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1.
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
2.
Many plant RNA isolation techniques aim to prevent contamination by means of secondary phenolics, carbohydrates, RNase, and
other chemicals. However, when applied in our laboratory to the isolation of RNA fromRumex obtusifolius, these protocols failed to produce good quality RNA. A major problem was contamination of the RNA samples with the secondary
metabolite oxalate. The relative quantities of guanidine isothiocyanate extraction buffer to plant tissue used in the protocol
had significant effects on oxalate contamination. An increase in extraction buffer, from 1.5 mL in the original method to
15 mL per 200–300 mg of tissue in our protocol, removed the oxalate from the RNA. This RNA was of a good quality and was suitable
for molecular biology applications. 相似文献
3.
Chris S. Jones Pietro P. M. Iannetta Mary Woodhead Howard V. Davies Ronnie J. McNicol Mark A. Taylor 《Molecular biotechnology》1997,8(3):219-221
Previous attempts to extract high-quality, total RNA from raspberry (Rubus idaeus) fruits using published protocols have proven to be unsuccessful. Even the use of protocols developed for the extraction
of RNA from other fruit tissue has resulted in low yields (1) or the isolation of degraded RNA (2). Here, we report on the development of a quick and simple method of extracting total RNA from raspberry fruit. Using this
method, high yields of good quality, undegraded RNA were obtained from fruit at all stages of ripening. The RNA is of sufficient
quality for northern analysis and cDNA library construction. 相似文献
4.
Detection and quantification of Entomophaga maimaiga resting spores in forest soil using real-time PCR 总被引:2,自引:0,他引:2
Louela A. Castrillo Lene Thomsen Punita Juneja Ann E. Hajek 《Mycological Research》2007,111(3):324-331
Environmental sampling to monitor entomopathogen titre in forest soil, a known reservoir of insect pathogens such as fungi and viruses, is important in the evaluation of conditions that could trigger epizootics and in the development of strategies for insect pest management. Molecular or PCR-based analysis of environmental samples provides a sensitive method for strain- or species-based detection, and real-time PCR, in particular, allows quantification of the organism of interest. In this study we developed a DNA extraction method and a real-time PCR assay for detection and quantification of Entomophaga maimaiga (Zygomycetes: Entomophthorales), a fungal pathogen of the gypsy moth, in the organic layer of forest soil. DNA from fungal resting spores (azygospores) in soil was extracted using a detergent and bead mill homogenization treatment followed by purification of the crude DNA extract using Sephadex–polyvinylpolypyrrolidone microcolumns. The purification step eliminated most of the environmental contaminants commonly co-extracted with genomic DNA from soil samples but detection assays still required the addition of bovine serum albumin to relieve PCR inhibition. The real-time PCR assay used primers and probe based on sequence analysis of the nuclear ribosomal ITS region of several E. maimaiga and two E. aulicae strains. Comparison of threshold cycle values from different soil samples spiked with E. maimaiga DNA showed that soil background DNA and remaining co-extracted contaminants are critical factors determining detection sensitivity. Based on our results from comparisons of resting spore titres among different forest soils, estimates were best for organic soils with comparatively high densities of resting spores. 相似文献
5.
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
6.
高通量测序分析DNA提取引起的对虾肠道菌群结构偏差 总被引:2,自引:0,他引:2
【目的】通过高通量测序技术,评价不同DNA试剂盒提取引起的对虾肠道菌群结构偏差,了解健康凡纳滨对虾肠道菌群结构特征。【方法】分别以细菌、粪便和组织DNA试剂盒3次重复提取凡纳滨对虾肠道总DNA(分别编号为SIB,SIS和SIT),检测DNA含量、纯度及其16S r DNA V4区可扩增性,进一步采用Illumina Mi Seq高通量测序比较SIB和SIS样品菌群组成和多样性。【结果】细菌试剂盒提取的虾肠总DNA效果最好,粪便试剂盒次之,而组织试剂盒所提DNA含量低且难以被扩增。从SIB和SIS样品分别获得52151±5085和55296±5147条有效序列,同一(46800条)测序深度下,SIS样品OTU(operational taxonomic unit)数量和Shannon多样性指数均显著高于SIB的,而SIB样品间OTU重复性则优于SIS样品间的。从SIB和SIS样品鉴定的优势门一致,均包括变形菌门(Proteobacteria)、厚壁菌门(Firmicutes)、拟杆菌门(Bacteroidetes)、浮霉菌门(Planctomycetes)、放线菌门(Actinobacteria)和蓝细菌门(Cyanobacteria),但不同分类水平上绝大多数优势菌群丰度在两种样品间差异明显。【结论】高通量测序分析表明对虾肠道菌群结构因DNA提取方法不同而呈现显著偏差;本研究健康凡纳滨对虾肠道核心菌群主要由发光杆菌属(Photobacterium),乳球菌属(Lactococcus),弧菌属(Vibrio),Aliivibrio和3个分类未定属构成。 相似文献
7.
