One-step isolation of plant DNA suitable for PCR amplification

We report a one-step extraction technique for the isolation of plant DNA, DNA suitable for amplification by PCR can be produced from leaf material smaller than 0.3 mm² in less than 20 min, with no tube changes. The method was tested on several plant specA00AK020ies. The described method was found to extract DNA that could be amplified without any further purification or treatment. The isolated DNA was amplified using a universal chloroplast primer set. The method was validated by comparing size of PCR products generated by the novel method to PCR products generated using standard DNA isolation techniques.     

Identification of seven species of hymenopteran parasitoids of Spodoptera frugiperda, using polymerase chain reaction amplification and restriction enzyme digestion

1 The fall armyworm Spodoptera frugiperda is a voracious pest of numerous crops of economic importance throughout the New World. In its native Mexico, larvae can be attacked by several species of parasitic wasps, which are candidate biological control agents against this and other lepidopteran pests. 2 We attempted to survey the parasitoid fauna on S. frugiperda in maize and sorghum fields throughout Mexico. However, our efforts have been hampered by the incomplete development of parasitoid larvae emerging from collected Spodoptera caterpillars. 3 This problem was solved by developing a method to identify seven species of parasitic wasps using polymerase chain reaction amplification and restriction enzyme digestion. This enables the precise determination of the species of those parasitoid larvae that are usually not morphologically identifiable.     

A simple extraction method suitable for PCR-based analysis of plant,fungal, and bacterial DNA

A simple and easy protocol for extracting high-quality DNA from microorganisms and plants is presented. The method involves inactivating proteins by using SDS/proteinase K and precipitating polysaccharides in the presence of high salt. Further purification is based on differential solubility of DNA and high-molecular-weight polysaccharides in aqueous media. The procedure does not use the toxic and potentially hazardous phenol and chloroform, and as many as 100 samples can be processed per day. Absorbency ratios (A₂₆₀/A₂₈₀) of 1.6–2.0 indicated a minimal presence of contaminating metabolites. The DNA was completely digested with 5 restriction enzymes:EcoR I,RsaI,TaqI,EcoR V, andHind III. PCR analysis using enterobacterial repetitive intergenic consensus (ERIC) sequence, sequence-characterized amplified region (SCAR), and random amplified microsatellite (RAMS) primers showed the DNA's compatibility with downstream applications. This procedure is applicable to a range of pathogens and plants and thus may find wide application in quarantine services and marker-assisted selection (MAS) breeding.     

Rapid isolation of fungal genomic DNA suitable for long distance PCR

A quick and reliable method for screening fungal transformants for specific genetic modifications is essential for many molecular applications. We have compared the applicability of a few rapid DNA extraction methods for Myrothecium and Aspergillus and tested the resulting DNA as to its suitability for PCR. For Myrothecium gramineum, the highest DNA concentration was obtained with the procedure described by N. Vanittanakom et al. (J Clin Microbiol 2002, 40: 1739–1742). For A. nidulans, concentrations higher than 100 ng/μl were reached with the glass bead, the LiCl, the boiling, the liquid N₂ and the protoplast-based method. Samples of M. gramineum resulting from the boiling and the liquid N₂ procedure were suitable for the amplification of fragments up to 2.3 kb. The direct use of mycelium from M. gramineum in the PCR tube can be employed for the reproducible amplification of fragments up to 1 kb. Amplification of fragments up to 4.3 kb requires the use of the Elongase Mix on samples extracted with the liquid N₂ procedure.     

Rapid isolation, purification and characterisation of type II restriction enzymes from Lactococcus spp.

