An efficient and good DNA extraction protocol should be simple, affordable and yield enough DNA with high quality. Rice(Oryza sativa L.) DNA extraction methods often use seedlings or leaves rather than the grains and ...An efficient and good DNA extraction protocol should be simple, affordable and yield enough DNA with high quality. Rice(Oryza sativa L.) DNA extraction methods often use seedlings or leaves rather than the grains and tend to be time-consuming, involve multiple steps, and use hazardous chemicals and expensive enzymes. Rice grains offer several benefits over seedlings and leaves as a source of DNA for genetic analysis. However, these benefits are underutilized because the bulk of a rice grain is made up of starch. It is particularly important, but difficult to get rid of the starch while extracting DNA from rice grains. This co-precipitated polysaccharide is a known inhibitor of DNA polymerase activity in polymerase chain reaction(PCR). We describe here a very simple and highly affordable Chelex~?-100 based DNA extraction method from rice grains. It does not require any hazardous chemicals or enzymes. This method reproducibly extracts DNA with good purity indices(A_(260)/A_(230) and A_(260)/A_(280) values), but requires only a few steps.展开更多
Melia dubia Cav. of family Meliaceae is a fast growing, high value tree species native to India. Isolating DNA from matured dried leaves of M. dubia was difficult due to accumulation of secondary metabolites, majorly ...Melia dubia Cav. of family Meliaceae is a fast growing, high value tree species native to India. Isolating DNA from matured dried leaves of M. dubia was difficult due to accumulation of secondary metabolites, majorly polyphenolics, which resulted in dark brown to black colour of the pellet. In this study, a modified STE-(Sucrose, Tris-HCl and Ethylene Diamine Tetra Acetic Acid) CTAB (hexadecyltrimethylammonium bromide) method was standardized for removal of polyphenolics. The protocol developed yielded 200 - 1000 ng/μl of quality DNA without any impurities as evident by A260/280 ratio ranging from 1.75 - 2.0. It was also suitable for extracting quality DNA from other members of Meliaceae like Azadirachta indica and Melia azedarach. In downstream applications, the extracted DNA was used for PCR amplification by using ISSR and SSR markers. ISSR PCR conditions were optimized in a reaction volume of 25 μl, consisting of 30 ng of template DNA, 1.5 mM MgCl<sub>2</sub>, 200 μM of each of dNTPs and 2 U of Taq polymerase. The best amplification was observed and the same was applicable for SSR markers.展开更多
Background:As an important player during food digestion,gut microbiota has attracted much attention in diet adaptation studies in birds.Microbiota extracted from feces has been widely used as a proxy for gut microbiot...Background:As an important player during food digestion,gut microbiota has attracted much attention in diet adaptation studies in birds.Microbiota extracted from feces has been widely used as a proxy for gut microbiota.Although several methods have been developed for microbial DNA extraction,their performances in the bird feces have not been systematacially evaluated yet.Methods:In this study,we applied three DNA extraction methods(Qiagen,MoBio and Bead)to extract DNA from feces of three avian dietary guilds(granivore,omnivore and carnivore),sequenced V4 region of 16S rRNA gene for each extract and evaluated the performances of DNA yield,DNA integrity,microbial composition,cell lysis capacity and alpha diversity for the three methods on each dietary guild.Results:Bead method was the best on the performance of both DNA yield and DNA integrity regardless of dietary guild.In granivore,microbial relative abundance at both species and phylum levels,alpha diversity and cell lysis capacity were comparable among all methods.In omnivore,Qiagen had the best performance on alpha diversity,fol-lowed by Bead and MoBio.There were small variations on microbial relative abundance at both species and phylum levels among different extraction methods.MoBio exhibited the best performance on cell lysis capacity.In carnivore,considerable variations were found on microbial relative abundance at both species and phylum levels.Qiagen had the best performance on alpha diversity,followed by MoBio and Bead.MoBio had the highest cell lysis capacity.Conclusions:DNA yield and integrity have no obvious impact on microbial composition,alpha diversity or cell lysis capacity.The microbiota results(e.g.,microbial composition,cell lysis capacity,alpha diversity)obtained from differ-ent methods are comparable in granivorous avian species but not in omnivorous or carnivorous birds.Either method could be used in granivore microbiota studies.For omnivores and carnivores,we recommend Qiagen method when the research purpose is microbial diversity and MoBio when gram-positive bacteria is the research target.展开更多
