共查询到20条相似文献,搜索用时 31 毫秒
1.
Cardamom is an important spice, condiment and medicine, and international commodity. DNA-based molecular profiling will be
aid in protecting the intellectual property rights of those who trade cardamom on the world market. Commercial cardamom has
so far proven recalcitrant to traditional DNA extraction methods. In this paper we report a protocol for the isolation of
amplifiable genomic DNA from traded cardamom. The method involves a modified CTAB (hexadecyltrimethylammonium bromide) extraction
step, followed by a purification step to remove polysaccharides, proteins, and polyphenols, which are abundant in storage
tissue such as cardamom capsules. The yield of DNA was 6–7 μg g−1 tissue. Spectrophotometric and electrophoretic analysis indicated that the isolated DNA was highly pure and of high molecular
weight. The isolated DNA could be amplified using different random decamer primers. The protocol has trade implications as
it will help in the PCR-based characterisation of traded cardamom. This protocol can be further extended to develop Sequence
Characterised Amplified Regions (SCAR) markers for profiling cardamoms. 相似文献
2.
High-quality plant DNA extraction for PCR: an easy approach 总被引:1,自引:0,他引:1
I. Ahmed M. Islam W. Arshad A. Mannan W. Ahmad B. Mirza 《Journal of applied genetics》2009,50(2):105-107
Polymerase chain reaction has found wide applications in modern research involving transformations and other genomic studies.
For reproducible PCR results, however, the quantity and quality of template DNA is of considerable importance. A simple and
efficient plant DNA extraction procedure for isolation of high-quality DNA from plant tissues is presented here. It requires
maceration of plant tissue of about 1.0 cm2 (e.g. of a leaf blade) in DNA extraction buffer (100 mM Tris-HCl, 100 mM EDTA, 250 mM NaCl) using 1.5-mL microfuge tubes,
followed by cell lysis with 20% SDS, and DNA extraction with phenol: chloroform: iso-amyl alcohol (25:24:1). Hydrated ether
is then used to remove polysaccharides and other contaminants from the DNA preparation. Average DNA yield is 20–30 μg cm−2 for fresh tissues, and ratio of absorbance at 260 nm to absorbance at 280 nm is 1.5–1.8. The DNA is quite suitable for PCR
using microsatellites, RAPD and specific markers for recombinant selection. Amplifications have been obtained for these markers
by using template DNA extracted from fresh as well as frozen leaf tissues of various plants, including barley, oat, potato
and tomato. DNA stored for more than 2 years has been successfully amplified with microsatellite markers, which shows suitability
of this method after long-term storage of DNA. Besides, the ease of use and cost-effectiveness make the procedure attractive. 相似文献
3.
Ranjana Bhattacharjee Maria Kolesnikova-Allen Peter Aikpokpodion Sunday Taiwo Ivan Ingelbrecht 《Plant Molecular Biology Reporter》2004,22(4):435-436
DNA extraction is a time-consuming and expensive component of molecular marker analysis, constituting about 30–60% of the
total time required for sample processing. Furthermore, the procedure for extracting high-quality DNA from tree species such
as cocoa differs from extraction protocols suitable for other crop plants. This is accompanied by problems in collecting leaf
tissues from field-grown cocoa trees, where storage facilities are not available and where transporting samples to laboratory
for immediate refrigeration is usually impossible. We preserved cocoa leaf tissues in the field in an NaCl-CTAB-azide solution
(as described in Rogstad, 1992), which did not require immediate refrigeration. This method also allowed preservation of leaf
tissues for a few days during transportation and protected leaf tissues from bacterial and fungal attacks. Once transported
to the laboratory, the samples were stored at 4°C for almost 1 y. To isolate good-quality DNA from stored leaf tissues, a
rapid semiautomated and relatively high-throughput protocol was established. The procedure followed a modified CTAB/β-mercaptoethanol
method of DNA extraction in a 96-well plate, and an automated system (i.e., GenoGrinder 2000) was used to grind the leaf tissues.