We applied human forensic techniques to the extraction of whole genomic DNA from processed wood samples to explore the possibility
of identifying an endangered tropical timber species by using DNA sequencing technology. High-yield and high-quality DNA samples
were obtained from 2 commercial wood and 3 herbarium samples. Large PCR fragments ranging from 500–800 bp were successfully
amplified from 2 chloroplast and 1 mitochondrial regions in all 5 samples, indicating limited degradation of the cytoplasmic
genomes. DNA extraction from stem wood taken from herbarium specimens appeared superior to that from stem wood with bark intact
or from leaf samples. DNA sequences from thetrn regions allowed for easy identification of the focal species based on GenBank Blast search. Little sequence variation was
observed in the 3 regions, with the mitochondrialcox3 region completely conserved. Extraction of high-quality and large intact DNA fragments makes dry wood materials amenable
to various DNA marker-based applications, including fingerprinting and historical approaches. By sampling stemwood, the wealth
of historical information housed in international herbaria can be explored with minimal damage to taxonomically important
features. 相似文献
8.
Restriction fragment length polymorphism (RFLP) analysis for DNA products amplified by the polymerase chain reaction (PCR)
was used for the direct detection ofRhizoctonia solani AG 1 IA and AG 2-2 IIIB,R. oryzae, R. oryzae-sativae andR. fumigata from the diseased rice sheaths. A rapid DNA extraction method with a solution of sodium hydroxide was conducted to extract
parasite DNA from diseased rice sheaths. 28S ribosomal DNA (rDNA) derived from fungal genomic DNA extracted by the alkaline
method was specifically PCR-amplified. The results of PCR-RFLP analysis for DNA samples from artificially inoculated rice
sheath tissues with eachRhizoctonia spp. and the corresponding culture on the medium using two restriction enzymes.HhaI andMspI, showed identical polymorphisms. PCR-RFLP analysis using DNA samples from naturally infected rice sheath tissues also revealed
the possibility of direct diagnosis ofR. solani AG 1 IA,R. oryzae andR. oryzae-sativae. 相似文献
9.
Heiko U. Kfferlein Jürgen Angerer 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》1999,734(2):2629
Two human urinary metabolites of the industrial solvent N,N-dimethylformamide (DMF), N-hydroxymethyl-N-methylformamide (HMMF) and N-acetyl-S-(N-methylcarbamoyl)cysteine (AMCC), were assayed using a new analytical method (gas chromatography and thermionic sensitive detection). Clean-up of urine samples includes a liquid–liquid extraction step followed by a solid-phase extraction step to separate HMMF and AMCC from other urine components. During clean-up, AMCC is converted into ethyl-N-methylcarbamate (EMC), and during gas chromatography, HMMF is degraded in the injector to N-methylformamide (NMF). All the validation data necessary for a quantitative procedure are given. The method was applied to urine samples from workers exposed to DMF and from the general population. The results were confirmed by mass spectrometric determination. For this purpose a further liquid–liquid extraction step was introduced in the clean-up procedure. Background levels of AMCC in the general population were identified. 相似文献
10.
Filippo Geuna Chiara Maitti Simona Digiuni Riccardo Banfi 《Plant Molecular Biology Reporter》2004,22(1):87-87
Isolating nucleic acids from sources rich in contaminants is particularly cumbersome when treating a large number of samples.
Several protocols have been published that address the problem of nucleic acid extraction and purification, but few address
sample number. We describe a method for extracting DNA from recalcitrant tree species by using a commercial grinding apparatus.
