A simple method for isolation of high-molecular-weight DNA from Rhodotorula gracilis

Unsheared DNA has been isolated from Rhodotorula and Rhodosporidium yeasts using a cell-wall-digesting enzyme preparation from Paecilomyces lilacinus. Pulsed-field gel electrophoresis indicated that at least 11 chromosomes were present in Rhodot. gracilis ATCC 90950. The DNA was amenable to digestion with restriction enzymes.The authors are with the Department of Microbiology, Central Food Technological Research Institute, Mysore-570 013, India.     

The isolation of high molecular weight DNA from wheat,barley and rye for analysis by pulse-field gel electrophoresis

A method is presented for the preparation of large DNA molecules from protoplasts embedded in agarose blocks of three different cereals-hexaploid bread wheat (Triticum aestivum), barley (Hordeum vulgare) and rye (Secale cereale). Pulse-field gel electrophoresis (PFGE) analysis of these DNA preparations using a contour-clamped homogeneous field (CHEF) apparatus indicated that the size of the DNA molecules was greater than 6 Mb. DNA samples prepared by this method were shown to be useful for restriction analysis using both frequent and rare cutting enzymes.     

PFGE analysis of the rice genome: estimation of fragment sizes,organization of repetitive sequences and relationships between genetic and physical distances

Pulsed-field gel electrophoresis (PFGE) has been applied to analyze the rice nuclear genome. Probing 56 RFLP probes selected from the 12 rice chromosomes to PFGE blots of nine rare-cutting restriction enzymes revealed that there are relatively high numbers of rare-cutting restriction sites in the rice genome. The average sizes of restriction fragments detected by single-copy probes are smaller than 200 kb for all of the rare-cutting restriction enzymes examined. Sizes of fragments detected by repetitive probes are variable, depending on the probes analyzed. By using PFGE, a tandemly repeated sequence, Os48, was found to be tightly linked to telomeric tandem repeats but not physically linked to r5s genes with which sequence homology had been observed. Relationships between genetic and physical distances have been established for three different chromosomal segments. In these regions 1 cm corresponds to ca. 260 kb on average. Analysis of a cluster of RFLP markers on chromosome 3 revealed that genetically clustered RFLP markers are also physically closely linked, suggesting that clustering of genetic markers may result in part from uneven distribution of single-copy sequences.     

A rapid and high yielding DNA miniprep for cotton (Gossypium spp.)

A rapid DNA minipreparation method was developed for cotton and yields 500–600 μg DNA from 1.0 g fresh leaf tissue. Cotton DNA extracted using this method is completely digested with restriction enzymes, supports PCR and Southern DNA analyses and was used successfully in these applications. An erratum to this article is available at .     

A rapid method for extraction of cotton (Gossypium spp.) genomic DNA suitable for RFLP or PCR analysis

Extraction of high-quality genomic DNA fromGossypium (cotton) species is difficult due to high levels of polysaccharide, oxidizable quinones, and other interfering substances. We describe a procedure that consistently permits isolation of cotton genomic DNA of satisfactory size and quality for RFLP and PCR analysis, as well as for most routine cloning applications. Several antioxidants, phenol-binding reagents, and phenol oxidase inhibitors are used throughout the procedure, and most polysaccharides are eliminated early in the procedure by isolation of nuclei.     

A simple and efficient method for isolation of DNA in high mucilaginous plant tissues

A protocol is described for rapid DNA isolation from Malvaceae plant species and different tissues of Bixaceae that contain large amounts of polysaccharides, polyphenols, and pigments that interfere with DNA extractions. The method is a modification of Dellaporta et al. The current protocol is simple, and no phenolchloroform extraction, ethanol, or isopropranol precipitation is required. The method is based in the incubation of soluble DNA with silica, mix in batch during the extraction. The procedure can be completed in 2 h and many samples can be processed at the same time. DNA of excellent quality was recovered and used for polymerase chain reaction (PCR) amplification, restriction enzyme digestion, and Southern blot analysis. The method was used with healthy Bixa orellana and virus-infected Malvaceae plants.     

