共查询到20条相似文献,搜索用时 31 毫秒
1.
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
2.
We have adapted a procedure for the isolation of genomic DNA from needles of silver fir (Abies alba) to meet the requirements for large-scale analysis of the population genetics of forest trees. Our modifications permit the
entire procedure to be carried out in Eppendorf tubes, which greatly minimizes time, plant material, and the amounts of chemicals.
DNA is recovered with a mean efficiency of 80 μg/g needles, is suitable for restriction by the common endonucleases, and serves
as a substrate for PCR. 相似文献
3.
Masaru Matsumoto Myo Zaw Seint San Aye 《Archives Of Phytopathology And Plant Protection》2013,46(13-16):777-784
A modified baiting technique was conducted for selective isolation, fungal DNA diagnosis and fungal cell lipid assay derived from Myanmar isolates of Rhizoctonia spp., causal agents of rice sheath diseases by trapping selective plant stem segments. Bait plant materials of rice, mat rush and cotton were successfully used to isolate R. solani AG1-IA, R. oryzae and R. oryzae-sativae. Moreover, the three plant materials were also effectively used to detect genomic DNA derived from all Rhizoctonia spp. obtained from Myanmar. Rice segment was the most successful materials for detection of fungal cell lipids including palmitic, stearic and linoleic acids. The results of this experiment demonstrate that bait plant materials of rice, mat rush and cotton were the best useful tools for not only direct isolation, but also fungal DNA diagnosis and cell lipid assay of Myanmar soil environmental conditions. 相似文献
4.
A High Through-put Procedure for Capturing Microsatellites from Complex Plant Genomes 总被引:4,自引:0,他引:4
Connell James P. Pammi Sujata Iqbal Muhammad J. Huizinga Tim Reddy Avutu S. 《Plant Molecular Biology Reporter》1998,16(4):341-349
A method is outlined for large-scale isolation and characterization of microsatellite sequences from complex plant genomes. The method presented here differs from the previously published procedures in the use of randomly sheared (nebulized) genomic DNA for adapter-ligation, rigorous removal of biotinylated oligos, and high-density colony blots for constructing enriched libraries. Using this method we have constructed cotton microsatellite enriched libraries with over 20% (high stringency screening) or 75% (by random sequencing). Thus far we have identified and sequenced over 500 cotton microsatellites using this procedure. The procedure can be used to generate enriched SSR libraries from genomic DNA in about one week. High throughput screening and automated DNA sequencing can be accomplished in less than one month. 相似文献
5.
Nivedita Bir A. Paliwal K. Muralidhar Prasad Reddy P. Usha Sarma 《Preparative biochemistry & biotechnology》2013,43(4):171-181
Abstract A majority of Aspergillus induced diseases are reported to be caused by Aspergillus fumigatus. In immunocompromized and post transplant cases it can lead to invasive aspergillosis. Due to this the molecular fingerprinting of aspergillus isolates by RFLP analysis and development of DNA diagnostic probes are gaining importance. Different methodologies are being adopted for extraction of the genomic DNA from fungus. The existing procedures for isolation of DNA are time consuming and range from several hours to few days. The most difficult step in the isolation of DNA from aspergillus species is to disrupt the tough chitin rich ceil wall without causing damage to genomic DNA. We report here a rapid method for extraction of genomic DNA based on the cleavage of chitin with chitinase. The subsequent modification steps included are lysis and microwave treatment. The chromosomal DNA obtained by this procedure is 1.5 – 2.0 μg per mg of wet weight of mycelia and is observed to be minimally sheared. It is pure enough for restriction analysis and for use in the PCR to detect the gene coding for 18 kDa allergen which has been identified in our laboratory using western blot analysis with human patient sera. 相似文献
6.
A simple and reliable method was developed for isolating pollen nuclei from Brassica napus and Triticum aestivum for DNA analysis using flow cytometry. The nuclei were released from pollen by ultrasonic treatment. The isolated nuclei following filtration through nylon mesh and a purification procedure were suitable for flow cytometric analysis as well as for isolating genomic DNA. Ultrasonic treatment time was optimized for B. napus pollen at different developmental stages. The method is effective and suitable for the preparation of many samples. We analyzed the nuclear DNA levels in pollen of B. napus at three major developmental stages as well as in mature wheat pollen. Only a single 1C peak representing the haploid DNA level was detected in the nuclei isolated from Brassica uninucleate microspores as well as in mature Triticum pollen. Interestingly, diploid nuclei were detected in both binucleate and mature pollen of B. napus. The possible origins of the diploid nuclei are discussed.Abbreviations DAPI 4,6-Diamidino-2-phenylindole - NIB Nuclear isolation buffer 相似文献
7.