Environmental DNA(eDNA)integrated with metabarcoding is a promising and powerful tool for species composition and biodiversity assessment in aquatic ecosystems and is increasingly applied to evaluate fish diversity.To...Environmental DNA(eDNA)integrated with metabarcoding is a promising and powerful tool for species composition and biodiversity assessment in aquatic ecosystems and is increasingly applied to evaluate fish diversity.To date,however,no standardized eDNA-based protocol has been established to monitor fish diversity.In this study,we investigated and compared two filtration methods and three DNA extraction methods using three filtration water volumes to determine a suitable approach for eDNA-based fish diversity monitoring in the Pearl River Estuary(PRE),a highly anthropogenically disturbed estuarine ecosystem.Compared to filtration-based precipitation,direct filtration was a more suitable method for eDNA metabarcoding in the PRE.The combined use of DNeasy Blood and Tissue Kit(BT)and traditional phenol/chloroform(PC)extraction produced higher DNA yields,amplicon sequence variants(ASVs),and Shannon diversity indices,and generated more homogeneous and consistent community composition among replicates.Compared to the other combined protocols,the PC and BT methods obtained better species detection,higher fish diversity,and greater consistency for the filtration water volumes of 1000 and 2000 mL,respectively.All eDNA metabarcoding protocols were more sensitive than bottom trawling in the PRE fish surveys and combining two techniques yielded greater taxonomic diversity.Furthermore,combining traditional methods with eDNA analysis enhanced accuracy.These results indicate that methodological decisions related to eDNA metabarcoding should be made with caution for fish community monitoring in estuarine ecosystems.展开更多
Myxosporidia constitute a major group of fish parasites which have a significant negative impact on wild and cultured fish. The used of DNA in Myxosporidia studies has progressed rapidly over the last twenty years, es...Myxosporidia constitute a major group of fish parasites which have a significant negative impact on wild and cultured fish. The used of DNA in Myxosporidia studies has progressed rapidly over the last twenty years, especially in their identification and characterization as well as determination of species diversity and investigation of their evolutionary relationships. Extraction and isolation of pure and high quality DNA are essential for any molecular study, but constitute a challenge for many laboratories especially in low and middle income countries. Myxosporidia plasmodia filled with mature myxospores were isolated from different tissues of <span style="white-space:nowrap;"><i></span>Labeo batesii<span style="white-space:nowrap;"></i></span> Boulenger, 1911. DNA from myxosporidia myxospores were extracted using a Livak optimized DNAs extraction protocol. Four particular phases of the original protocol were optimized. Yield and absorbance ratios of extracted DNA were determined using spectrophotometer. DNA samples were used as template for the amplification of the 18S rDNA region and amplicons resolved on 1.5% agarose gel for determination of fragment sizes and purity evaluation. The concentration of extracted DNA from all Myxosporidia species ranged from 4.6 to 26 ng/μl with purity indices ranging from 1.88 to 2.12. We successfully amplified the 1050 bp DNA fragment as targeted. The intensity, thickness and clarity of the bands were evidences of non-degradation of DNA. The optimized Livak protocol is simple, low-cost and manageable. Regarding the quantity, purity and quality of extracted DNA, the optimized Livak protocol is highly recommended for Myxosporidia studies.展开更多
[Objectives]This study was conducted for the purpose of widely using molecular biology techniques in the detection of seafood species.[Methods]In this study,such four kinds of seafood as fish,shrimps,crabs and shellfi...[Objectives]This study was conducted for the purpose of widely using molecular biology techniques in the detection of seafood species.[Methods]In this study,such four kinds of seafood as fish,shrimps,crabs and shellfish were used as samples to compare three DNA extraction methods,including Tiangen marine animal tissue genome extraction kit method(DP324)(a),SN/T 3589.6-2013/8.3(b)method and modified SN/T 3730.8-2013/8.1(c)method.The purity and concentrations of DNA and the amplification effects of real-time fluorescene PCR were compared.[Results]The three methods had different extraction effects while satisfying the real-time fluorescent PCR detection.The DNA templates extracted by the SN/T 3730.8-2013/8.1 standard method had higher purity and concentrations,and showed clearer bands in gel electrophoresis,indicating that the method has less effect on real-time fluorescent PCR and less inhibition,and can meet the detection needs of the four types of seafood,including fish,shrimps,crabs and shellfish.[Conclusions]This study provides a theoretical basis for the detection of seafood animal-derived components.展开更多