A simple protocol for isolation of fungal DNA

Genomic DNA was isolated from as little as 2 mg dry biomass of Magnaporthe grisea by microwave treatment within 30 s. The quantity of DNA was good enough for PCR analysis and Dot blot hybridization. This technique can be used for various studies, such as DNA fingerprinting to study the population structure of the phytopathogen in different regions, and for a quick screening of M. grisea transformants.     

用脉冲电场凝胶电泳分析棉病囊霉及其突变体的染色体DNA

采用交变脉冲电场凝胶电泳和碱变性交变脉冲电场凝胶电泳方法,分析了棉病囊霉酵母菌及其2个不同的突变菌株的核型,得知此菌株含有5条染色体 DNA,而2株突变体的染色体 DNA 都没有大片段的缺失或双链断裂,但其稳定性不如野生型菌株的 DNA,而且存在单链断裂等碱不稳定性位点.     

Analysis of Vibrio cholerae O139 Bengal Isolated from Different Geographical Areas Using Macrorestriction DNA Analysis

Vibrio cholerae O139 isolated from different countries, as well as from different locations within a country, were examined using macrorestriction DNA analysis to determine the clonality of the O139 strains. NotI digests of genomic DNA of representative strains from Nepal, India, Bangladesh, China, Thailand, and Malaysia revealed very similar but not identical patterns. Examinations of the banding patterns generated by pulsed-field gel electrophoresis of strains isolated within countries revealed complete homogeneity. These results further reiterate the spread of an identical clone of V. cholerae O139 although it appears that genetic polymorphism among the O139 strains is becoming apparent.     

Alternative approach for consistent yields of total genomic DNA from cotton (Gossypium hirsutum L.)

A persistent limitation to molecular biological research on cotton (Gossypium spp.) has been the difficulty in isolation of total genomic DNA from the plant tissue. This report describes a reliable strategy for isolation of genomic DNA from cotton. The mini-preparation procedure involves use of lyophilized, etiolated cotyledons and an anion exchange column kit. The isolated DNA had a molecular weight in excess of 50 kb with minimal degradation or shearing. Routine yields ranged from 5 to 7 μg DNA per etiolated cotyledon pair (corresponding to 100 ng/mg dry weight), in contrast to little or no DNA from equivalent amounts of either green cotyledons or mature leaf tissue. The decreased yields from the latter tissues appeared to be correlated with increased afmounts of flavonoid. The DNA was amenable to routine molecular applications as demonstrated by: digestibility with a number of restriction enzymes (Eco RI,HindIII,Sau 3A), and hybridization of a tomato genomic clone containing the gene for S-adenosylmethionine synthetase to a 13.3-kbEco RI fragment of cotton. Using DNA from an isoline immune to root-knot nematodes, we observed no impediment to genomic cloning.     

A simple and rapid method for preparing genomic DNA from gram-positive bacteria

Molecular epidemiologic and other studies may require preparation of genomic DNA from large numbers of bacteria in sufficiently pure form for restriction endonuclease digestion, cloning, RAPD-PCR, Southern hybridization, and so on.Staphylococcus and other Gram-positive bacteria have a rigid cell wall and can be difficult to lyse. Here, a simple and rapid method for the preparation of genomic DNA from multiple samples is reported. This method produces clean DNA for use in most molecular biology methods in <90 min.     

从植物细胞核分离大分子量核DNA

研究了从植物中分离百万碱基对级大分子量核DNA的方法。该方法利用差速离心分离植物细胞核,经低熔点琼脂糖块或低熔点琼脂糖微珠包埋,蛋白酶K原位裂解后制备大分子量核DNA。结果表明,选择不同生长时期的材料和不同的包埋细胞核方式对大分子量核DNA的制备有很大的影响,由黄化苗或幼嫩的绿叶为材料分离细胞核,进行胶块包埋是制备大分子量核DNA的最佳条件。利用该法获得的DNA分子量在200kb－5．7Mb之间,主要集中在2．2～5．7Mb之间;每一胶块DNAE量为18～20μg。与包埋原生质体制备大分子量核DNA的方法相比,该方法获得的DNA纯度较高,去除了大部分细胞器DNA的污染;易于被限制性内切酶部分和完全消化,其消化结果具可重复性。该方法操作简单、适用植物种类广泛,用该方法从水稻（OryzasativaL．）、苹果（MaluspumilaMill．）、大豆（Glycinemax（L．）Merr．）、玉米（ZeamaysL．）等多种植物材料中成功地制备了大分子量核DNA。该方法制备的核DNA适用于植物的脉冲交变电泳基因组分析和构建人工细菌染色体文库和人工酵母染色体文库。     

