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1.
A rapid and hazardous reagent free protocol for genomic DNA extraction suitable for genetic studies in plants 总被引:2,自引:0,他引:2
Protocols for genomic DNA extraction from plants are generally lengthily, since they require that tissues be ground in liquid
nitrogen, followed by a precipitation step, washing and drying of the DNA pellet, etc. This represents a major challenge especially
when several hundred samples must be screened/analyzed within a working day. There is therefore a need for a rapid and simple
procedure, which will produce DNA quality suitable for various analyses. Here, we describe a time and cost efficient protocol
for genomic DNA isolation from plants suitable for all routine genetic screening/analyses. The protocol is free from hazardous
reagents and therefore safe to be executed by non-specialists. With this protocol more than 100 genomic DNA samples could
manually be extracted within a working day, making it a promising alternative in genetic studies of large-scale genomic screening
projects. 相似文献
2.
Large amounts of polyphenolics in dove tree leaves make it difficult to obtain high-quality genomic DNA during extraction.
A rapid DNA minipreparation method was developed for dove tree (Davidia involucrata) and yields 40–50 μg genomic DNA from 0.1 g fresh matured and young leaves and bracts. The yield and quality of the resulting
DNA is satisfactory, and the protocol can be scaled up according to sample size. The obtained DNA is suitable for PCR and
the restriction enzyme digestion needed for Southern blotting. 相似文献
3.
Lingling Zhang Lijing Dai Junbo Gou Junhua Peng 《Journal of plant biochemistry and biotechnology.》2013,22(4):492-497
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. 相似文献
4.
Arun Dev Sharma Prabhjot Kaur Gill Prabhjeet Singh 《Plant Molecular Biology Reporter》2002,20(4):415-415
DNA extraction is difficult in a variety of plants because of the presence of metabolites that interfere with DNA isolation
procedures and downstream applications such as DNA restriction, amplification, and cloning. The chemotypic heterogeneity among
species may not permit optimal DNA yield with a single protocol; thus, even closely related species may require different
isolating protocols. Here we describe a modified procedure based on the hexadecyltrimethylammonium bromide (CTAB) method to
isolate DNA from tissues containing high levels of polysaccharides. The procedure is applicable to both dry and fresh tissues
and was tested on chickpea seeds, soybean, and wheat leaves. This method solved the problems of DNA degradation, contamination,
and low yield due to binding and/or coprecipitation with starches and polysaccharides. The isolated DNA proved amenable to
PCR amplification and restriction digestion. 相似文献
5.
Echevarría-Machado I Sánchez-Cach LA Hernández-Zepeda C Rivera-Madrid R Moreno-Valenzuela OA 《Molecular biotechnology》2005,31(2):129-135
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. 相似文献
6.
Michiels A Van den Ende W Tucker M Van Riet L Van Laere A 《Analytical biochemistry》2003,315(1):85-89
The isolation of intact, high-molecular-mass genomic DNA is essential for many molecular biology applications including long PCR, endonuclease restriction digestion, Southern blot analysis, and genomic library construction. Many protocols are available for the extraction of DNA from plant material. However, for latex-containing Asteraceae (Cichorioideae) species, standard protocols and commercially available kits do not produce efficient yields of high-quality amplifiable DNA. A cetyltrimethylammonium bromide protocol has been optimized for isolation of genomic DNA from latex-containing plants. Key steps in the modified protocol are the use of etiolated leaf tissue for extraction and an overnight 25 degrees C isopropanol precipitation step. The purified DNA has excellent spectral qualities, is efficiently digested by restriction endonucleases, and is suitable for long-fragment PCR amplification. 相似文献
7.
Mustapha Aitchitt Charles C. Ainsworth Madan Thangavelu 《Plant Molecular Biology Reporter》1993,11(4):317-319
A method is presented for the rapid isolation of high-molecular-weight DNA from mature leaves of date palm (Phoenix dactylifera L.), using a CTAB-based buffer. The method yields up to 800 μg of DNA from 1 g of leaf tissues. The procedure was also suitable
for DNA extraction from callus or buds from tissue culture. The DNA obtained through this method was a good substrate for
at least seventeen restriction endonucleases. This method was also used to extract DNA from mature leaves of coconut and may
be applicable to other species of palms. 相似文献
8.