DNA extraction from degraded skeletal samples is often particularly challenging. The difficulty derives from the fact that variable environment has a significant effect on DNA preservation. During the years 2002-2015 ...DNA extraction from degraded skeletal samples is often particularly challenging. The difficulty derives from the fact that variable environment has a significant effect on DNA preservation. During the years 2002-2015 unidentified degraded skeletal remains were accumulated at our institute, National Institute of Forensic Medicine (NIFM), most of them with none or partial DNA profile. As new methods rapidly emerge, we revisited these samples with partial DNA profiles in the hope to add additional alleles and eventually be able to identify these previously unidentifiable samples. We have chosen to use these samples to compare two automated methods: Prepfiler Express BTA (Applied Biosystems) and QIAcube (Quiagen), in hope of acquiring a more complete DNA profile and eventually make new identifications possibly comparing these profiles with missing person database. In both methods, a preparation step is required, after which the samples undergo automatic DNA extraction. The two protocols are based on different extraction methods. Fresh or non-problematic bone samples as the positive control gave the same results in both methods. In the degraded skeletal samples, the results were significantly better using the QIAcube method in our hands, but since degraded samples are highly variable the combination of both methods could be useful to receive better and more reliable profiles.展开更多
Among many reports investigating microbial diversity from environmental samples with denaturing gradient gel electrophoresis (DGGE), limited attention has been given to the effects of universal primers and DNA extract...Among many reports investigating microbial diversity from environmental samples with denaturing gradient gel electrophoresis (DGGE), limited attention has been given to the effects of universal primers and DNA extraction on the outcome of DGGE analysis. In this study, these effects were tested with 16S rRNA gene-based DGGE on a bacterial community from farming water samples. The results indicate that the number of discernable bands in the DGGE fingerprint differed with the primer pairs used; the bands produced by 63f/518r, 341f/926r and 933f/1387r primer pairs were obviously fewer than those by 968f/1401r. Also, we found that each DNA extraction method resulted in different community profiles, reflected by the number and intensity of bands in the DGGE fingerprint. Furthermore, the main bands (theoretically representing dominant bacteria) differed with the extraction methods applied. It is therefore believed that the effects of universal primers and DNA extraction should be given more attention and carefully chosen before performing an investigation into a new environment with DGGE.展开更多
The basic method of DNA extraction (CTAB) was improved as the multi-times STE-CTAB extraction method and used to extract the DNA of birch leaved in this experiment. Results showed that the improved method is suitable ...The basic method of DNA extraction (CTAB) was improved as the multi-times STE-CTAB extraction method and used to extract the DNA of birch leaved in this experiment. Results showed that the improved method is suitable not only for genomic DNA extraction of birch but also for that of other plants. The purity of genomic DNA extracted by the multi-times STE-CTAB extraction method is higher than that by one time STE-CTAB method, and it does not need the process of RNase. The factors of influencing ISSR system were explored based on the genomic DNA of birch extracted by the two methods. The optimal conditions for ISSR system were determined as follows: Mg2+ concentration is 1.5–3.0 mmol·L-1, dNTP concentration 0.10–0.25 mmol·L-1, the quantity of Taq polymerase 0.5–2.0 U, template DNA 30–100 ng, and the concentration of primer is 0.2?0.4 μmmol·L-1, and the reaction program was as: initial denaturation for 5 min at 94oC, 30 cycles of denaturation for 30 s at 94oC, annealing for 30 s at 51 oC, extension for 30 s at 72oC, and a final 7 min extension at 72 oC.展开更多
A new method CTAB-silica for DNA extraction and purification from the leaves and buds of Castanea mollissima and Ginkgo biloba was tested. The method is based on the silica-based purification protocol developed by Boo...A new method CTAB-silica for DNA extraction and purification from the leaves and buds of Castanea mollissima and Ginkgo biloba was tested. The method is based on the silica-based purification protocol developed by Boom et al. (1990). By modifying the protocol, plant genome DNA could be extracted easily from dormant buds, mature leaves, and other parts of plant. Our results showed that the purified DNA was of high purity and could be analyzed by PCR. Furthermore, this CTAB-silica method took much less time for a successful DNA purification process compared to the traditional methods (CTAB and SDS). By our method, the suitable DNA can be extracted and purified from over 10 plant samples by one person in an hour.展开更多