A rapid method for tissue collection and high-throughput isolation of genomic DNA from mature trees

Collection of tissue and subsequent isolation of genomic DNA from mature tree species often proves difficult. DNA extraction from needles, leaves, or buds is recommended in many protocols. Collecting these tissues from mature trees generally requires the use of firearms or climbing if sampling is to be nondestructive. As a result, sample collection is a major expense of many tree-based projects. Tree (and plant) tissues generally contain large amounts of polysaccharides and phenolic compounds that are difficult to separate from DNA. Many methods aim to overcom these problems, with most involving extraction in buffers containing the nonionic detergent cetyltrimethyl-ammonium bromide (CTAB), followed by numerous steps to clean contaminants from the DNA, using organic solvents and differential salt precipitation. These steps are time-consuming, such that isolation of DNA becomes the bottleneck in many molecular studies. This paper presents a new, efficient, cambium collection method for tree species and a DNA extraction protocol based on that of Doyle and Doyle (1987), with follow-up purification using the Wizard nuclei lysis and protein precipitation solutions (Promega). Results show a significant improvement in yield and DNA purity compared with other published methods, with consistently high yields of pure genomic DNA and high sample throughput. The relatively low cost per extraction, no requirement for use of liquid nitrogen, no requirement for freezer storage, and long-term sample stability after collection are important additional benefits.     

Analysis of rice (Oryza sativa L.) genome using pulsed-field gel electrophoresis and rare-cutting restriction endonucleases

TheOryza sativa (rice) genome is small (600 to 900 megabase pairs) when compared to that of other monocotyledonous plants. Rice was the first of the major cereals to be successfully transformed and regenerated. An RFLP map with approximately 300 markers is readily available, and the DNA content per map unit is only two to three times that ofArabidopsis thaliana. Rice is also the main staple food for the majority of peoples in the world. We developed techniques for the preparation of intact genomic DNA from Indica and Japonica subspecies of rice, used statistical methods to determine which restriction endonucleases are rare-cutting, and used pulsed-field gel electrophoresis (PFE) to separate large fragments of rice DNA. Southern hybridization to blotted rice PFE gels was used to show that the digests were complete. The long-term goal of our work is to generate an integrated genetic/physical map for the rice genome, as well as helping to establish rice as a model for map-based gene cloning and genome analysis.     

A rapid and efficient method for the isolation of restrictable total DNA from plants of the genusAbelmoschus

A rapid, simple and efficient protocol is given for the extraction of restrictable total DNA from plants of the genusAbelmoschus, for which the main obstacle is the stickiness of the solution, after grinding of green leaves. This problem is resolved using cotyledons of dark-grown seedlings.     

DNA isolation by a rapid method from human blood samples: Effects of MgCl2, EDTA,storage time,and temperature on DNA yield and quality

The isolation of DNA from whole blood by a modified rapid method (RM) was tested using various detergents and buffer conditions. Extraction of DNA with either NP-40 or Triton X-100 gave a high yield of undegraded DNA in less than an hour. The concentration of magnesium ion in the buffers was critical to obtaining intact, high molecular weight (HMW) DNA. Greater than 10 mM MgCl₂ led to degradation. Addition of EDTA to the buffer inhibits this degradation. Preparation of DNA from blood stored at room temperature or incubated at 37°C for 24 hr resulted in the same amount and quality of DNA as from samples frozen at −70°C. DNA from blood samples that had undergone more than four freeze-thaw cycles was found to be partially degraded. The modified RM can be applied to extract DNA from as little as 10 μl of blood (340 ng of DNA) and from dried blood samples. DNA samples remained intact and undegraded for longer times when DNA was dissolved in higher concentrations of EDTA. This work was supported by grants from the Indiana Department of Mental Health and PHS RO1 AG10297.     

