An improved method to extract DNA from mango Mangifera indica

High quality genomic DNA is the first step in the development of DNA-based markers for fingerprinting and genetic diversity of crops, including mango (Mangifera indica L.), a woody perennial. Poor quality genomic DNA hinders the successful application of analytical DNA-based tools. Standard protocols for DNA extraction are not suitable for mango since the extracted genomic DNA often contains secondary metabolites that interfere with analytical applications. In this study, we employed an additional step to remove polysaccharides, polyphenols and secondary metabolites from genomic DNA extracted from young or mature leaf tissue; then a modified traditional cetyl trimethyl ammonium bromide (CTAB) method was applied. The use of 0.4 M glucose improved DNA quality and avoided contamination and browning by polyphenolics, relative to the traditional CTAB method. This is an easy and efficient method for genomic DNA extraction from both young and mature leaves of mango. The isolated DNA was free of polysaccharides, polyphenols, RNA and other major contaminants, as judged by its clear colour, its viscosity, A²⁶⁰/A²⁸⁰ ratio and suitability for PCR-based reactions. This modified protocol was also used to extract high quality genomic DNA from other woody perennials, including walnut, guava, lychee, pear, grape and sugarcane.     

DNA isolation protocol for the medicinal plant lemon balm (Melissa officinalis, Lamiaceae)

Lemon balm (Melissa officinalis) is a medicinal plant that is widely used as a sedative or calmant, spasmolytic and antibacterial agent and sleep aid. This has led to a high demand for lemon balm products, resulting in the extinction of this species in some of its natural habitats. Molecular techniques have increasingly been used in plant diversity conservation and isolation of PCR amplifiable genomic DNA is an important pre-requisite. Lemon balm contains high levels of polyphenols and polysaccharides, which pose a major challenge for the isolation of high-quality DNA. We compared different genomic DNA extraction protocols, including traditional phenol-chloroform DNA extraction protocols and two commercial kits for DNA purification for their ability to produce good-quality DNA from fresh leaves of five lemon balm genotypes. Quality and quantity of the DNA samples were determined using 0.8% agarose gel electrophoresis and a spectrophotometer. The DNA purity was further confirmed by PCR amplification using barley retrotransposon LTR base primers. The spectral quality of DNA as measured by the A(260)/A(280) ratio ranged from 1.46 to 2.37. The Fermentase genomic DNA purification kit and the CTAB extraction protocol using PVP and ammonium acetate to overcome the high levels of polyphenols and polysaccharides yielded high-quality DNA with a mean A(260)/A(280) ratio of 1.87. The quantity of DNA and its PCR purity were similar with all the protocols, but considering the time and cost required for extraction of DNA from a large number of samples, the CTAB protocol using PVP and ammonium acetate is suitable for lemon balm.     

Comparison of Metagenomic DNA Extraction Methods for Soil Sediments of High Elevation Puga Hot Spring in Ladakh,India to Explore Bacterial Diversity

Extraction of good-quality metagenomic DNA from extreme environments is quite challenging, particularly from high elevation hot spring sediments. Low microbial load, high humic acid content and other contaminants complicate the process of extraction of metagenomic DNA from hot spring sediments. In the present study, efficacy of five manual DNA extraction protocols with modifications has been evaluated for metagenomic DNA extraction from boron–sulfur rich high elevation Puga hot spring sediments. Best suited protocol was identified based on the cell lysis efficiency, DNA yield, humic acid content, PCR reproducibility and representation of bacterial diversity. Quantity as well as quality of crude metagenomic DNA differed remarkably between various protocols used and were not pure enough to give PCR amplification using 16S rRNA bacterial and archaeal primers. Crude metagenomic DNA extracted using five different DNA extraction protocols was purified using spin column based purification method. Even after purification, only three protocols C, D and E yielded metagenomic DNA that could be amplified using both archaeal and bacterial primers. To evaluate the degree of microbial diversity represented by protocols C, D and E, phylogenetic genes amplified were subjected to amplified ribosomal DNA restriction analysis (ARDRA) and denaturing gradient gel electrophoresis analysis (DGGE) analysis. ARDRA banding pattern of amplicons generated for all the three extraction protocols, i.e., C, D and E were found to be similar. DGGE of protocol E derived amplicons resulted in the similar number of dominant bands but a greater number of non-dominant bands, i.e., the highest microbial diversity in comparison to protocols C and D, respectively. In the present study, protocol E developed from Yeates et al. protocol has been found to be best in terms of DNA yield, DNA purity and bacterial diversity depiction associated with boron–sulfur rich sediment of high elevation hot springs.     

