共查询到20条相似文献,搜索用时 15 毫秒
1.
Isolation of high-quality DNA from plants, especially plants from the Cerrado, is notoriously difficult because of polysaccharides and secondary compounds produced by plants from this biome. DNA isolation and its quality may be compromised by chemical defenses such as tannins and phenols. Quantitative plant defenses tend to have a cumulative effect, increasing in concentration during leaf development, reducing DNA quality extracted in mature compared to young leaves. We report the effect of leaf age on DNA extraction of Dimorphandra mollis. Our working hypothesis was that the young leaves have more DNA than old leaves of the same individual because chemical defenses accumulate in older leaves. Young and old leaves were sampled from eight mature trees as well as leaves from eight seedlings in the north region of Minas Gerais State. Genomic DNA extraction followed the standard CTAB procedure. DNA isolation was very successful from young leaves of 16 individuals of D. mollis. The extracted DNA exhibited high quality and the DNA quantity was also high, with an A(260)/A(280) ratio above 1.8, which is within the optimal sample range. In contrast, DNA isolation from old leaves was not successful. When the DNA was extracted from old leaves, the DNA was brownish, indicating contamination by phenolic compounds. These metabolites oxidize the DNA irreversibly, which hinders amplification of DNA by PCR by inhibiting the action of enzymes such as Taq polymerase. PCR performed with DNA from young leaves of D. mollis was successful and produced strong bands for RAPD markers. 相似文献
2.
Mohammad S. Uddin Wenli Sun Xinhua He Jaime A. Teixeira da Silva Qi Cheng 《Biologia》2014,69(2):133-138
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. 相似文献
3.
Modification of a CTAB DNA extraction protocol for plants containing high polysaccharide and polyphenol components 总被引:57,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
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.
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. 相似文献
6.
In order to investigate the mutation characteristics and to further examine the genetic variation of mutant sunflower (Helianthus annuus) obtained in plants grown from seeds exposed to space conditions, only the mature tissues such as leaf and flower could be used for DNA extraction after mutation characteristics were confirmed. The rich contents of polysaccharides, tannins, secondary metabolites, and polyphenolics made it difficult to isolate high-quality DNA from mature leaves of sunflower according to previous reports. Based on the comparison of the differences in previously reported protocols, a modified method for the extraction of high-quality DNA was developed. Using this protocol, the DNA isolated from sunflower was high in quality and suitable for restriction digestion (EcoRI, HindII, BamHI), random amplified polymorphic DNA study and further molecular research. Therefore, the modified protocol was suitable for investigating the genetic variation of sunflower using mature leaf DNA. 相似文献
7.
Extraction of total RNA from leaves of Eucalyptus and other woody and herbaceous plants using sodium isoascorbate 总被引:3,自引:0,他引:3
Rapid extraction of total RNA from Eucalyptus leaves is difficult due to the high content of polyphenolics and polysaccharides. A rapid and simple method was developed by using an extraction buffer containing sodium isoascorbate at a concentration of 500 mM. This method consisted of one or two chloroform extractions, one acid guanidium-phenol-chloroform extraction, and isopropanol precipitation alone. The yields of the RNA fractions were 246-1750 micrograms/g fresh weight when leaves of Eucalyptus, five other woody plants, and four herbaceous plants were used as samples. The contamination of the RNA fractions by proteins and polysaccharides was very limited as judged spectrophotometrically. When the RNA fractions were subjected to agarose gel electrophoresis, intact rRNA bands were detected. The RNA fractions could be used for RT-PCR. These results indicate that our new method achieves a simple and rapid preparation of high-quality RNA from leaves of Eucalyptus and other plant species. 相似文献
8.
Josquin F. G. Tibbits Luke J. McManus Antanas V. Spokevicius Gerd Bossinger 《Plant Molecular Biology Reporter》2006,24(1):81-91
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. 相似文献
9.
Comparative analysis of different DNA extraction protocols in fresh and herbarium specimens of the genus Dalbergia 总被引:1,自引:0,他引:1
Five published DNA extraction protocols were compared for their ability to produce good quality DNA from fresh and herbarium leaves of several species of the genus Dalbergia. The leaves of these species contain high amounts of secondary metabolites, which make it difficult to perform a clean DNA extraction and thereby interfering with subsequent PCR amplification. The protocol that produced the best DNA quality in most of the Dalbergia species analyzed, utilizes polyvinylpyrrolidone to bind the phenolic compounds, a high molar concentration of NaCl to inhibit co-precipitation of polysaccharides and DNA, and LiCl for removing RNA by selective precipitation. The DNA quality of herbarium specimens was worse than that for fresh leaves, due to collecting conditions and preservation of samples. We analyzed 54 herbarium specimens, but the recovered DNA allowed successful PCR amplification in only eight. For the genus Dalbergia, the herbarium is an important source of material for phylogenetic and evolutionary studies; due to the occurrence of the different species in various geographical regions in Brazil, it is difficult to obtain fresh material in nature. Our results demonstrated that for Dalbergia species the methods used for the collection and preservation of herbarium specimens have a mayor influence on DNA quality and in the success of phylogenetic studies of the species. 相似文献
10.
