羊毛织品的DNA,用18S rDNA片段、山羊和绵羊源性成分PCR扩增结果来比较提取效果。结果：试剂盒法提取DNA的效果优于chelex-100法,整个提取过程约需2h。9种供试材料均提取到DNA,且含有山羊和/或绵羊源性成分,与显微镜观察结果的符合率为100%。结论：建立的试剂盒法是一种快速、高效的适用于羊绒羊毛织品DNA提取的方法,为应用分子生物学方法鉴别山羊绒和绵羊毛奠定了基础。     

锈菌夏孢子DNA的微量快速提取方法

采用4种方法对毛白杨锈病单个夏孢子堆DNA提取和ITS—PCR扩增表明,钢珠法、玻片法均是锈菌基因组DNA微量快速提取的合适方法,以钢珠法最优,整个过程仅需30min,并且可以获得与CTAB法、氯化苄法相同的PCR扩增结果。钢珠法是在提取液中加入2-3颗钢珠,靠钢珠在涡旋中的相互碰撞将夏孢子破壁,以同时加入NaOH(终浓度3．3％)和Chelex-100(终浓度1．6％)效果最好,ITS-PCR扩增能稳定得到700bp片段。玻片法也能得到相同的结果。分析比较了4种方法的优缺点。     

Systematic Cross-biospecimen Evaluation of DNA Extraction Kits for Long- and Short-read Multi-metagenomic Sequencing Studies

High-quality DNA extraction is a crucial step in metagenomic studies. Bias by different isolation kits impairs the comparison across datasets. A trending topic is, however, the analysis of multiple metagenomes from the same patients to draw a holistic picture of microbiota associated with diseases. We thus collected bile, stool, saliva, plaque, sputum, and conjunctival swab samples and performed DNA extraction with three commercial kits. For each combination of the specimen type and DNA extraction kit, 20-gigabase (Gb) metagenomic data were generated using short-read sequencing. While profiles of the specimen types showed close proximity to each other, we observed notable differences in the alpha diversity and composition of the microbiota depending on the DNA extraction kits. No kit outperformed all selected kits on every specimen. We reached consistently good results using the Qiagen QiAamp DNA Microbiome Kit. Depending on the specimen, our data indicate that over 10 Gb of sequencing data are required to achieve sufficient resolution, but DNA-based identification is superior to identification by mass spectrometry. Finally, long-read nanopore sequencing confirmed the results (correlation coefficient > 0.98). Our results thus suggest using a strategy with only one kit for studies aiming for a direct comparison of multiple microbiotas from the same patients.     

土壤细菌DNA提取及多样性分析的T-RFLP方法

获得高质量的土壤总DNA是土壤细菌生态学的关键步骤之一.实验通过综合应用两个试剂盒(Soilmaster kit和DNA IQTM系统)的优点进行土壤样品总DNA的提取,结果证明该方法是一种快速、有效、灵敏、稳定的土壤DNA提取方法.另外尝试将16S rDNA序列和T-RFLP(Terminal restriction fragment 1ength polymorphism)技术引入土壤细菌DNA群落多样性的研究中,证明T-RFLP是一种有力的土壤细菌多样性分析工具.     

A Simple Method for Isolating DNA of High Yield and Quality from Cotton (shape Gossypium hirsutum L.) Leaves

An easy, reproducible and fast procedure to isolate DNA from cotton leaves is described. The addition of 0.5 M glucose in the extraction buffer avoids browning by polyphenolic compounds and improves the quality of DNA for molecular analysis. The DNA yield ranged between 150–400 mg per gram of fresh tissue. The DNA was suitable for digestion by restriction enzymes and amplificatiion by Taq DNA polymerase.     

A Simple and Rapid Extraction of High Molecular Weight Chromosomal DNA from Bacillus subtilis Protoplasts for Cosmid Cloning and Interspecific Transformation

After conversion of Bacillus subtilis vegetative cells to protoplasts, a simple and rapid method for extracting high-molecular-weight chromosomal DNA was devised with the inclusion of bovine serum albumin and phenol-chloroform treatments. The DNA sample thus prepared was the size of 100-450 kb and could be used for cosmid cloning and interspecific transformation.     

不同方法提取组培百里香精油质量及成分的比较分析

采用水蒸气蒸馏法、有机溶剂萃取法、CO2超临界萃取法3种提取方法提取组培百里香精油,比较分析精油得率、精油化学成分以及相对含量,以期得出最佳提取方法。结果表明：水蒸气蒸馏法提取的百里香精油得率为O．21％,主要化学成分为：百里酚（36．53％）、间伞花烃（14．13％）、松油烯（8．09％）和石竹烯（4．14％）;有机溶剂萃取法提取的精油得率为0．19％,主要化学成分为：1,2-苯二甲酸-单-2-乙基己基酯（55．23％）、百里酚（873％）、松油烯（5．23％）;CO2超临界萃取法提取的精油得率为0．27％,主要化学成分为：百里酚（26．68％）、3-苯基-2-丙烯酸-甲酯（21．55％）、间伞花烃（9．69％）。从精油得率、精油质量以及精油主要化学成分综合比较3种方法,水蒸气蒸馏法是提取百里香精油的最佳方法。     

