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1.
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
2.
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
3.
A rapid DNA extraction method for sugarcane and its relatives 总被引:3,自引:0,他引:3
Rhonda J. Honeycutt Bruno W. S. Sobral Paul Keim James E. Irvine 《Plant Molecular Biology Reporter》1992,10(1):66-72
A simple DNA extraction method based on CTAB precipitation was used to obtain DNA from members of the genusSaccharum and related species. DNA yields and purities were similar for allSaccharum species sampled. The method described here resulted in high quality total DNA suitable for polymerase chain reaction (PCR)-based
techniques as well as restriction endonuclease digestion, Southern hybridization, and DNA cycle-sequencing. 相似文献
4.
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
5.
Ichiro Kasajima Yoko Ide Naoko Ohkama-Ohtsu Hiroaki Hayashi Tadakatsu Yoneyama Toru Fujiwara 《Plant Molecular Biology Reporter》2004,22(1):49-52
We present a method for instant DNA extraction fromArabidopsis thaliana based on a simple DNA extraction method (Edwards et al., 1991). A piece of rosette leaf (typically 3–5 mg) was ground in
a centrifuge tube in extraction solution. Extracted DNA was suitable for PCR analysis, without centrifugation. The feasibility
of this method was confirmed by testing 24 primer sets. This method requires less than 1 mg of plant tissue and is useful
for genetic mapping, transgene detection, and other experiments. 相似文献
6.
Following basal stem rot in young oil palm plantings 总被引:1,自引:0,他引:1
The PCR primer GanET has previously been shown to be suitable for the specific amplification of DNA from Ganoderma boninense. A DNA extraction and PCR method has been developed that allows for the amplification of the G. boninense DNA from environmental samples of oil palm tissue. The GanET primer reaction was used in conjunction with a palm-sampling programme to investigate the possible infection of young palms through cut frond base surfaces. Ganoderma DNA was detected in frond base material at a greater frequency than would be expected by comparison with current infection levels. Comparisons are made between the height of the frond base infected, the number of frond bases infected, and subsequent development of basal stem rot. The preliminary results suggest that the development of basal stem rot may be more likely to occur when young lower frond bases are infected. 相似文献
7.
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
8.
With recent advances in molecular biology, it is now possible to use the trace amounts of DNA in faeces to non-invasively
sample endangered species for genetic studies. A highly vulnerable population of approximately 100 great bustards (Otis tarda) exists in Morocco necessitating the use of non-invasive protocols to study their genetic structure. Here we report a reliable
silica-based method to extract DNA from great bustard faeces. We found that successful extraction and amplification correlated
strongly with faeces freshness and composition. We could not extract amplifiable DNA from 30% of our samples as they were
dry or contained insect material. However 100% of our fresh faecal samples containing no obvious insect material worked, allowing
us to assess the levels of genetic variation among 25 individuals using a 542 bp control region sequence. We were able to
extract DNA from four out of five other avian species, demonstrating that faeces represents a suitable source of DNA for population
genetics studies in a broad range of species.
This revised version was published online in July 2006 with corrections to the Cover Date. 相似文献
9.
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
10.
Bruno D’Alessandro Karina Antúnez Claudia Piccini Pablo Zunino 《World journal of microbiology & biotechnology》2007,23(4):593-597
Summary
Paenibacillus larvae causes American foulbrood (AFB), a severe disease that affects the brood of honey bee Apis mellifera. AFB is worldwide distributed and causes great economic losses to beekeepers, but in many cases early diagnosis could help
in its prevention and control. The aim of the present work was to design a reliable protocol for DNA extraction of P. larvae spores from naturally contaminated honey and adult bees. A novel method that includes a step of spore-decoating followed
by an enzymatic spore disruption and DNA purification was developed. Also a freeze-thaw cycle protocol was tested and the
results were compared. The DNA extracted was used as template for specific bacterial detection by amplification of a 16S rDNA
fragment. Both methods allowed the direct detection by polymerase chain reaction (PCR) of P. larvae spores present in naturally contaminated material. The spore-decoating strategy was the most successful method for DNA extraction
from spores, allowing specific and remarkably sensitive PCR detection of spores in all honey and bees tested samples. On the
other hand freeze-thawing was only effective for detection of spores recovered from bees, and extensive damage to DNA affected
detection by PCR. This work provides new strategies for spore DNA extraction and detection by PCR with high sensitivity, and
brings an alternative tool for P.
larvae detection in natural samples. 相似文献
11.