Modified CTAB Procedure for DNA Isolation from Epiphytic Cacti of the Genera Hylocereus and Selenicereus (Cactaceae) 总被引:4,自引:0,他引:4
We present a simple protocol for DNA isolation from climbing cacti, genera Hylocereus and Selenicereus. The abundant polysaccharides present in Hylocereus and Selenicereus species interfere with DNA isolation, and DNA extracts, rich in polysaccharides, are poor templates for amplification using polymerase chain reaction (PCR). We used roots as the source tissue due to the lower viscosity of the extracts relative to that of other tissues. The extraction and isolation procedure we devised consists of the following steps: (1) three washes of ground tissue with the extraction buffer to remove the polysaccharides; (2) extraction with high-salt (4 M NaCl) cetyltrimethylammonium bromide (CTAB) buffer to remove the remaining polysaccharides; (3) removal of RNA by RNase; (4) phenol:chloroform extraction to remove proteins; (5) chloroform extraction to remove remaining phenols. The yields ranged from 10 to 20 g DNA/g fresh roots. DNA samples prepared by our method were consistently amplifiable in the RAPD reaction and gave reproducible profiles. 相似文献
11.
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. 相似文献
12.
顽拗植物类群的总DNA制备 总被引:43,自引:0,他引:43
从富含多糖的顽拗植物类群提取与纯化DNA是许多研究领域例如居群生物学、生物多样性、分子标记辅助育种研究普遍遇到的难题。以西南桦 (Betulaalnoides)为例发展了一套改进的方案 ,有效地从这种顽拗植物的干叶和鲜叶中制备了DNA。此方案包括 3个关键步骤 :首先从植物细胞匀浆中用不含CTAB的缓冲液洗去大部分多糖和其他次生物质 ;在提取介质中采用 3%CTAB而不是通常用的 2 %CTAB ;将常用的高盐去糖的纯化操作提前到用异丙醇沉淀DNA之前进行。从西南桦提取的DNA已成功地用于RAPD_PCR扩增和限制性酶切。这个简单、经济和可靠的改进方案也适用于许多其他的顽拗植物类群。 相似文献
13.
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
14.
Juan F. Fern’andez Victoria L. Sork Gerardo Gallego Jershon L’opez Adriana Bohorques Joe Tohme 《Plant Molecular Biology Reporter》2000,18(4):397-397
Conservation of 15 out of 24 previously identified microsatellite loci (62.5%), was found in a survey of the South American
oak,Quercus humboldtii. The number of alleles per locus varied from 2 to 20, detecting at least 5 microsatellite loci with 5 or more alleles. This
number of loci and alleles is adequate for most studies of genetic diversity and gene flow analysis. In addition, a method
for extracting DNA from mature oak leaves is described that minimizes oxidation of tannins, a common problem in silica-gel-dried
samples. The microsatellite markers detected in this study and the DNA extraction protocol may be applied to more than 30
species ofQuercus that exist in tropical America. 相似文献
15.
Drosera rotundifolia, Drosera capensis, and Drosera regia are carnivorous plants of the sundew family, characterized by the presence of stalked and sticky glands on the upper leaf surface, to attract, trap, and digest insects. These plants contain exceptionally high amounts of polysaccharides, polyphenols, and other secondary metabolites that interfere with DNA isolation and subsequent enzymatic reactions such as PCR amplification. We present here a protocol for quick isolation of Drosera DNA with high yield and a high level of purity, by combining a borate extraction buffer with a commercial DNA extraction kit, and a proteinase K treatment during extraction. The yield of genomic DNA is from 13.36 μg/g of fresh weight to 35.29 μg/g depending of the species of Drosera, with a A???/A??? ratio of 1.43-1.92. Moreover, the procedure is quick and can be completed in 2.5 h. 相似文献
16.