Molecular microbiological methods, such as competetive PCR, real-time PCR, denaturing gradient gel electrophoresis (DGGE) and large-scale parallel-pyrosequencing, require the extraction of sufficient quantity of high ...Molecular microbiological methods, such as competetive PCR, real-time PCR, denaturing gradient gel electrophoresis (DGGE) and large-scale parallel-pyrosequencing, require the extraction of sufficient quantity of high quality DNA from microbiologically and chemically complex matrices. Due to difficulties in the field to standardize/select the optimum DNA preservation-extraction methods in view of laboratories differences, this article attempts to present a straight-forward mathematical framework for comparing some of the most commonly used methods. To this end, as a case study, the problem of selecting an optimum sample preservation-DNA extraction strategy for obtaining total bacterial DNA from swine feces was considered. Two sample preservation methods (liquid nitrogen and RNAlater?) and seven extraction techniques were paired and compared under six quantitative DNA analysis criteria: yield of extraction, purity of extracted DNA (A260/280 and A 260/230 ratios), duration of extraction, degradation degree of DNA, and cost. From a practical point of view, it is unlikely that a single sample preservation-DNA extraction strategy can be optimum for all selected criteria. Hence, a systematic multi-criteria decision-making (MCDM) approach was used to compare the methods. As a result, the ZR Fecal DNA MiniPrepTM DNA extraction kit for samples preserved either with liquid nitrogen or RNAlater? were identified as potential optimum solutions for obtaining total bacterial DNA from swine feces. Considering the need for practicality for in situ applications, we would recommend liquid nitrogen as sample preservation method, along with the ZR Fecal DNA MiniPrepTM kit. Total bacterial DNA obtained by this strategy can be suitable for downstream PCR-based DNA analyses of swine feces.展开更多
There is an increased interest in the extraction of nucleic acids from various environmental samples since culture-independent molecular techniques contribute to deepen and broaden the understanding of a greater porti...There is an increased interest in the extraction of nucleic acids from various environmental samples since culture-independent molecular techniques contribute to deepen and broaden the understanding of a greater portion of uncultivable microorganisms. Due to difficulties to select the optimum DNA extraction method in view of downstream molecular analyses, this article presents a straightforward mathematical framework for comparing some of the most commonly used methods. Four commercial DNA extraction kits and two physical-chemical methods (bead-beating and freeze-thaw) were compared for the extraction of DNA under several quantitative DNA analysis criteria: yield of extraction, purity of extracted DNA (A260/280 and A260/230 ratios), degradation degree of DNA, easiness of PCR amplification, duration of extraction, and cost per extraction. From a practical point of view, it is unlikely that a single DNA extraction strategy can be optimum for all selected criteria. Hence, a systematic Technique for Order Preference by Similarity to Ideal Solution (TOPSIS) was employed to compare the methods. The PowerSoil? DNA Isolation Kit was systematically defined as the best performing method for extracting DNA from soil samples. More specifically, for soil:manure and soil:manure:biochar mixtures, the PowerSoil?DNA Isolation Kit method performed best, while for neat soil samples its alternative version gained the first rank.展开更多
There have been many arguments on the classification of Eriocaulon Linn. by morphology so far, and little is known about the use of molecular marker for genetic for genetic diversity of Eriocaulon plants. To apply the...There have been many arguments on the classification of Eriocaulon Linn. by morphology so far, and little is known about the use of molecular marker for genetic for genetic diversity of Eriocaulon plants. To apply the technique of molecular marker to the research of genetic diversity of Eriocaulon plants, the study of the extraction method of DNA from the Eriocaulon plants and the RAPD system are essential for researchers. In this paper, the extraction of genome DNA from the silica-gel-dried leaves of several species of Eriocaulon distributed in China was studied, and the best RAPD analysis technique condition of Eriocaulon plants was analyzed.展开更多
Extraction of DNA from fresh tissues is routine in studies of tropical forest species, but DNA extraction from wood is considered as difficult due to its highly degraded nature and adequate quality of genomic DNA extr...Extraction of DNA from fresh tissues is routine in studies of tropical forest species, but DNA extraction from wood is considered as difficult due to its highly degraded nature and adequate quality of genomic DNA extraction is essential for molecular studies. Very few studies have validated the potential for isolating DNA from dried wood (Heartwood and Sapwood). Wood genomic DNA extraction is difficult from mature timber (Teak (Tectona grandis f;verbanaceae), Black Rosewood (Dalbergia latifolia f;Fabaceae) Ben Teak (Lagerstroemia lanceolata f;Lytheraceae) tissues due to presence of high quantity of secondary metabolites