An inexpensive procedure that uses small volumes (5–10 ml) of cell culture for the rapid isolation of restriction enzymes, sufficiently pure to allow preliminary characterisation, is presented. The method was designed initially to screen for Type II restriction enzymes, but different assays can be devised to screen for other types of restriction enzymes. Although initially optimised in Lacotococcus lactis subsp. cremoris LC17-1, this method potentially holds wider applications in other lactococcal species as was shown by its successful application to Lactococcus lactis subp. lactis. Without the necessity for chromatographic techniques that are often expensive and time consuming, the convenience of the technique makes it suitable for rapid, routine screening of a large number of lactic acid bacterial strains, or restriction and modification systems cloned into them, for restriction enzyme activity.     

A rapid and cost-effective method of genomic DNA isolation from cyanobacterial culture,mat and soil suitable for genomic fingerprinting and community analysis

This study presents a phenol and lysozyme free protocol for genomic DNA isolation of cyanobacteria from culture, mats and soil. For an efficient and pure DNA isolation from cyanobacteria having tough cell wall, extra steps of glass beading and Sepharose 4B purification were added. The modified method gave a higher yield of DNA than the phenol: chloroform extraction method. Four parameters selected for purity testing of the isolated DNA were: (i) restriction digestion with Hind III, (ii) randomly amplified polymorphic DNA-PCR of axenic culture of cyanobacteria to assess phylogenetic relatedness, (iii) denaturing gradient gel electrophoretic (DGGE) analysis of cyanobacterial mat and soil to ascertain the applicability of the isolated DNA for community analysis, and (iv) sequencing of partial 16S rDNA of Hapalosiphon intricatus BHULCR1, Anabaena doliolum LCR1, Anabaena oryzae LCR2, Aulosira fertilissima LCR4, and Tolypothrix tenuis LCR7 and BLAST analysis to confirm their cyanobacterial identity. Data generated from above analyses lead us to conclude that the modified method in question is rapid, cost effective, health and time conscious and promising for genetic fingerprinting and community analysis of cyanobacteria from diverse habitats.     

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.     

Characterization of Aspergillus flavus strains from Brazilian Brazil nuts and cashew by RAPD and ribosomal DNA analysis

Aims:  The aim of this study was to determine the genetic variability in Aspergillus flavus populations from Brazil nut and cashew and develop a polymerase chain reaction (PCR) detection method.
Methods and Results:  Chomatography analysis of 48 isolates identified 36 as aflatoxigenic (75%). One hundred and forty-one DNA bands were generated with 11 random amplified polymorphic DNA (RAPD) primers and analysed via unweighted pair group analysis, using arithmetic means (UPGMA). Isolates grouped according to host, with differentiation of those from A. occidentale also according to geographical origin. Aspergillus flavus -specific PCR primers ASPITSF2 and ASPITSR3 were designed from ribosomal DNA internal transcribed spacers (ITS 1 and 2), and an internal amplification control was developed, to prevent false negative results. Specificity to only A. flavus was confirmed against DNA from additional aspergilli and other fungi.
Conclusions:  RAPD-based characterization differentiated isolates according to plant host. The PCR primer pair developed showed specificity to A. flavus , with a detection limit of 10 fg.
Significance and Impact of the Study:  Genetic variability observed in A. flavus isolates from two Brazilian agroecosystems suggested reproductive isolation. The PCR detection method developed for A. flavus represents progress towards multiplex PCR detection of aflatoxigenic and nonaflatoxigenic strains in Hazard Analysis Critical Control Point systems.     

Simple and efficient genomic DNA extraction protocol for molecular characterisation of Phytophthora colocasiae causing taro leaf blight

Genomic DNA extraction protocol with relatively high quantity and purity is prerequisite for the successful molecular identification and characterisation of plant pathogens. Conventional DNA extraction methods are often time-consuming and yield only very poor quantity of genomic DNA for samples with higher mycelial age. In our laboratory, we have aimed at establishing an efficient DNA isolation procedure, exclusively for the oomycete pathogen Phytophthora colocasiae causing serious leaf blight disease in taro. For this a phenol free protocol was adopted, which involves SDS/Proteinase K-based inactivation of protein contaminants, extraction of nucleic acids using chloroform: isoamyl alcohol and later precipitation of genomic DNA using isopropanol and sodium acetate. The purity of the isolated DNA was analysed by A_260/280 and A_260/230 spectrophotometric readings and confirmed by restriction digestion with restriction enzyme Eco RI. In this study, a comparative assessment was done with CTAB method and the commercial genomic DNA purification kit (Thermo Fisher Scientific, Fermentas, EU). The extracted DNA was found to be suitable for further downstream applications like ITS amplification of the rDNA ITS region and PCR amplification with species-specific primers.     