The quality of DNA was not affected by long storage, and the quantity obtained per sample was adequate for about 1000 PCR
reactions. Thus, this method allowed isolation of about 200 samples per day at a cost of $0.60 per sample and is a relatively
high-throughput, low-cost extraction compared with conventional methods that use manual grinding and/or expensive kits. 相似文献
4.
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
5.
A Modified Protocol for RNA Extraction from Different Peach Tissues Suitable for Gene Isolation and Real-Time PCR Analysis 总被引:1,自引:0,他引:1
RNA extraction is the first step in the study of gene isolation and expression. However, it is difficult to extract high quantity
and quality RNA from tissues containing large quantities of polysaccharides and polyphenols. Peach (Prunus persica), in addition to containing high levels of polysaccharides and polyphenols, is a challenging starting material for RNA isolation
using a single method because of different amounts of those substances in diverse tissues. Based on three reported methods,
we developed a modified RNA isolation protocol to solve this problem, leading to high quality and quantity of total RNA from
peach mesocarp tissues of fruits which were sampled from all developmental stages and different storage periods, as well as
from other tissues including flowers, leaves, stems, and roots. With our modified method, 28–650 μg of total RNA was routinely
obtained from per gram of fresh material, gave at least a 1.16-fold improvement by compared with those isolated by other seven
methods. The RNA extracts were successfully used in downstream applications such as RT-PCR, RACE, and real-time PCR. 相似文献
6.
We describe a simple and efficient method for genomic DNA extraction from woody fruit crops containing high polysaccharide
levels. This method involves a modified CTAB or SDS procedure employing a purification step to remove polysaccharides by using
water-saturated ether and 1.25 M NaCl. Precipitation with an equal volume of isopropanol caused a DNA pellet to form. After
being washed with 70% ethyl alcohol, the pellet easily dissolved in TE buffer. Using this method, DNA was extracted from samples
of more than 1000Citrus spp., including young leaves, old leaves, frosted old leaves, withered old leaves, and callus lines. The average yield of
DNA ranged from 50–500 μg/g of sample. DNA was suitable for PCR and RFLP analyses and long-term storage. Recently, the procedure
was used to isolate DNA from withered old leaves of more than 20 tropical and subtropical fruit crops. 相似文献
7.
A qualitative procedure of purified DNA/RNA co-extraction from complex organic matter, used as biofilter support for removing
volatile organic compounds, was set up and applied to detect xylene monooxygenase gene expression by RT-PCR. A DNA/RNA extraction
protocol based on a combination of sample lyophilization pre-treatment and CTAB––phenol/chloroform extraction procedure was
optimized for the recovery of purified nucleic acids [100–500 ng DNA (10 kb) and 0.5–2 μg of rRNA 16S from 100 mg matrix].
PCR and RT-PCR protocols were established to detect xylene monooxygenase gene expression starting from differentially induced
organic matrices obtained by biofiltration technology. This work allowed the microbial degradation activities in heterogeneous
organic solid media to be studied and suggests a rapid method to follow specific biological activities during solid and/or
semisolid organic substrates biotransformation. 相似文献
8.
Yanti Rachmayanti Ludger Leinemann Oliver Gailing Reiner Finkeldey 《Plant Molecular Biology Reporter》2006,24(1):45-55
A successful DNA extraction from wood yielding appropriate DNA quality for PCR amplification allows molecular genetic investigations
of wood tissue. Genotypes, the origin of sampled material, and species can be identified based on an investigation of wood
if suitable information on genetic variation patterns within and among species is available. Potential applications are in
forensics and in the control of the timber and wood trade. We extracted DNA from wood of Dipterocarpaceae, a family that dominates
rainforests and comprises many important timber species in Southeast Asia. Several different DNA isolation techniques were
compared and optimized for wood samples from natural populations and from wood processing enterprises. The quality of the
DNA was tested by spectrophotometry, PCR amplification, and PCR inhibitor tests. An average DNA yield of 2.2 μg was obtained
per 50–100 mg of dried wood sample. Chloroplast DNA (cpDNA) regions of different length were amenable to PCR amplification
from the extracted DNA. Modification of DNA isolation techniques by the addition of polyvinylpyrrolidone (PVP) addition up
to 3.1% into lysis buffer reduced PCR inhibition effectively. In order to evaluate the extraction method, we analyzed leaves
and wood from the same tree by PCR amplification, genotyping and sequencing of chloroplast microsatellites. 相似文献
9.