A simple protocol for isolating genomic DNA from chestnut rose (<Emphasis Type="Italic">Rosa roxburghii</Emphasis> tratt) for RFLP and PCR analyses

Isolation of high-quality DNA from rosaceous species is particularly difficult because of their high levels of polyphenols, polysaccharides, and other compounds. The yields and quality of genomic DNA are considerably affected when the common protocol for DNA isolation is applied to the chestnut rose (Rosa roxburghii Tratt). A simple, rapid, and efficient protocol for the extraction of DNA from the chestnut rose is described. The modified hexadecyltrimethylammonium bromide (CTAB) procedure, which uses phenol-absent extraction to enhance the yield, involves a washing step before extraction for the removal of organic molecules and excessive water; the use of high concentrations of polyvinylpyrrolidone (2% [w/v]), CTAB (3% [w/v]), and β-mercaptoethanol (3% [v/v]) in the high-salt-concentration extraction buffer to remove polyphenols and polysaccharides; and the combined use of potassium acetate and chloroform to remove proteins and polysaccharides. Finally, DNA is precipitated with an equal volume of isopropanol and 0.1 vol of sodium acetate. This protocol results in high yields of DNA. The average yield of DNA ranged from 980–1800 μg/g of fresh weight of leaves. Downstream results indicate that DNA quality is sufficient for restriction fragment length polymorphism (RFLP) and polymerase chain reaction (PCR) analyses.     

An effective protocol to solve the problem in genomic DNA isolation of tung tree

Tung tree (Vernicia fordii) is a native and oil-producing woody plant in China. The oil is industrially important and promising biodiesel raw material. However, until recently the lack of effective protocols for the extraction of genomic DNA had made DNA-based molecular studies of tung tree difficult. Here, four conventional protocols and one novel protocol were compared for their capacity in isolating DNA from tung tree leaves of different age. Our results showed that all the four conventional protocols could isolate DNA from old leaves, two from matured leaves, but none from young leaves. However, the detectable DNA samples contained many contaminations, leading to overestimation of DNA concentration measured by ultraviolet spectrophotometer, also interfering with the downstream PCR reaction. The novel protocol could produce high-yield and good-quality DNA from tung tree leaves regardless of leaf age. Its key steps were that a single leaf tissue sample could be recycled for DNA extraction for up to four times, and correspondingly four DNA precipitations (termed as the 1st, 2nd, 3rd and 4th DNA sample, respectively) were conducted. All the four DNA samples of a single tissue were good template for PCR reaction. The novel protocol is an effective method for genomic DNA isolation of tung tree.     

Isolation of High-Quality RNA from <Emphasis Type="Italic">Reaumuria soongorica</Emphasis>, a Desert Plant Rich in Secondary Metabolites

RNA isolation is a prerequisite for the study of the molecular mechanisms of stress tolerance in the desert plant Reaumuria soongorica, an extreme xeric semi-shrub. However, R. soongorica that contains high levels of secondary metabolites that co-precipitate with RNA, making RNA isolation difficult. Here the authors propose a new protocol suitable for isolating high-quality RNA from the leaves of R. soongorica. Based on a CTAB method described by Liu et al., the protocol has been improved as follows: the samples were ground with PVPP to effectively inhibit the oxidation of phenolics, contaminating DNA was removed with DNase I, and NaAc was used along with ethanol for precipitation to enhance the RNA yield and shorten the precipitation time. Gel electrophoresis and spectrophotometric analysis indicated that this isolation method provides RNA with no DNA contamination. Moreover, the yield (183.79 ± 40.36 μg/g) and quality were superior to those using the method of Liu et al., which yields RNA with significant DNA contamination at 126.30 ± 29.43 μg/g. Gene amplification showed that the RNA obtained using this protocol is suitable for use in downstream molecular procedures. This method was found to work equally well for isolating RNA from other desert plants. Thus, it is likely to be widely applicable.     