Evaluation of amplified ribosomal DNA restriction analysis as a method for the identification of Botryosphaeria species

The polymerase chain reaction was used to amplify a rDNA fragment containing the internal transcribed spacers (ITS1-5.8S-ITS2) and the D1/D2 variable domains of the 28S rDNA from 10 species of the genus Botryosphaeria (Fungi, Ascomycota). Restriction analysis of the amplicons with frequent-cutting endonucleases (amplified ribosomal DNA restriction analysis) allowed the definition of 12 rDNA haplotypes. Each of the rDNA haplotypes could be unambiguously assigned to a single Botryosphaeria species, thus allowing clear identification of all the species tested. Intraspecific polymorphism was very low and detected only in Botryosphaeria parva and Botryosphaeria dothidea. Cluster analysis of banding patterns of the isolates corresponded well with known species delineations. The method described in this paper provides a simple and rapid procedure for the differentiation and identification of Botryosphaeria isolates at the species level.     

DNA isolation from forest soil suitable for PCR assays of fungal and plant rRNA genes

This protocol for DNA isolation from forest soil samples is advantageous because it uses only one liquid transference step and can process several samples with minimal time and equipment. The use of benzyl chloride early in the extraction protocol increases DNA yield and purity. The obtained DNA is useful for PCR amplification of nuclear and mitochondrial ribosomal related sequences from fungi and ribosomal DNA from plant chloroplasts. Isolated DNA can be used either undiluted or at low dilutions in PCR assays. A final glassmilk treatment of isolated DNA is useful to recover high molecular weight DNA fractions from agarose gel. DNA losses during glassmilk treatment can generate negative PCR results.     

A simple and efficient method of DNA isolation from orchid species and hybrids

A simple and reliable method for extracting DNA has been developed for orchid species and hybrids. The high quality of DNA obtained is suitable for amplification via the polymerase chain reaction (PCR) for producing random amplified polymorphic DNA (RAPD) markers. This revised version was published online in July 2006 with corrections to the Cover Date.     

Rapid and simple preparation of mushroom DNA directly from colonies and fruiting bodies for PCR

We have optimized a simple and rapid preparation procedure for mushroom DNA extraction from colonies on media or from fruiting bodies for PCR amplification. The protocol combines microwaving twice for 1 min, cooling for 10 min, and centrifuging for 5 min. By using this procedure, more than 100 samples of mushroom DNA can be prepared within 1 h. The DNA obtained can be used for (1) identifying mushroom species by PCR and subsequent sequencing, (2) amplifying low copy number genes (at least 2,000 bp), and (3) screening genetic transformants. This technique will contribute to the mycology of mushroom species.     

Rapid and simple method for DNA extraction from plant and algal species suitable for PCR amplification using a chelating resin Chelex 100

A DNA extraction method using Chelex 100 is widely used for bacteria, Chlamydomonas, and animal cell lines, but only rarely for plant materials due to the need for additional time-consuming and tedious steps. We have modified the Chelex 100 protocol and successfully developed a rapid and simple method of DNA extraction for efficient PCR-based detection of transgenes from a variety of transgenic plant and algal species. Our protocol consists of homogenizing plant tissue with a pestle, boiling the homogenized tissue in a microfuge tube with 5% Chelex 100 for 5 min, and centrifuging the boiled mixture. The supernatant, which is used for PCR analysis, was able to successfully amplify transgenes in transgenic tobacco, tomato, potato, Arabidopsis, rice, strawberry, Spirodela polyrhiza, Chlamydomonas, and Porphyra tenera. The entire DNA extraction procedure requires <15 min and is therefore comparable to that used for bacteria, Chlamydomonas, and animal cell lines.     