polyphenols, tannins and terpenoids and protein inhibitors. Mostly in laboratories DNA extraction kits are available but by using kits, DNA yield is very low and it is quite expensive too. We have standardized and validated the DNA extraction through manual protocol which is applicable for Bark, Sapwood and Heartwood samples of tree species which contains huge amount of inflexible tissues and fibers. The quality of the DNA was tested by spectrophotometer, gel electrophoresis and PCR (ISSR and SSR) amplification. An avrage DNA yield for heartwood ranges from 0.186 - 0.166 μg/μL and sapwood was ranges from 0.26 - 0.244 μg/μL. Modification of CTAB method was by addition of polyvinylpyrrolidone (PVP) appx 0.25%, variation in Rnase concentration, proteinase treatment with different concentration and incubation time. In order to evaluate the standardized wood genomic DNA extraction protocol, we compared it with the mature leaf and core samples (heartwood and sapwood) of the same timber species. The outcome was also quantified and proved by means of polymerase chain reaction analysis by using ISSR and SSR microsatellite markers conducted with isolated pure DNAs. This modified protocol made increased yield and purity of wood total genomic DNA and facilitate the important application of forensic timber species effort.展开更多
For first-line non-small-cell lung cancer(NSCLC) therapy,detecting mutation status of the epidermal growth factor receptor(EGFR) gene constitutes a prudent test to identify patients who are most likely to benefit from...For first-line non-small-cell lung cancer(NSCLC) therapy,detecting mutation status of the epidermal growth factor receptor(EGFR) gene constitutes a prudent test to identify patients who are most likely to benefit from EGFR-tyrosine kinase inhibitor(TKI) therapy.Now,the material for detecting EGFR gene mutation status mainly comes from formalin-fixed and paraffin-embedded(FFPE) tissues.DNA extraction from FFPE and the amplification of EGFR gene by polymerase chain reaction(PCR) are two key steps for detecting EGFR gene mutation.We showed a simple method of DNA extraction from FFPE tissues for the effective amplification of EGFR gene.Extracting DNA from the FFPE tissues of NSCLC patients with 1% Triton X-100(pH=10.0) was performed by heating at 95 °C for 30 min.Meanwhile,a commercial kit was used to extract DNA from the same FFPE tissues of NSCLC patients for comparison.DNA extracted products were used as template for amplifying the exons 18,19,20 and 21 of EGFR by PCR for different amplified fragments.Results show that DNA fragment size extracted from FFPE tissues with 1% Triton X was about 250―500 base pairs(bp).However,DNA fragment size extracted from FFPE tissues via commercial kit was about from several hundreds to several thousands bp.The DNA yield extracted from FFPE tissues with 1% Triton X was larger than that via commercial kit.For about 500 bp fragment,four exons of EGFR could not be amplified more efficiently from extracted DNA with 1% Triton X than with commercial kit.However,for about 200 bp fragment.This simple and non-laborious protocol could successfully be used to extract DNA from FFPE tissue for the amplification of EGFR gene by PCR,further screening of EGFR gene mutation and facilitating the molecular analysis of a large number of FFPE tissues from NSCLC patients.展开更多
Approximately 200 mg of fin tissue from each specimen was cut into pieces and treated with a gradient of ethanol (65%, 70%, 75%, 80%, 85%, 90%, 95% and 100%) and then DNA was extracted from formalin fish fin tissues. ...Approximately 200 mg of fin tissue from each specimen was cut into pieces and treated with a gradient of ethanol (65%, 70%, 75%, 80%, 85%, 90%, 95% and 100%) and then DNA was extracted from formalin fish fin tissues. Agarose gel visualization of the DNA confirmed DNA extracted. MtDNA cyt b gene banding pattern of samples with agarose gel was clearly visible and could be used to be sequenced. In terms of DNA purity, the 260/280 ratio of 87.5% samples ranged between 1.8 and 2.0, indicating that DNA of the majority of the samples was pure. The developed DNA extraction procedure from formalin fish fin tissues by pretreatment with a gradient of ethanol system will be a useful tool to study the genetic structure and phylogenesis of endangered fish, by specimens preserved formalin-fixed in museum and herbarium.展开更多
A simple protocol was established for DNA extraction using etiolated rice seedlings, whereby rice DNA was directly extracted in 0.5 mol/L NaOH solution in a single eppendorf tube. Results of comparative PCR analyses a...A simple protocol was established for DNA extraction using etiolated rice seedlings, whereby rice DNA was directly extracted in 0.5 mol/L NaOH solution in a single eppendorf tube. Results of comparative PCR analyses and electrophoresis showed that the DNA extracted using this method was as good and useful as that using standard CTAB method.展开更多