Isolation and Amplification of Genomic DNA from Recalcitrant Dried Berries of Black Pepper (<Emphasis Type="Italic">Piper nigrum</Emphasis> L.)—A Medicinal Spice

Black pepper is an important medicinal spice traded internationally. The extraction of high quality genomic DNA for PCR amplification from dried black pepper is challenging because of the presence of the exceptionally large amount of oxidized polyphenolic compounds, polysaccharides and other secondary metabolites. Here we report a modified hexadecyl trimethyl ammonium bromide (CTAB) protocol by incorporating potassium acetate and a final PEG precipitation step to isolate PCR amplifiable genomic DNA from dried and powdered berries of black pepper. The protocol has trade implication as it will help in the PCR characterization of traded black peppers from different countries.     

DNA Isolation and Amplification from Cacti

The cacti family is a morphologically heterogeneous group comprising 100 genera and about 1500 species (Hernandez and Barcenas, 1996). With the exception of one genus, all members of this family are native to America (Hernandez and Barcenas, 1996). There are three subfamilies, Opuntioideae, Cactoideae, and Pereskioideae (Gibson and Nobel, 1986). DNA isolation from cacti is notoriously difficult because they contain high amounts of polysaccharides and secondary metabolites which form insoluble complexes with nucleic acids during extraction (Guillemaut and Marechal-Drouard, 1992). Like in other groups of plants, the secondary metabolites and polysaccharides in cacti inhibit enzyme action (Porebski et al., 1997). The polysaccharides are visually evident by their viscous, glue-like texture and they make the DNA unmanageable when pipeting and hard to amplify by the polymerase chain reaction (PCR) (Poresbski et al., 1997). We report an easy and inexpensive protocol to isolate DNA from cacti. We used this method to isolate DNA from 85 species (170 individuals) of 39 genera of the subfamilies Pereskioideae, Opuntioidea, and Cactoideae. This procedure is a modification of a protocol described by De la Cruz et al. (1995) for the Cacti family. It requires only a few grams of tissue and does not require destruction of the whole plant to produce high molecular weight genomic DNA. The DNA from this procedure can be amplified consistently by PCR and used for RAPD analysis.     

A rapid and inexpensive method for isolation of total DNA from Trichoderma spp (Hypocreaceae)

Extraction of high-quality genomic DNA for PCR amplification from filamentous fungi is difficult because of the complex cell wall and the high concentrations of polysaccharides and other secondary metabolites that bind to or co-precipitate with nucleic acids. We developed a modified sodium dodecyl sulfate/phenol protocol, without maceration in liquid nitrogen and without a final ethanol precipitation step. The A(260/280) absorbance ratios of isolated DNA were approximately 1.7-1.9, demonstrating that the DNA fraction is pure and can be used for analysis. Additionally, the A(260/230) values were higher than 1.6, demonstrating negligible contamination by polysaccharides. The DNA isolated by this protocol is of sufficient quality for molecular applications; this technique could be applied to other organisms that have similar substances that hinder DNA extraction. The main advantages of the method are that the mycelium is directly recovered from culture medium and it does not require the use of expensive and specialized equipment.     

Rapid and simple methodology for isolation of high quality genomic DNA from coniferous tissues (<Emphasis Type="Italic">Taxus baccata</Emphasis>)

Various investigations have been so far performed for extraction of genomic DNA from plant tissues, in which the extracted intact DNA can be exploited for a diverse range of biological studies. Extraction of high quality DNA from leathery plant tissues (e.g., coniferous organs) appears to be a critical stage. Moreover, for some species such as Taxus trees, bioprocess engineering and biosynthesis of secondary metabolites (e.g., paclitaxel) is a crucial step due to the restrictions associated with extinction of these species. However, extraction of intact genomic DNA from these plants still demands a rapid, easy and efficient protocol. To pursue such aim, in the current work, we report on the development of a simple and highly efficient method for the extraction of DNA from Taxus baccata. Based upon our protocol, interfering phenolic compounds were removed from extraction using polyvinylpyrrolidone and RNA contamination was resolved using LiCl. By employing this method, high quality genomic DNA was successfully extracted from leaves of T. baccata. The quality of extracted DNA was validated by various techniques such as RAPD marker, restriction digestions and pre-AFLP. Upon our findings, we propose this simple method to be considered for extraction of DNA from leathery plant tissues.     