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
12.
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
13.
Crowley Tamsyn M. Muralitharan Morley S. Stevenson Trevor W. 《Plant Molecular Biology Reporter》2003,21(1):97-97
Genomic DNA was isolated from frozen needles of maturePinus radiata clones using a modified extraction technique incorporating cetyltrimethylammonium bromide (CTAB) for cell lysis. A high sodium
chloride concentration (2 M) was used at 2 stages of the extraction procedure to eradicate polysaccharides, yielding pure
genomic DNA suitable for restriction enzyme digestion and PCR amplification. Extractions were scaled down to suit 1.5-mL Eppendorf
tubes, allowing easier handling and enhanced sterility. 相似文献
14.
Deborah A. Berthold Barbara A. Best Richard Malkin 《Plant Molecular Biology Reporter》1993,11(4):338-344
A method for preparing DNA for PCR has been adapted from the forensic work of Walsh et al. (Biotechniques 10:506–513) for
use withChlamydomonas reinhardtii andArabidopsis thaliana. The method consists of a short incubation of cells or tissue in ethanol, followed by addition of Chelex-100 and heat treatment.
Following centrifugation, the supernatant is added directly to the PCR reaction; forChlamydomonas, amplification product is visible over a range of four orders of magnitude of starting cells. Using this method, DNA suitable
for PCR template can also be obtained fromArabidopsis leaf tissue without grinding, organic extraction or precipitation steps. This method may prove to be useful for other plant
and algal species. 相似文献
15.
A protocol is described for the extraction of geminiviral DNA from bhendi yellow vein mosaic virus-infectedAbelmoschus esculentus (known as bhendi or okra) containing high amounts of mucilage and other phenolic compounds. This method involves extraction
with a buffer containing sodium citrate at pH 6 and PEG precipitation of the virus followed by alkali lysis. The extraction
buffer eliminates the mucilage and other polyphenols, PEG precipitates the viral particles and DNA and the alkali lysis enriches
the replicative forms of the viral DNA. The extracted DNA could be digested with restriction enzymes and cloned without any
interference from chromosomal DNA. The quality of the DNA extracted by this method was compared to three other common plant
DNA extraction protocols and was found superior. This method was used for PCR amplification and cloning of the 2.7 kbp DNA-A
of BYVMV. 相似文献
16.
Use of a 'Miniprep' for rapid extraction of plasmids from vancomycin- and gentamicin-resistant Enterococcus faecium 总被引:1,自引:0,他引:1
Al-Doori Zainab Hill R.L.R. Casewell M.W. 《World journal of microbiology & biotechnology》2001,17(5):517-521
Conventional methods of plasmid extraction are largely unsuited to diagnostic laboratories. The 'Miniprep' is a rapid method that utilises a centrifugable column to separate plasmid DNA from chromosomal DNA. We have modified this technique to extract plasmid DNA from seven strains of vancomycin- and gentamicin-resistant Enterococcus faecium (VGREF): 1% mannitol was added to the growth medium and cell lysis was achieved by incubation in 10 mg of lysozyme/ml in 10 mM Tris, 1 mM EDTA and 25% sucrose at pH 8.0. RNase A was added to plasmid eluate rather than at the lytic step. In comparison to a standard phenol/chloroform method, Miniprep completely eliminated chromosomal interference in gel electrophoresis but otherwise produced identical plasmid profiles. Plasmids obtained from the VGREF ranged from 42 to 1.3 Md. Band densities on a single elution from the Miniprep varied from 8.3 to 106.3 relative units. Double elution increased band densities from the same preparation from 30.4 to 196 relative units; mean percentage increases per track between 7.0 and 34.6%. This method is suitable to achieve plasmid DNA extraction from VGREF within 1 h, making the process more suitable for diagnostic laboratories. 相似文献
17.