Use of a single seed is very useful for genetic studies on Vitis vinifera. However, molecular markers require a fair amount of high purity DNA. Grapevine contains high concentrations of polysaccharides, polyphenols, t...Use of a single seed is very useful for genetic studies on Vitis vinifera. However, molecular markers require a fair amount of high purity DNA. Grapevine contains high concentrations of polysaccharides, polyphenols, tannins and other secondary metabolites. These compounds may hamper the DNA isolation processes and subsequent analysis. In this study we have compared two DNA isolation methods: the NucleoSpin Plant II method and a modified protocol from Doyle and Doyle. The average value of 260/280 nm absorption ratio, which is used to assess the purity of DNA and RNA was 1.8 (accepted as “pure” DNA) and 0.9 (presence of protein or other contaminants) for the first and second method, respectively. Using the NucleoSpin protocol, from a single seed (20 - 35 mg) we obtained an average yield of extracted DNA of 24.8 ± 5.2 to 38.4 ± 11.5 ng·mg-1 dry weight. Among the two protocols examined, the NucleoSpin method was more efficient and gave better quality of DNA values compared to those from the modified Doyle and Doyle procedures.展开更多
Our previously described environmental DNA extraction method has been widely used in environmental microbial community analysis.However,residual humic substances may remain with obtained environmental DNA,which interf...Our previously described environmental DNA extraction method has been widely used in environmental microbial community analysis.However,residual humic substances may remain with obtained environmental DNA,which interferes downstream molecular analyses.To remedy this situation,two DNA extraction buffers (PIPES and Tris-HCl) and four purification strategies including our new modified low melting point gel purification method and three commercial kits from QIAEX,Omega and Promega were evaluated with diverse soil samples.The PIPES buffer (pH 6.5) is found to be more effective for removing the humic substances,but it leads to lower DNA yield and causes more severe DNA shearing than using the Tris-HCl buffer (pH 8.0).Gel purification and the Promega purification kit achieve much higher DNA recoveries than QIAEX or Omega kit,and higher purity of DNA is obtained by gel purification than by the Promega kit with both DNA extraction buffers mentioned above.Considering all results together,two alternative methods for DNA extraction and purification are proposed:one uses Tris-HCl buffer extraction and gel purification as the primary approach when the amount of soil or biomass is not a major concern,and the other uses PIPES buffer extraction and the Promega kit purification when severe DNA shearing and/or limited biomass occurs.Purified DNA samples by both methods are amenable for use as templates for whole community genome amplifications and PCR amplifications of bacterial 16S rRNA genes.It is demonstrated that these two alternative methods could be applied to a wide variety of environmental samples.展开更多
[Objective] The paper aimed to get a efficient,stable and low-priced DNA extraction method of mulberry leaves. [Method] With tender leaves of mulberry varieties in Anhui Province as materials,genomic DNA was extracted...[Objective] The paper aimed to get a efficient,stable and low-priced DNA extraction method of mulberry leaves. [Method] With tender leaves of mulberry varieties in Anhui Province as materials,genomic DNA was extracted with CTAB method,and further identified by ultraviolet spectrophotometer,agarose gel electrophoresis and PCR methods. [Result] The A260/A280 ratio of extracted DNA was 1. 80-1. 90,and the yield was 0. 187-0. 275 μg/mg. With extracted DNA as the template,clear target bands were obtained by PCR. [Conclusion]Extracted DNA was good in quality,which could satisfy the needs of follow-up molecular biological operation.展开更多
基金conducted partly with the Regional Cooperative Agreement project(RAS5062)grant from the International Atomic Energy Agencythe University Grants Commission’s fund(2015–2016)for research projects in the University of Dhaka+1 种基金International Atomic Energy Agencythe University Grants Commission,Bangladesh for their support。
文摘An efficient and good DNA extraction protocol should be simple, affordable and yield enough DNA with high quality. Rice(Oryza sativa L.) DNA extraction methods often use seedlings or leaves rather than the grains and tend to be time-consuming, involve multiple steps, and use hazardous chemicals and expensive enzymes. Rice grains offer several benefits over seedlings and leaves as a source of DNA for genetic analysis. However, these benefits are underutilized because the bulk of a rice grain is made up of starch. It is particularly important, but difficult to get rid of the starch while extracting DNA from rice grains. This co-precipitated polysaccharide is a known inhibitor of DNA polymerase activity in polymerase chain reaction(PCR). We describe here a very simple and highly affordable Chelex~?-100 based DNA extraction method from rice grains. It does not require any hazardous chemicals or enzymes. This method reproducibly extracts DNA with good purity indices(A_(260)/A_(230) and A_(260)/A_(280) values), but requires only a few steps.