Modification of a CTAB DNA extraction protocol for plants containing high polysaccharide and polyphenol components 总被引:58,自引:1,他引:57
A relatively quick, inexpensive and consistent protocol for extraction of DNA from expanded leaf material containing large
quantities of polyphenols, tannins and polysaccharides is described. Mature strawberry leaves, which contain high levels of
these secondary components, were used as a study group. The method involves a modified CTAB extraction, employing high salt
concentrations to remove polysaccharides, the use of polyvinyl pyrrolidone (PVP) to remove polyphenols, an extended RNase
treatment and a phenol-chloroform extraction. Average yields range from 20 to 84 μg/g mature leaf tissue for both wild and
cultivated octoploid and diploidFragaria species. Results from 60 plants were examined, and were consistently amplifiable in the RAPD reaction with as little as 0.5
ng DNA per 25-μL reaction. Presently, this is the first procedure for the isolation of DNA from mature strawberry leaf tissue
that produces consistent results for a variety of different species, both octoploid and diploid, and is both stable and PCR
amplifiable before and after extended storage. This procedure may prove useful for other difficult species in the family Rosaceae. 相似文献
9.
Rodrigo Hasbún Luís Valledor Jos L. Rodríguez Estrella Santamaria Darcy Ríos Manuel Sanchez María J. Caal Roberto Rodríguez 《Plant Physiology and Biochemistry》2008,46(8-9):815-822
Quantification of deoxynucleosides using micellar high-performance capillary electrophoresis (HPCE) is an efficient, fast and inexpensive evaluation method of genomic DNA methylation. This approach has been demonstrated to be more sensitive and specific than other methods for the quantification of DNA methylation content. However, effective detection and quantification of 5-methyl-2′-deoxycytidine depend of the sample characteristics. Previous works have revealed that in most woody species, the quality and quantity of RNA-free DNA extracted that is suitable for analysis by means of HPCE varies among species of the same gender, among tissues taken from the same tree, and vary in the same tissue depending on the different seasons of the year. The aim of this work is to establish a quantification method of genomic DNA methylation that lends itself to use in different Castanea sativa Mill. materials, and in other angiosperm and gymnosperm woody species. Using a DNA extraction kit based in silica membrane has increased the resolutive capacity of the method. Under these conditions, it can be analyzed different organs or tissues of angiosperms and gymnosperms, regardless of their state of development. We emphasized the importance of samples free of nucleosides, although, in the contrary case, the method ensures the effective separation of deoxynucleosides and identification of 5-methyl-2′-deoxycytidine. 相似文献
10.
A simple and efficient method for DNA extraction from grapevine cultivars andVitis species 总被引:3,自引:0,他引:3
Muhammad A. Lodhi Guang-Ning Ye Norman F. Weeden Bruce I. Reisch 《Plant Molecular Biology Reporter》1994,12(1):6-13
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. 相似文献
11.
12.
A DNA extraction procedure that does not require hazardous materials, such as CTAB, phenols, or liquid nitrogen, was optimized
forAnthurium andreanum, a plant rich in polysaccharides and polyphenolics. Three DNA isolation techniques were tested. The modified Rowhani protocol
(1983), with slight modifications, was found to yield up to milligrams of DNA suitable for RAPD from spathe and leaf tissues.
High-quality DNA was obtained readily from spathe tissues, while a spermine precipitation step was found to be essential when
DNA was extracted from the leaf tissues. 相似文献
13.
An improved protocol for the isolation of DNA from dry material of someHesperis specimens is described. The isolated DNA is suitable for random amplification of polymorphic DNA (RAPD) analysis. Different
DNA extraction protocols were examined to determine which might yield DNA from dry leaf tissue ofHesperis specimens. The methods examined include the protocols with hexadecyltrimethylammonium bromide (CTAB) described by Doyle and
Doyle (1987); sodium dodecyl sulfate (SDS) by Dellaporta et al. (1983); and CTAB and SDS, the modified minipreparation, by
Dellaporta et al (1983). None of these procedures yielded DNA of suitable purity for RAPD assay. We established an improved
procedure involving CTAB and enzymatic digestion of proteins and RNA. The recovery of DNA with an average yield of 25 mg/g
of leaf material was possible with this procedure. RAPD bands, which could be used to distinguish amongHesperis specimens, were generated. 相似文献
14.
High-throughput DNA extraction from forest trees 总被引:2,自引:1,他引:1
Mervyn Shepherd Michael Cross Rhonda L. Stokoe Leon J. Scott Megan E. Jones 《Plant Molecular Biology Reporter》2002,20(4):425-425
It is difficult to extract pure high-quality DNA from trees, which may not be amenable to advances in extraction methods suitable
for other plants. A new commercial high-throughput DNA extraction system, using a silica binding matrix for purification and
a multisample mixer mill for tissue disruption, was evaluated for its suitability withEucalyptus spp.,Pinus spp., andAraucaria cunninghamii (hoop pine). DNA suitable for a range of molecular biology applications was successfully extracted from all genera. The method
was highly reliable when tested in more than 500 preparations and could be adapted to different tree species with relatively
minor modifications. 相似文献
15.