Dnyaneshwar Warude Preeti Chavan Kalpana Joshi Bhushan Patwardhan 《Plant Molecular Biology Reporter》2003,21(4):467-467
Current DNA isolation methods are limited in their ability to obtain quality and/or quantity DNA from plants, such asEmblica officinalis, Terminalia belerica, andTerminalia chebula, which have low pH and high amounts of secondary metabolites in tissue extracts. Our modified DNA isolation method yields
good-quality, high-molecular-weight DNA that is free of contaminants and colored pigments and is suitable for PCR amplification.
This method is also useful for isolating DNA from dry powders. 相似文献
18.
Leuko S Goh F Ibáñez-Peral R Burns BP Walter MR Neilan BA 《Extremophiles : life under extreme conditions》2008,12(2):301-308
The extraction of nucleic acids from a given environment marks a crucial and essential starting point in any molecular investigation.
Members of Halococcus spp. are known for their rigid cell walls, and are thus difficult to lyse and could potentially be overlooked in an environment.
Furthermore, the lack of a suitable lysis method hinders subsequent molecular analysis. The effects of six different DNA extraction
methods were tested on Halococcus hamelinensis, Halococcus saccharolyticus and Halobacterium salinarum NRC-1 as well as on an organic rich, highly carbonated sediment from stromatolites spiked with Halococcus hamelinensis. The methods tested were based on physical disruption (boiling and freeze/thawing), chemical lysis (Triton X-100, potassium
ethyl xanthogenate (XS) buffer and CTAB) and on enzymatic lysis (lysozyme). Results showed that boiling and freeze/thawing
had little effect on the lysis of both Halococcus strains. Methods based on chemical lysis (Triton X-100, XS-buffer, and CTAB) showed the best results, however, Triton X-100
treatment failed to produce visible DNA fragments. Using a combination of bead beating, chemical lysis with lysozyme, and
thermal shock, lysis of cells was achieved however DNA was badly sheared. Lysis of cells and DNA extraction of samples from
spiked sediment proved to be difficult, with the XS-buffer method indicating the best results. This study provides an evaluation
of six commonly used methods of cell lysis and DNA extraction of Halococcus spp., and the suitability of the resulting DNA for molecular analysis. 相似文献
19.
C. El Adlouni M. J. Mukhopadhyay P. Walsh G. G. Poirier D. Nadeau 《Molecular and cellular biochemistry》1995,142(1):19-23
Our interest in detecting genotoxic exposure in earthworms led us to isolate high quality DNA from theEisenia fetida species. For that, we compared a modification of the conventional phenol-chloroform extraction procedure, usually refered to as the Maniatis procedure, to two commercially available kits reportedly eliminating multiple partitions in phenol and chloroform, namely the Qiagen and Nucleon protocols. From the 260 nm optical density values, the commercial kits extracts hinted toward higher DNA recovery with those procedures. However, the 260/280 nm ratios indicated that the quality of the DNA isolated with the modified Maniatis procedure was purer than that isolated with the commercial kits, the latter being most probably contaminated by proteins and/or RNA. The Maniatis procedure was slightly modified by the introduction of a potassium acetate step for protein precipitation and by shortening the proteinase K treatment from 12–18 h to only 2 h. The higher quality of the DNA isolated by phenol-chloroform extraction was confirmed by quantification with the fluorescent 3,5-diaminobenzoic acid assay. Preliminary results suggest that the modified Maniatis procedure herein described is not only applicable for DNA adducts studies using32P-postlabelling techniques but is also suitable for DNA extraction from other earthworm species such asLumbricus terrestris. 相似文献
20.
A rapid, inexpensive and reliable method for total RNA extraction from fruiting bodies of Lentinula edodes containing large quantities of polysaccharides and secondary metabolites is described. An initial extraction step using saturated NaCl solution facilitates the separation of nucleic acids from contaminants and, after further extraction with organic solvents and precipitation with 2-propanol, total RNA of high purity and suitable for applications such as cDNA synthesis, RT-PCR and Northern blot hybridization was obtained. The procedure may also have wider applicability for total RNA extraction from the tissues of other mushrooms. 相似文献