This alleviates the hard work of sample preparation prior to lysis and purification. Our method has been tested extensively
on different fruit tree species and in projects that require the simultaneous processing of hundreds of samples. Moreover,
it does not require the availability of robotic workstations. 相似文献
11.
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
12.
Successful amplification of rice chloroplast microsatellites from century-old grass samples from the park grass experiment 总被引:3,自引:0,他引:3
We report the successful amplification of microsatellite markers for the chloroplast genome from century-old samples of 2
grasses growing in the Park Grass Experiment (PGE):Anthoxanthum ordoratum andFestuca rubra. This opens the possibility of establishing a long-term genetic time series for the PGE, which began in 1856 and is believed
to be the oldest ecological experiment in existence. Although the plant samples used were not originally prepared or stored
with molecular analysis in mind, the hexadecyltrimethylammonium bromide (CTAB) method of DNA extraction was successfully used.
Obtained DNA was degraded but could be amplified by means of PCR. It produced bands around the expected size for chloroplast
microsatellite primers derived from rice. When sequenced, bands showed good homology with sequences from rice chloroplast
genomes listed in GenBank (accession No X15901). 相似文献
13.
A rapid, simple and efficient protocol is given for the extraction of restrictable total DNA from plants of the genusAbelmoschus, for which the main obstacle is the stickiness of the solution, after grinding of green leaves. This problem is resolved
using cotyledons of dark-grown seedlings. 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.
Genomic DNA was extracted from 13 samples of Sargassum polycystum and S. siliquosum collected from various localities around Peninsular Malaysia and Singapore by using four different extraction methods. The
yields and the suitability of the DNA to be used as template for the polymerase chain reaction (PCR) was compared. DNA samples
were subjected to PCR analysis by using random primers. Only DNA samples that were extracted using the CTAB method were successfully
amplified by random amplified polymorphic DNA (RAPD)-PCR. Five of 31 random primers (OPA02, OPA03, OPA04, OPA13 and OPM10)
tested amplified sequences of DNA from the DNA samples. Reproducible, amplified products were obtained using these primers
and showed some potential to be useful in discriminating individual samples within the genus, in determining relationships
between species within a genus and in developing individual fingerprints for individual samples. 相似文献
16.
Lara-Reyna J. Olalde-Portugal V. Olmedo-Alvarez G. 《World journal of microbiology & biotechnology》2000,16(4):345-351
DNA extraction techniques for endospore-forming bacteria in soil are often labour-intensive and unreliable. Our objective in this work was to investigate whether good quality DNA could be obtained from spores germinated in soil. To this end, endospores from Bacillus subtilis, B. megaterium and B. thuringiensis were inoculated into soil microcosms and germination was induced by addition of LB medium supplemented with l-alanine, glucose, fructose and KCl. Heat resistance count was reduced to 80% for B. subtilis and more than 90% for B. thuringiensis and B. megaterium after a few minutes. Isolation of DNA from soil with a procedure which did not work on spores was shown to be as efficient for in situ-germinated spores as for inoculated vegetative cells. Furthermore, we developed a simple procedure that allowed us to use the recovered DNA in PCR amplifications. The present methodology is simple and efficient; it avoids the use of special equipment and harsh spore rupturing methods and can be carried out with multiple samples. 相似文献
17.
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
18.
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
19.
Mtambo J Van Bortel W Madder M Roelants P Backeljau T 《Experimental & applied acarology》2006,38(2-3):189-199
Five differently preserved groups of adult Rhipicephalus appendiculatus specimens were compared for quality of DNA extracted. Three methods were used to extract DNA from specimens i.e. two simple
mosquito validated DNA extraction methods and a tick validated method. Extraction of DNA from tick legs was attempted. The
quality of DNA extracted was evaluated by the success of PCR amplification of the ITS2 gene and the mitochondrial COI gene
fragment. Fresh specimens (i.e. killed just before extraction) had the highest success of DNA amplification followed by specimens
killed in ethanol and subsequently stored in the refrigerator (4 °C). There was no significant difference in amplification
success between cryopreserved and 70% ethanol preserved specimens. It was possible to amplify DNA from legs of ticks. Sequenced
ITS2 amplicon of template obtained from legs of ticks was as legible as those from whole tick extract. The two mosquito validated
DNA extraction methods showed a significantly lower amplification success than the tick validated protocol. 相似文献
20.
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