Mithun Raj Vishnu S. Nath M. Senthil @ Sankar M.L. Jeeva 《Archives Of Phytopathology And Plant Protection》2013,46(6):753-760
The methods employed for DNA extraction from many plants is difficult because of the metabolites that interfere with DNA isolation procedures. We have developed a reliable and efficient method for isolating genomic DNA free from polysaccharide, polyphenols and protein contaminants from Dioscorea spp. The method involves inactivation of contaminant proteins by using CTAB/Proteinase K and precipitation of polysaccharides in the presence of high concentration of salt. The purity of genomic DNA was confirmed by A260/280 and A260/230 ratios calculated from the spectrophotometric readings and further by restriction analysis of the isolated DNA using restriction enzymes Eco RI. The total genomic DNA extracted by the new protocol was used for polymerase chain reaction amplification, RAPD analysis, restriction digestion and pathogen screening. The new protocol can be successfully used for both small- and large-scale preparation of genomic DNA from different tissues of Dioscorea spp. The quarantine of seed tubers and use of pathogen-free tubers for planting is a prerequisite for integrated disease management strategy. The protocol can be used for the isolation of genomic DNA from other crop plants too. 相似文献
16.
Dimorphandra mollis (Leguminosae), known as faveiro and fava d'anta, is a tree that is widely distributed throughout the Brazilian Cerrado (a savanna-like biome). This species is economically valuable and has been extensively exploited because its fruits contain the flavonoid rutin, which is used to produce medications for human circulatory diseases. Knowledge about its genetic diversity is needed to guide decisions about the conservation and rational use of this species in order to maintain its diversity. DNA extraction is an essential step for obtaining good results in a molecular analysis. However, DNA isolation from plants is usually compromised by excessive contamination by secondary metabolites. DNA extraction of D. mollis, mainly from mature leaves, results in a highly viscous mass that is difficult to handle and use in techniques that require pure DNA. We tested four protocols for plant DNA extraction that can be used to minimize problems such as contamination by polysaccharides, which is more pronounced in material from mature leaves. The protocol that produced the best DNA quality initially utilizes a sorbitol buffer to remove mucilaginous polysaccharides. The macerated leaf material is washed with this buffer until there is no visible mucilage in the sample. This protocol is adequate for DNA extraction both from young and mature leaves, and could be useful not only for D. mollis but also for other species that have high levels of polysaccharide contamination during the extraction process. 相似文献
17.
Isolating RNA from dry conifer seeds can be difficult because of a number of interfering compounds present in seeds. We describe
a protocol for total RNA isolation from black spruce dry seeds, which is an adaptation of a method used for mouse myeloma
tissues. The extraction relies on selective precipitation of RNA by using lithium chloride. 相似文献
18.
Alternative approach for consistent yields of total genomic DNA from cotton (Gossypium hirsutum L.) 总被引:1,自引:1,他引:0
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. 相似文献
19.
DNA extraction is difficult in many plants because of metabolites that interfere with DNA isolation procedures and subsequent
applications such as DNA restriction, amplification, and cloning. We developed the first reliable and efficient method for
isolatingVictoria amazonica genomic DNA that is free from polysaccharides and polyphenols. This protocol uses 1.5 M NaCl, 2% polyvinylpyrrolidone (PVP)
(Mr 1000), 5% mercaptoethanol, 0.12% sodium sulfite, and an incubation at 65°C for 4 h. The purity of isolated genomic DNA
was confirmed by means of high-performance liquid chromatography (HPLC) profile and spectrophotometric analyses (A260/230 ratio of 1.836, A260/280 of 1.842). DNA was obtained in the amount of 387 μg per gram of leaf material, and it proved amenable to restriction digestion. 相似文献
20.
A simple procedure for the isolation of DNA from mature leaf tissue was developed. This procedure purified DNA using Sephacryl
S-1000 column and PEG 8000 precipitation. Polysaccharide-like components were successfully removed from DNA samples from species
in which polysaccharides were found to be difficult to remove by phenol/chloroform extraction. The DNA obtained by this method
was suitable for PCR, RAPD, enzyme digestion, and Southern-blot analyses. 相似文献