RAPD analysis in flax: Optimization of yield and reproducibility using klenTaq 1 DNA polymerase,chelex 100, and gel purification of genomic DNA 总被引:1,自引:0,他引:1
We have developed an optimized RAPD analysis approach using the unusually heat-stable KlenTaq1 DNA polymerase. This enzyme is used in conjunction with a genomic DNA isolation method that includes a modified CTAB DNA
isolation protocol, ethanol re-precipitation of resuspended nucleic acids from 2M NaCl, and Chelex 100 treatment. When needed,
additional gel purification and isolation of high molecular weight DNA for use as a template in RAPD analysis is shown to
remove amplification product ambiguity from within isolates of the same line as well as from between lines. This optimized
RAPD analysis was used to define polymorphisms in lines of flax nearly isogenic for rust resistance at theL locus. It should also be useful for any plant species. 相似文献
8.
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
9.
Improved high-throughput sunflower and cotton genomic DNA extraction and PCR fidelity 总被引:1,自引:0,他引:1
The extraction of high-quality genomic DNA for PCR amplification from sunflower (Helianthus annuus) and cotton (Gossypium spp.) is challenging because of the presence of polysaccharides, secondary metabolites, and polyphenolics in the tissues.
A high-throughput DNA extraction protocol was needed in our laboratory for simple sequence repeats (SSR)-marker screening
and other molecular analyses that do not require organic extraction steps of phenol or chloroform. Here we describe 2 improved
highthroughput protocols for DNA extraction and in-PCR modification that result in successful PCR amplification of sunflower
and cotton. While the sunflower DNA extraction protocol uses reducing agents such as sodium metabisulfite and dithiothreitol
(DTT), the cotton protocol uses polyvinylpyrrolidone (PVP) in PCR reactions and reducing agents in the DNA extraction procedure. 相似文献
10.
De S Kaur G Roy A Dogra G Kaushik R Yadav P Singh R Datta TK Goswami SL 《Indian journal of microbiology》2010,50(4):412-418
A simple, inexpensive and effective genomic DNA isolation procedure for Lactobacillus isolates from traditional Indian fermented milk (dahi) is described. A total of 269 Lactobacillus isolates from fermented milk collected from four places in North and west India were tested for lysis by an initial weakening
of the Gram positive cell wall with Ampicillin followed by Lysozyme treatment. The average genomic DNA yield was ~50 μg/ml
log phase culture. Quality and repeatability of the method was found to be adequate for subsequent molecular applications.
The quality of the genomic DNA isolated by this method was verified by restriction digestion and polymerase chain reaction
(PCR). No inhibition was observed in subsequent PCR amplification and restriction digestion. The presented method is rapid,
cheap and useful for routine DNA isolation from gram positive bacteria such as Lactobacillus. 相似文献
11.
R. Arredondo-Peter A. Bonic G. Sarath R. V. Klucas 《Plant Molecular Biology Reporter》1995,13(2):138-146
Phage DNA isolation from genomic and cDNA libraries is time-consuming and cumbersome. Our work reports on the use of a pair
of λ gt11 primers to amplify by PCR any insert DNA that is cloned into theEco RI restriction site. Using crude phage pools and direct phage plaques from a corn root cDNA library, we amplified and cloned
DNA fragments. The amount of amplification products was similar when phage DNA and phage pools were used as templates. Amplified
fragments could be purified, cloned and sequenced by a relatively simple procedure, thus allowing rapid detection and analysis
of inserts in a large number of phage plaques. 相似文献
12.
Repetitive sequences constitute a significant component of most eukaryotic genomes, and the isolation and characterization
of repetitive DNA sequences provide an insight into the organization of the genome of interest. Here, we report the isolation
and the molecular analysis and methylation status of a novel tandemly organized repetitive DNA sequence from the genome of
Poncirus trifoliata. Digestion of P. trifoliata DNA with Afa I produced a prominent fragment of approximately 400 bp. Southern blotting analysis of genomic DNA digested with the same
enzyme revealed a ladder composed of DNA fragments that are multimers of the 400-bp Afa I band, indicating that the repetitive DNA is arrayed in tandem. This suggests that Afa I isolated a novel satellite that we have called Poncirus trifoliata satellite DNA 400 (PN400). This satellite composes 25% of the genome and it is also present in lemon, sour orange and kumquat.
Analysis of the methylation status demonstrated that the cytosines in CCGG sequences in this satellite were methylated. 相似文献
13.
Isolation of genomic DNA from defatted oil seed residue of opium poppy (Papaver sominiferum) 总被引:1,自引:0,他引:1
A simple protocol for obtaining pure, restrictable and amplifiable megabase genomic DNA from oil free seed residue ofPapaver sominiferum, an important oil seed and morphanian alkaloid yielding plant, has been developed. Fixed oil from the dry seeds was completely
recovered in an organic solvent and quantified gravimetrically followed by processing of the residual biomass (defatted seed
residue) for genomic DNA isolation. The isolated DNA could be cut by a range of restriction enzymes. The method enables simultaneous
isolation and recovery of lipids and genomic DNA from the same test sample, thus allowing two independent analyses from a
single sample. 相似文献
14.