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
10.
Alasaad S Soglia D Maione S Sartore S Soriguer RC Pérez JM Rasero R Rossi L 《Experimental & applied acarology》2009,47(2):173-178
The aim of the present study was to assess whether individual Sarcoptes mites collected from frozen skin (‘postponed isolation’ method) are suitable sources of PCR-quality genomic DNA, and to test
the effectiveness of this method in comparison with the ‘direct isolation’ method, often used through force of habit. Hundreds
of single Sarcoptes scabiei samples, resulting from direct (live) or postponed (post-frozen) isolation, were tested using a ~450 bp product (ITS-2) and
multi-locus 10× genotyping with microsatellite markers. No statistical difference in yield of soluble DNA was found between
the two isolation methods. Nevertheless, 19% of the reactions were classified as failed preparations in the direct isolation
method, whereas the rate of unsuccessful reactions was 34% in the postponed isolation method. Consequently, post-frozen isolation
is suitable and recommendable for Sarcoptes mite gDNA preparation, particularly when performing a balancing act among safety, practicability and profitability. These
results have implications for mite collection for DNA extraction, and hence the needed wider leap of Sarcoptes into the genetic era. 相似文献
11.
Abolghasem Abbasi Kejani Sayed Ali Hosseini Tafreshi Sayed Mojtaba Khayyam Nekouei Mohammad Reza Mofid 《Molecular biology reports》2010,37(2):797-800
Improved and efficient methods were developed for isolating high quality DNA and RNA from different sources of Iranian Yew
(Taxus baccata L.). The methods were based on CTAB extraction buffer added with high levels of polyvinylpyrrolidone (PVP) and β-mercaptoethanol to properly remove polysaccharides and prevent oxidation of phenolics. The pellets obtained by ethanol precipitation
were washed only with Chloroform: isoamyl alcohol (24:1). So, we could successfully eliminate the dangerous phenol/chloroform
extraction steps from the isolation procedure. Both spectrophotometric (A260/A280 and A260/A230 ratios) and agarose electrophoresis analysis of isolated nucleic acids (DNA and RNA) indicated good results. DNA with the
average yield of 100–300 μg/g leaf and stem tissue and total RNA with an average yield of 20–30 μg/g cell culture and 80–100 μg/g
leaf and stem tissue of Iranian yew could be obtained. Successful amplification of pam and pds by PCR and RT-PCR, showed the integrity of isolated DNA and RNA, respectively. 相似文献
12.
For several years Physcomitrella patens Hedw. was the only land plant allowing targeted gene knockout via homologous recombination, which provides an efficient and
elegant tool for the analysis of gene functions. The moss Ceratodon purpureus Hedw. was recently shown to have similar capabilities. However, extraction of high quality total DNA from both moss species
— necessary for unambiguous proof of successful gene targeting events — is still a severe problem. Here, we report an improved
DNA isolation protocol for moss filaments, which is fast, reliable, cheap, quick, and easy. It results in satisfying yields
of DNA suitable for PCR and Southern blotting. The modified extraction procedure was additionally tested successfully for
the alga Mougeotia scalaris Hass. as well as the higher plant Arabidopsis thaliana L.
Published in Russian in Fiziologiya Rastenii, 2007, Vol. 54, No. 4, pp. 634–638.
The text was submitted by the authors in English. 相似文献
13.
Zinger L Gury J Giraud F Krivobok S Gielly L Taberlet P Geremia RA 《Microbial ecology》2007,54(2):203-216
The molecular signature of bacteria from soil ecosystems is an important tool for studying microbial ecology and biogeography.