Chitosan functionalized magnetic particle-assisted detection of genetically modified soybeans based on polymerase chain reaction and capillary electrophoresis

The high quality of DNA template is one of the key factors to ensure the successful execution of polymerase chain reaction (PCR). Therefore, development of DNA extraction methods is very important. In this work, chitosan modified magnetic particles (MPs) were synthesized and employed for extraction of genomic DNA from genetically modified (GM) soybeans. The extraction protocol used aqueous buffers for DNA binding to and releasing from the surface of the MPs based on the pH inducing the charge switch of amino groups in chitosan modified MPs. The extracted DNA was pure enough (A(260)/A(280)=1.85) to be directly used as templates for PCR amplification. In addition, the PCR products were separated by capillary electrophoresis for screening of GM organisms. The developed DNA extraction method using chitosan modified MPs was capable of preparation of DNA templates, which were PCR inhibitor free and ready for downstream analysis. The whole process for DNA extraction and detection was preferable to conventional methods (phenol-chloroform extraction, PCR, and gel electrophoresis) due to its simplicity and rapidity as well as its avoiding the use of toxic reagents and PCR inhibitors.     

改良CTAB法用于多年生植物组织基因组DNA的大量提取*

根据多年生植物组织富含多酚、多糖的具体特性,对现有的DNA提取方法进行了改进。通过增加提取缓冲液中b-巯基乙醇用量,简化氯仿/异戊醇抽提液步骤,改用经-20℃预冷异丙醇沉淀DNA等,对CTAB法加以改进。改进后方法具有以下优点：（1）获得的DNA质量良好,提取过程无明显的DNA降解,基本上排除了多酚物质的干扰;（2）用获得的DNA进行Southern杂交,可得到理想的杂交信号,可满足相关的分子研究要求;（3）操作简便。Abstract It is a difficult problem to isolate high quality DNA from plants containing a high contents of polyphenolics and polysaccharose, such as Actinidia plant. The protocol described in this paper is a modified CTAB (hexadecyltrimethylammonium bromide) method. High quality genomic DNA can be isolated from Actinidia plant using the improved method. The DNA is good enough for Southern blot and other uses in DNA research. The protocol is also efficient for quick and macro-DNA extraction.     

A simplified protocol for preparing DNA from filamentous cyanobacteria

The preparation of good quality genomic DNA from microalgae and plants is often time-consuming because of the need to remove contaminants that may interfere with the downstream enzymatic manipulation of the DNA. Simpler protocols have been reported but these are applicable only to a few species and in many cases are not effective for removing trace contaminants. In this report, we describe a modification of existing protocols that significantly simplified the preparation of genomic DNA from cyanobacteria and plants. A key step in our protocol is the precipitation of DNA in a high concentration of salt (2–2.5 M NaCl) in the presence of isopropanol, immediately following phenol and chloroform extractions. The preparation and enzymatic digestion of the DNA can be performed in a single day. The DNA was easily digested in 2 h at normal restriction enzyme concentrations, and is highly suitable for PCR and Southern hybridization. We successfully used this simplified protocol to prepare genomic DNA from several filamentous cyanobacteria, such asAnabaena sp. PCC 7120,Anabaena siamensis, andSpirulina strains M2 and Kenya. This protocol may also be useful for preparing genomic DNA from other algae and from higher plants.     