Protocols are presented for preparing DNA from a genomic library in λ phage and for synthesizing genomic fragments using PCR
with nested vector- and gene-specific primers and linker-primers. Library DNA, isolated fromE. coli liquid lysates by a simple protocol, is used as template in PCR following a commercial protocol. The method produces library
DNA sufficient for several hundred PCRs, incorporates nested primers to reduce nonspecific product formation, and enables
the synthesis of linker-containing DNA fragments containing selected restriction sites to simplify subsequent cloning. The
isolation of 5′ upstream sequences of three different arabidopsis genes by this methodod is described. 相似文献
15.
Y. Al-Shayji M. Saleem S. Al-Amad S. Al-Awadhi F. Al-Salameen 《Engineering in Life Science》1994,14(2):163-168
The objective of this study was to adapt a plant DNA preparation procedure for the isolation of biologically active DNA and DNA with a high molecular weight from the date palm and other related palms. Mature leaf tissue extractions of the date palm, Phoenix dactylifera L., the coconut tree, Cocos nucifera, and the Mexican Fan Palm, Washingtonia robusta, were characterized for total genomic DNA yield, purity, integrity, as well as restriction digestion and ligation capabilities. It is demonstrated here that the DNA isolation procedure, modified for use with various palm leaf tissues, met the criteria for simplicity and low costs, and yielded DNA of high molecular weight (~50 Kbp) and of sufficient purity suitable for molecular studies. 相似文献
16.
Mini-scale Genomic DNA Extraction from Cotton 总被引:8,自引:1,他引:7
Chaudhry Bushra Yasmin Afshan Husnain Tayyib Riazuddin S. 《Plant Molecular Biology Reporter》1999,17(3):280-280
Large amounts of polyphenolics in cotton leaves make it difficult to obtain high-quality genomic DNA during extraction. A procedure to isolate nuclear DNA from local cotton leaves (gossypium hirsutum, MNH93, CIM443, FH672) was therefore developed. It consists of rapid isolation of stable nuclei, which hinders covalent interactions with phenolics, followed by DNA extraction. The yield and quality of the resulting DNA is satisfactory and the protocol can be scaled up or down according to sample size. It is suitable for PCR and the restriction enzyme digestion needed for Southern and RFLP analysis. 相似文献
17.
Bipinchandra B. Kalbande Anita S. Patil Pranjib K. Chakrabarty 《Journal of plant biochemistry and biotechnology.》2016,25(4):437-441
Cotton is a stubborn plant for genomic DNA isolation due to its high-level of polyphenolics, polysaccharides and secondary metabolites. Genomics and molecular biology studies require high quality and large quantity of DNA. We have standardized an efficient miniprep protocol for cotton genomic DNA isolation, which not only provides higher yield from 800 to 1400 µg of DNA from 200 to 300 mg of fresh leaf tissue but also provide excellent purity. The DNA is amenable to all elementary enzymatic preparations, PCR techniques, Southern blotting and to high end genomic studies. The technique does not require liquid nitrogen, needs small amount of sample, less time, fewer chemicals and one can process up to 100 samples per day. The genomic DNA extracted was good for transgenic event characterization and marker assisted selection. 相似文献
18.
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
19.
Athanasios Tsaftaris Konstantinos Pasentzis Anagnostis Argiriou 《Biotechnology letters》2010,32(1):157-161
We have devised an improved method of genome walking, named rolling circle amplification of genomic templates for Inverse
PCR (RCA–GIP). The method is based on the generation of circular genomic DNA fragments, followed by rolling circle amplification
of the circular genomic DNA using ϕ29 DNA polymerase without need for attachment of anchor sequences. In this way from the
circular genomic DNA fragments, after RCA amplification, a large amount of linear concatemers is generated suitable for Inverse
PCR template that can be amplified, sequenced or cloned allowing the isolation of the 3′- and 5′- of unknown ends of genomic
sequences. To prove the concept of the proposed methodology, we used this procedure to isolate the promoter regions from different
species. Herein as an example we present the isolation of four promoter regions from Crocus sativus, a crop cultivated for saffron production. 相似文献
20.
We describe a rapid and efficient procedure for the isolation of chloroplast (and nuclear) DNA from walled cells ofChlamydomonas reinhardtii. Total nucleic acids are prepared and separated by equilibrium centrifugation in CsCl-bisbenzimide gradients using a high-speed
table-top ultracentrifuge. Chloroplast DNA sufficient for several restriction analysis is obtained from 1 liter of cells in
one to two days. 相似文献