Molecular differentiation of Bifidobacterium species with amplified ribosomal DNA restriction analysis and alignment of short regions of the ldh gene

The differentiation of Bifidobacterium species was performed with specific primers using the PCR technique, the amplified ribosomal DNA restriction analysis (ARDRA) technique based on reports on the sequence of the 16S rRNA gene and speciation based on a short region of the ldh gene. Four specific primer sets were developed for each of the Bifidobacterium species, B. animalis, B. infantis and B. longum. The use of the ARDRA method made it possible to discriminate between B. infantis, B. longum and B. animalis with the combination of BamHI, TaqI and Sau3AI restriction enzymes. The ldh gene sequences of 309-312 bp were determined for 19 Bifidobacterium strains. Alignment of these short regions of the ldh gene confirmed that it is possible to distinguish between B. longum and B. infantis but not between B. lactis and B. animalis.     

A rapid mini-prep method for isolation of ectomycorrhizal DNA from Tuber species

A rapid procedure has been developed to isolate DNA from the ectomycorrhizae of Tuber spp. for use in PCR experiments. The method described is fast and sensitive and can overcome the amplification problems that can arise in the presence of inhibitors. For this reason it can be used to type ectomycorrhizae even starting from a single root tip and make mycorrhizae identification much more rapid.     

Sulfonation of polyvinylidene difluoride resin and its application in extraction of restriction enzymes from DNA digestion solutions

Sulfonation of polyvinylidene difluoride (PVDF) resin was achieved by incubation of the resin with sulfuric acid at a moderately high temperature. The sulfonated PVDF (SPVDF) resin was studied for its ability to extract restriction enzymes from DNA digestion solutions. The SPVDF resin was effective in adsorbing restriction enzymes such as EcoRI and BamHI and the extraction procedure was easy and simple to perform. The adsorption depended upon the amount of the resin added. We found that 1 mg of the SPVDF resin could completely remove all restriction enzyme activity routinely used in DNA digestion within 2 min after its addition. Treatment of a digestion solution with the SPVDF resin did not change the reaction solution and the same digestion buffer could be used for another digestion of the same DNA with other enzymes. We also found that, in comparison with normal PVDF, the SPVDF resin adsorbed less DNA, resulting in less loss of DNA in the extraction step. The potential application of the SPVDF resin in other procedures of molecular cloning and enzyme purification is discussed.     

Genetic diversity study of Chromobacterium violaceum isolated from Kolli Hills by amplified ribosomal DNA restriction analysis (ARDRA) and random amplified polymorphic DNA (RAPD)

Aim: Chromobacterium are saprophytes that cause highly fatal opportunistic infections. Identification and strain differentiation were performed to identify the strain variability among the environmental samples. We have evaluated the suitability of individual and combined methods to detect the strain variations of the samples collected in different seasons. Methods and Results: Amplified ribosomal DNA restriction analysis (ARDRA) and random amplified polymorphic DNA (RAPD) profiles were obtained using four different restriction enzyme digestions (AluI, HaeIII, MspI and RsaI) and five random primers. A matrix of dice similarity coefficients was calculated and used to compare these restriction patterns. ARDRA showed rapid differentiation of strains based on 16S rDNA, but the combined RAPD and ARDRA gave a more reliable differentiation than when either of them was analysed individually. Conclusion: A high level of genetic diversity was observed, which indicates that the Kolli Hills’C. violaceum isolates would fall into at least three new clusters. Significance and Impact of the Study: Results showed a noteworthy bacterial variation and genetic diversity of C. violaceum in the unexplored, virgin forest area.