Plant highly repeated satellite DNA: Molecular evolution,distribution and use for identification of hybrids

Relationships among genomes are often revealed by the occurrence of common or related satellite DNA (satDNA) types. A typical satDNA characterized by specific sites for one (or more) restriction endonuclease(s) is called ‘restriction satellite DNA’. Restriction satDNA comprises ‐ in addition to transposons and retrotransposable elements ‐ often highly repeated genome components present in most higher plants. Large arrays of satDNA elements are concentrated at subtelo‐meric and/or centromeric regions (intermingled with other retrotransposon‐derived elements), however, they can be also located as large intercalating blocks along the chromosome. The head‐to‐tail tandemly arranged repeat units (monomers) of satDNA mostly exhibit lengths of 160 to 180 bp or 320 to 370 bp, but other lengths were also found in plants. In particular, in interspecific hybrids between more distantly related species, which often exist only after polyploidization, the individual repetitive DNA of the crossing partners contribute to recombination and rearrangement processes in the hybrids, thereby stimulating genome evolution. Here, we concentrate on the possible origin, molecular evolution, organization and distribution of highly repeated satDNA in various higher plants with emphasis on hybrids and allopolyploids.     

Comparative Analysis of Genetic Chemotyping Methods for Fusarium: Tri13 Polymorphism Does not Discriminate between 3‐ and 15‐acetylated Deoxynivalenol Chemotypes in Fusarium graminearum

Genetic chemotyping is an essential tool for characterizing Fusarium populations causing head blight on wheat and other cereals. Three PCR methods, based on tri cluster polymorphism, were optimized and compared on 94 single‐spore isolates obtained from three continents belonging to F. gramineaurm, F. culmorum, F. poae, F. avenaceum and Microdochium nivale. While the methods based on the tri3, tri7 and tri12 polymorphism correctly identified all the tested strains, the method based on tri13 polymorphism was unable to discriminate between the 3‐ and 15‐acetylated DON forms in F. graminearum. It is advised to avoid the use of tri13 polymorphism for genetic chemotyping of the two acetylated chemotypes.     

Polymorphisms of glutathione S-transferases M1, T1, P1 and A1 genes in the Tunisian population: an intra and interethnic comparative approach

Genetic polymorphisms in glutathione S-transferases (GSTs) genes might influence the detoxification activities of the enzymes predisposing individuals to cancer risk. Owing to the presence of these genetic variants, inter-individual and ethnic differences in GSTs detoxification capacity have been observed in various populations. Therefore, the present study was performed to determine the prevalence GSTM1 0/0, GSTT1 0/0, GSTP1 Ile(105)Val, and GSTA1 A/B polymorphisms in 154 healthy individuals from South Tunisia, and to compare them with those observed in North and Centre Tunisian populations and other ethnic groups. GSTM1 and GSTT1 polymorphisms were analyzed by a Multiplex-PCR approach, whereas GSTP1 and GSTA1 polymorphisms were examined by PCR-RFLP. The frequencies of GSTM10/0 and GSTT1 0/0 genotypes were 53.9% and 27.9%, respectively. The genotype distribution of GSTP1 was 47.4% (Ile/Ile), 40.9% (Ile/Val), and 11.7% (Val/Val). For GSTA1, the genotype distribution was 24.7% (A/A), 53.9% (A/B), and 21.4% (B/B). The combined genotypes distribution of GSTM1, GSTT1, GSTP1 and GSTA1 polymorphisms showed that thirty one of the 36 possible genotypes were present in our population; eight of them have a frequency greater than 5%. To the best of our knowledge, this is the first report of GSTs polymorphisms in South Tunisian population. Our findings demonstrate the impact of ethnicity and reveal a characteristic pattern for Tunisian population. The molecular studies in these enzymes provide basis for further epidemiological investigations in the population where these functional polymorphisms alter therapeutic response and act as susceptibility markers for various clinical conditions.     

Intra-specific hybridization: generator of genetic diversification and heterosis in Andrographis paniculata Nees. A bridge from extinction to survival

Andrographis paniculata (AP) has been stated as a low-diverse, endangered and red-listed plant species. Self-pollinated mating system, being an introduced species and experiencing a bottleneck as well as over exploitation cause such a consequence. Inter and intra-specific hybridizations have been suggested as essential techniques for generating genetic diversity. To test the effect of intra-specific hybridization on diversification and heterosis of AP, seven accessions were outcrossed manually in all 21 possible combinations. Three types of markers including morphological, phytochemical and RAPD markers were employed to evaluate the mentioned hypothesis. The results revealed that hybridization acted as a powerful engine for diversification of AP as it caused heterotic expression of the studied traits, simultaneously. Initially, it seems that additive and non-additive gene effects both can be considered as the genetic basis of heterosis in AP for the investigated traits. Agronomic and morphological traits were differentiated from each other, while positive heterosis was recorded mainly for agronomic traits but not for the morphological traits. Intra-specific hybridization increased the genetic diversity in AP population. Nevertheless, a part of this variation could also be attributed to the negative heterosis. The current exploration demonstrated the first ever conducted manual intra-specific hybridization among AP accessions in a mass scale. However, the 17 RAPD primers produced a monomorph pattern, but perhaps increasing the number of markers can feature a new genetic profile in this plant.