文摘Melia dubia Cav. of family Meliaceae is a fast growing, high value tree species native to India. Isolating DNA from matured dried leaves of M. dubia was difficult due to accumulation of secondary metabolites, majorly polyphenolics, which resulted in dark brown to black colour of the pellet. In this study, a modified STE-(Sucrose, Tris-HCl and Ethylene Diamine Tetra Acetic Acid) CTAB (hexadecyltrimethylammonium bromide) method was standardized for removal of polyphenolics. The protocol developed yielded 200 - 1000 ng/μl of quality DNA without any impurities as evident by A260/280 ratio ranging from 1.75 - 2.0. It was also suitable for extracting quality DNA from other members of Meliaceae like Azadirachta indica and Melia azedarach. In downstream applications, the extracted DNA was used for PCR amplification by using ISSR and SSR markers. ISSR PCR conditions were optimized in a reaction volume of 25 μl, consisting of 30 ng of template DNA, 1.5 mM MgCl<sub>2</sub>, 200 μM of each of dNTPs and 2 U of Taq polymerase. The best amplification was observed and the same was applicable for SSR markers.
基金This study was supported by National Natural Science Foundation of China(No.31930013,31872240)the National Key Program of Research and Development,Ministry of Science and Technology(2016YFC0503200)Youth Innovation Promotion Association of Chinese Academy of Sciences(2020086)to SP.
文摘Background:As an important player during food digestion,gut microbiota has attracted much attention in diet adaptation studies in birds.Microbiota extracted from feces has been widely used as a proxy for gut microbiota.Although several methods have been developed for microbial DNA extraction,their performances in the bird feces have not been systematacially evaluated yet.Methods:In this study,we applied three DNA extraction methods(Qiagen,MoBio and Bead)to extract DNA from feces of three avian dietary guilds(granivore,omnivore and carnivore),sequenced V4 region of 16S rRNA gene for each extract and evaluated the performances of DNA yield,DNA integrity,microbial composition,cell lysis capacity and alpha diversity for the three methods on each dietary guild.Results:Bead method was the best on the performance of both DNA yield and DNA integrity regardless of dietary guild.In granivore,microbial relative abundance at both species and phylum levels,alpha diversity and cell lysis capacity were comparable among all methods.In omnivore,Qiagen had the best performance on alpha diversity,fol-lowed by Bead and MoBio.There were small variations on microbial relative abundance at both species and phylum levels among different extraction methods.MoBio exhibited the best performance on cell lysis capacity.In carnivore,considerable variations were found on microbial relative abundance at both species and phylum levels.Qiagen had the best performance on alpha diversity,followed by MoBio and Bead.MoBio had the highest cell lysis capacity.Conclusions:DNA yield and integrity have no obvious impact on microbial composition,alpha diversity or cell lysis capacity.The microbiota results(e.g.,microbial composition,cell lysis capacity,alpha diversity)obtained from differ-ent methods are comparable in granivorous avian species but not in omnivorous or carnivorous birds.Either method could be used in granivore microbiota studies.For omnivores and carnivores,we recommend Qiagen method when the research purpose is microbial diversity and MoBio when gram-positive bacteria is the research target.