Rapid isolation of high-quality total RNA from taxus and ginkgo

An easy and efficient protocol was developed for isolating good-quality total RNA from various tissues including fruits, leaves, stems, and roots of ancient gymnosperm species, taxus and ginkgo. The protocol was developed based on the CTAB method with modifications, including higher-strength CTAB to help the lysis of plant cells, more PVP, and beta-mercaptoethanol to prevent oxidation of phenolic complexes, and higher-centrifugation force to get rid of most cell debris and to ensure RNA quality. In RNA isolation, chloroform/isoamyl alcohol was used to remove proteins, genomic DNA, and secondary metabolites and lithium chloride was subsequently adopted to concentrate total RNA away from most of the cytoplasmic components. Good-quality total RNA from various tissues of native taxus and ginkgo could be easily isolated within 24 hr by this protocol which avoided the limitation of plant materials and the usage of dangerous chemicals, such as phenol, and could provide total RNA for all kinds of further molecular studies.     

DNA isolation protocol for red seaweed (rhodophyta)

We report a DNA isolation protocol for red seaweed. The method is a modification of the Dellaporta et al. (1983) protocol for land plants. Our simplified version can be used to process large sample numbers and to minimise polysaccharide co-isolation. The protocol was applied to 12 red seaweed species as well as one green alga and one land plant. The protocol yields about 5 μg of high molecular weight DNA from 10 mg of dried material, with no RNA. No sign of degradation was observed after agarose gel electrophoresis for both freshly extracted DNA and DNA stored for 18 months at 4°C. DNA isolated by our protocol was suitable for genomic library construction (tested for one species), endonuclease restriction, and PCR amplification for all species.     

Two measurement methods of leaf dry matter content produce similar results in a broad range of species

BACKGROUND AND AIMS: Leaf dry matter content (LDMC) is widely used as an indicator of plant resource use in plant functional trait databases. Two main methods have been proposed to measure LDMC, which basically differ in the rehydration procedure to which leaves are subjected after harvesting. These are the 'complete rehydration' protocol of Garnier et al. (2001, Functional Ecology 15: 688-695) and the 'partial rehydration' protocol of Vendramini et al. (2002, New Phytologist 154: 147-157). METHODS: To test differences in LDMC due to the use of different methods, LDMC was measured on 51 native and cultivated species representing a wide range of plant families and growth forms from central-western Argentina, following the complete rehydration and partial rehydration protocols. KEY RESULTS AND CONCLUSIONS: The LDMC values obtained by both methods were strongly and positively correlated, clearly showing that LDMC is highly conserved between the two procedures. These trends were not altered by the exclusion of plants with non-laminar leaves. Although the complete rehydration method is the safest to measure LDMC, the partial rehydration procedure produces similar results and is faster. It therefore appears as an acceptable option for those situations in which the complete rehydration method cannot be applied. Two notes of caution are given for cases in which different datasets are compared or combined: (1) the discrepancy between the two rehydration protocols is greatest in the case of high-LDMC (succulent or tender) leaves; (2) the results suggest that, when comparing many studies across unrelated datasets, differences in the measurement protocol may be less important than differences among seasons, years and the quality of local habitats.     

Optimization of DNA extraction from fresh leaf tissues of Melanoxylon brauna (Fabaceae)

Melanoxylon brauna (Fabaceae - Caesalpinioideae) is an endemic and valuable hardwood tree species in the Brazilian Atlantic rainforest; it is comparable to African ebony wood. We tested three protocols of DNA extraction based on the citrimonium bromide (CTAB) method and evaluated the quantity, purity and integrity of the DNA. We also determined whether these procedures interfere with PCR amplification in order to develop a protocol for M. brauna. We found that the quality and integrity of DNA were improved with the use of proteinase K in the extraction buffer and by modifications in the centrifugation speed. The lowest concentration of DNA was obtained with Doyle and Doyle's protocol (5.42 ng/μL). Ferreira and Grattapaglia's protocol modified for M. brauna provided the most DNA (36.89 ng/μL) and the highest quality DNA (purity ratio of 1.80 nm). The original Ferreira and Grattapaglia protocol provided 13.42 ng/μL DNA; however, the purity ratio (1.44 nm) indicates protein contamination. PCR results showed that Ferreira and Grattapaglia's protocol modified for M. brauna gave satisfactory quantity and purity of DNA for molecular studies.