However, a high-throughput technology is needed for such studies. In this article, we tested the suitability of available
methods ranging from soil DNA extraction to capillary electrophoresis single-strand conformation polymorphism (CE–SSCP) for
high-throughput studies. Our results showed that the extraction method does not dramatically influence CE–SSCP profiles, and
that DNA extraction of a 0.25 g soil sample is sufficient to observe overall bacterial diversity in soil matrices. The V3
region of the 16S rRNA gene was amplified by PCR, and the extension time was found to be critical. We have also found that
proofreading DNA polymerases generate a better signal in CE–SSCP profiles. Experiments performed with different soil matrices
revealed the repeatability, efficiency, and consistency of CE–SSCP. Studies on PCR and CE–SSCP using single-species genomic
DNA as a matrix showed that several ribotypes may migrate at the same position, and also that single species can produce double
peaks. Thus, the extrapolation between number of peaks and number of species remains difficult. Additionally, peak detection
is limited by the analysis software. We conclude that the presented method, including CE–SSCP and the analyzing step, is a
simple and effective technique to obtain the molecular signature of a given soil sample. 相似文献
14.
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
15.
A rapid and efficient DNA minipreparation suitable for screening transgenic plants 总被引:10,自引:0,他引:10
Rong-Cheng Lin Zai-Song Ding Liang-Bi Li Ting-Yun Kuang 《Plant Molecular Biology Reporter》2001,19(4):379-379
We present a modified method for DNA minipreparation suitable for large-scale screening of transgenic plants. The method is
rapid and efficient—one person can prepare DNA from approximately 50 samples per day. The average yield was about 40 μg DNA
per 100 mg of fresh tissue, and the A260/A280 was 1.89–2.03. The total DNA extracted by this method could be used for PCR, restriction enzyme digestion, and Southern blotting. 相似文献
16.
17.
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
18.
DNA is one of the most basic and essential genetic materials in the field of molecular biology.To date,isolation of sufficient and good-quality DNA is still a challenge for many plant species,though various DNA extraction methods have been published.In the present paper,a recycling DNA extraction method was proposed.The key step of this method was that a single plant tissue sample was 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.This recycling step was integrated into the conventional CTAB DNA extraction method to establish a recycling CTAB method.This modified CTAB method was tested in eight plant species,wheat,sorghum,barley,corn,rice,Brachypodium distachyon,Miscanthus sinensis and tung tree.The results showed that high-yield and good-quality DNA samples could be obtained by using this new method in all the eight plant species.The DNA samples were good templates for PCR amplification of both ISSR and SSR markers.The recycling method can be used in multiple plant species and can be integrated with multiple conventional DNA isolation methods,and thus is an effective and universal DNA isolation method. 相似文献
19.
Optimization and scale-up of a new photobleaching agar extraction process from Gracilaria lemaneiformis 总被引:1,自引:0,他引:1
Haiyan Li Jianyu Huang Yanjuan Xin Biaoming Zhang Yan Jin Wei Zhang 《Journal of applied phycology》2009,21(2):247-254
An eco-friendly photobleaching extraction process for agar extraction from the red alga Gracilaria lemaneiformis was developed for the benefit of workers’ health and environmental safety. Here we report the optimization of key process
parameters (alkali modification concentration, photobleaching duration, algal length and screen filter opening size) in order
to scale up this new technique. The optimal conditions were found to be modification by 3–5% NaOH, photobleaching for 5 h,
using algal fragments 2 –4 cm in length, and a filter screen with a 6 μm opening. A 20-L agar extraction reactor was thus
constructed, and the scale-up of the agar extraction process was tested in six batch experiments. The resulting agar quality
was similar to that of the laboratory-scale extraction. In addition, batch-to-batch reproducibility was excellent. The results
demonstrate the excellent scale-up ability and potential application of this new photobleaching agar extraction process on
a commercial scale. The agar yield and gel strength for 5% NaOH modified agar were 26.8% and 1,897 g cm−2, while those for 3% NaOH modified agar were 28.2% and 1,287 g cm−2, respectively. It is clear that the agar yield and quality can be manipulated via alkali modification in this new eco-friendly
extraction to meet market demands. 相似文献