A simple and efficient method for DNA extraction from grapevine cultivars andVitis species

A quick, simple, and reliable method for the extraction of DNA from grapevine species, hybrids, andAmpelopsis brevipedunculata (Vitaceae) has been developed. This method, based on that of Doyle and Doyle (1990), is a CTBA-based extraction procedure modified by the use of NaCl to remove polysaccharides and PVP to eliminate polyphenols during DNA purification. The method has also been used successfully for extraction of total DNA from other fruit species such as apple (Malus domestica), apricot (Prunus armeniaca), cherry (Prunus avium), peach (Prunus persica), plum (Prunus domestica), and raspberry (Rubus idaeus). DNA yield from this procedure is high (up to 1 mg/g of leaf tissue). DNA is completely digestible with restriction endonucleases and amplifiable in the polymerase chain reaction (PCR), indicating freedom from common contaminating compounds.     

Restriction Profiles of the Chromosomal DNA from Acidithiobacillus ferrooxidans Strains Adapted to Different Oxidation Substrates

Restriction profiles of chromosomal DNA were studied in different Acidithiobacillus ferrooxidans strains grown on medium with Fe²⁺ and further adapted to another oxidation substrate (S⁰, FeS₂, or sulfide ore concentrates). The restriction endonuclease XbaI digested the chromosomal DNA from different strains into different numbers of fragments of various sizes. Adaptation of two strains (TFBk and TFN-d) to new oxidation substrates resulted in structural changes in XbaI-restriction patterns of their chromosomal DNA. Such changes in the DNA restriction patterns occurred in strain TFBk after the adaptation to precyanidated gravitational pyrite-arsenopyrite concentrate (no. 1) from the Nezhdaninskoe deposit or to copper-containing ore from the Udokanskoe deposit and also in strain TFN-d adapted to untreated pyrite-arsenopyrite concentrate (no. 2) from the Nezhdaninskoe deposit. No changes in the number or size of the XbaI-restriction patterns of chromosomal DNA were revealed in either strain TFBk cultivated on media with pyrite from the Angren and Tulun deposits or in strains TFN-d and TFO grown on media with S⁰ and pyrite. Neither were changes observed in the XbaI-restriction patterns of the DNA from strain TFV-1, isolated from the copper ore of the Volkovskoe deposit, when Fe²⁺ was substituted with alternative substrates—S⁰, pyrite or concentrate no. 2 from the ore of the Nezhdaninskoe deposit. In strain TFO, no differences in the XbaI-restriction patterns of the chromosomal DNA were revealed between the culture grown on medium containing concentrate no. 2 or the concentrate of surface-lying ore from the Olimpiadinskoe deposit and the culture grown on medium with Fe²⁺. When strain TFO was cultivated on the ore concentrate from deeper horizons of the Olimpiadinskoe deposit, which are characterized by lower oxidation degrees and high antimony content, mutant TFO-2 differing from the parent strain in the chromosomal DNA structure was isolated. The correlation between the lability of the chromosomal DNA structure in A. ferrooxidans strains and the physical and chemical peculiarities of the isolation substrate and habitat is discussed.     

A method for the ligation of DNA following isolation from low melting temperature agarose

A rapid, simple, and reliable method is presented for the isolation and subsequent ligation of DNA from agarose gels. The technique involves the use of low melting temperature agarose, but with the inclusion of bovine serum albumin or gelatin to the ligation reaction.     

Biotyping,serotyping and genotyping of aeromonads from environmental and clinical samples

Pulsed-field gel electrophoresis (PFGE) and biochemical–serological assays were used to characterize environmental and clinical aeromonads. On the basis of their biochemical characteristics, 31 strains were assigned to one of the recognized groups or species within the Aeromonas genus and 11 different serogroups were detected. Low correlation between molecular and traditional typing methods was observed. The results obtained showed that the genomic analysis performed by PFGE can be a more effective means for distinguishing between Aeromonas isolates than conventional biochemical methods.