Genomic DNA of high quality and quantity is needed to analyze genetic diversity with AFLP.Carpobrotus plant species, like most succulents, contain high amounts of polysaccharides and polyphenols, making PCR amplification difficult.
Our protocol eliminates contaminants before DNA isolation by using leaf callus as plant material. This simple and inexpensive
technique gives an average DNA yield of 1800 ng/g of callus and high reproducible profiles in AFLP. Our results indicate that
no genetic variability is associated with callus culture conditions. This technique is suitable for studying genomic polymorphism
in succulents and other plants when classic DNA extraction procedures fail. 相似文献
17.
A protocol for the extraction of DNA from ancient skeletal material was developed. Bone specimen samples (powder or slice), buffer, pretreatment, and extraction methodologies were compared to investigate the best conditions yielding the highest concentration of DNA. The degree of extract contamination by polymerase chain reaction (PCR) inhibitors was compared as well. Pretreatment was carried out using agitation in an incubator shaker and microwave digestion. Subsequently, DNA from bones was isolated by the classical organic phenol–chloroform extraction and silica-based spin columns. Decalcification buffer for total demineralization was required as well as lysis buffer for cell lysis to obtain DNA, whereas microwave-assisted digestion proved to be very rapid, with an incubation time of 2 min instead of 24 h at an incubator shaker without using lysis buffer. The correction of isolated DNA was detected using real-time PCR with melt curve analysis, which was 82.8 ± 0.2 °C for highly repetitive α-satellite gene region specific for human chromosome 17 (locus D17Z1). Consequently, microwave-based DNA digestion followed by silica column yielded a high-purity DNA with a concentration of 19.40 ng/μl and proved to be a superior alternative to the phenol–chloroform method, presenting an environmentally friendly and efficient technique for DNA extraction. 相似文献
18.
Isolation of plant nuclei suitable for flow cytometry from recalcitrant tissue by use of a filtration column 总被引:4,自引:0,他引:4
Flow cytometry is widely applied in the determination of nuclear DNA content and ploidy level in many organisms. However,
a difficulty with flow cytometry is the method's intrinsic inability to tolerate large particles that associate with the isolated
nuclei. A suspension of plant nuclei can often contain a high level of crystalline calcium oxalate, which blocks the fluidics
system of the flow cytometer. We designed a cotton column and added polyvinylpyrrolidone-40 to the buffer to remove phenolic
impurities and cytoplasmic compounds from plant nuclei, making the suspension suitable for flow cytometry. This simple and
highly efficient protocol enables isolation of intact nuclei from plant tissues containing high levels of polysaccharides,
calcium oxalate crystals and other metabolites. Our protocol resulted in the isolation of intact nuclei from mature orchid
leaves. This method can be used on recalcitrant tissues and is particularly effective on plants containing calcium oxalate
crystals. 相似文献
19.
Sina Atashpaz Sajjad Khani Abolfazl Barzegari Jaleh Barar Sepideh Zununi Vahed Reza Azarbaijani Yadollah Omidi 《Microbiology》2010,79(4):538-542
The intactness of DNA is the keystone of genome-based clinical investigations, where rapid molecular detection of life-threatening
bacteria is largely dependent on the isolation of high-quality DNA. Various protocols have been so far developed for genomic
DNA isolation from bacteria, most of which have been claimed to be reproducible with relatively good yields of high-quality
DNA. Nonetheless, they are not fully applicable to various types of bacteria, their processing cost is relatively high, and
some toxic reagents are used. The routine protocols for DNA extraction appear to be sensitive to species diversity, and may
fail to produce high-quality DNA from different species. Such protocols remain time-consuming and tedious, thus to resolve
some of these impediments, we report development of a very simple, rapid, and high-throughput protocol for extracting of high-quality
DNA from different bacterial species. Based upon our protocol, interfering phenolic compounds were removed from extraction
using polyvinylpyrrolidone (PVP) and RNA contamination was precipitated using LiCl. The UV spectrophotometry and gel electrophoresis
analysis resulted in high A
260/A
280 ratio (>1.8) with high intactness of DNA. Subsequent evaluations were performed using some quality-dependent techniques (e.g.,
RAPD marker and restriction digestions). The isolated DNA from 9 different bacterial species confirmed the accuracy of this
protocol which requires no enzymatic processing and accordingly its low-cost making it an appropriate method for large-scale
DNA isolation from various bacterial species. 相似文献
20.
Borges DB Amorim MB Waldschmidt AM Mariano-Neto E Vivas CV Pereira DG 《Genetics and molecular research : GMR》2012,11(2):1586-1591
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. 相似文献