基金supported by the National Natural Science Foundation of China(32102793)National Key R&D Program of China(2018YFD0900802)+4 种基金Central Public-Interest Scientific Institution Basal Research FundSouth China Sea Fisheries Research Institute,CAFS(2019TS13,2021SD18)Key Special Project for Introduced Talents Team of Southern Marine Science and Engineering Guangdong Laboratory(Guangzhou)(GML2019ZD0605)Open Fund Project of Key Laboratory of Offshore Fishery Development of Ministry of Agriculture and Rural Affairs(LOF 2020-02)China-ASEAN Maritime Cooperation Fund(CAMC-2018F)。
文摘Environmental DNA(eDNA)integrated with metabarcoding is a promising and powerful tool for species composition and biodiversity assessment in aquatic ecosystems and is increasingly applied to evaluate fish diversity.To date,however,no standardized eDNA-based protocol has been established to monitor fish diversity.In this study,we investigated and compared two filtration methods and three DNA extraction methods using three filtration water volumes to determine a suitable approach for eDNA-based fish diversity monitoring in the Pearl River Estuary(PRE),a highly anthropogenically disturbed estuarine ecosystem.Compared to filtration-based precipitation,direct filtration was a more suitable method for eDNA metabarcoding in the PRE.The combined use of DNeasy Blood and Tissue Kit(BT)and traditional phenol/chloroform(PC)extraction produced higher DNA yields,amplicon sequence variants(ASVs),and Shannon diversity indices,and generated more homogeneous and consistent community composition among replicates.Compared to the other combined protocols,the PC and BT methods obtained better species detection,higher fish diversity,and greater consistency for the filtration water volumes of 1000 and 2000 mL,respectively.All eDNA metabarcoding protocols were more sensitive than bottom trawling in the PRE fish surveys and combining two techniques yielded greater taxonomic diversity.Furthermore,combining traditional methods with eDNA analysis enhanced accuracy.These results indicate that methodological decisions related to eDNA metabarcoding should be made with caution for fish community monitoring in estuarine ecosystems.
文摘Myxosporidia constitute a major group of fish parasites which have a significant negative impact on wild and cultured fish. The used of DNA in Myxosporidia studies has progressed rapidly over the last twenty years, especially in their identification and characterization as well as determination of species diversity and investigation of their evolutionary relationships. Extraction and isolation of pure and high quality DNA are essential for any molecular study, but constitute a challenge for many laboratories especially in low and middle income countries. Myxosporidia plasmodia filled with mature myxospores were isolated from different tissues of <span style="white-space:nowrap;"><i></span>Labeo batesii<span style="white-space:nowrap;"></i></span> Boulenger, 1911. DNA from myxosporidia myxospores were extracted using a Livak optimized DNAs extraction protocol. Four particular phases of the original protocol were optimized. Yield and absorbance ratios of extracted DNA were determined using spectrophotometer. DNA samples were used as template for the amplification of the 18S rDNA region and amplicons resolved on 1.5% agarose gel for determination of fragment sizes and purity evaluation. The concentration of extracted DNA from all Myxosporidia species ranged from 4.6 to 26 ng/μl with purity indices ranging from 1.88 to 2.12. We successfully amplified the 1050 bp DNA fragment as targeted. The intensity, thickness and clarity of the bands were evidences of non-degradation of DNA. The optimized Livak protocol is simple, low-cost and manageable. Regarding the quantity, purity and quality of extracted DNA, the optimized Livak protocol is highly recommended for Myxosporidia studies.
文摘[Objectives]This study was conducted for the purpose of widely using molecular biology techniques in the detection of seafood species.[Methods]In this study,such four kinds of seafood as fish,shrimps,crabs and shellfish were used as samples to compare three DNA extraction methods,including Tiangen marine animal tissue genome extraction kit method(DP324)(a),SN/T 3589.6-2013/8.3(b)method and modified SN/T 3730.8-2013/8.1(c)method.The purity and concentrations of DNA and the amplification effects of real-time fluorescene PCR were compared.[Results]The three methods had different extraction effects while satisfying the real-time fluorescent PCR detection.The DNA templates extracted by the SN/T 3730.8-2013/8.1 standard method had higher purity and concentrations,and showed clearer bands in gel electrophoresis,indicating that the method has less effect on real-time fluorescent PCR and less inhibition,and can meet the detection needs of the four types of seafood,including fish,shrimps,crabs and shellfish.[Conclusions]This study provides a theoretical basis for the detection of seafood animal-derived components.
文摘DNA extraction from degraded skeletal samples is often particularly challenging. The difficulty derives from the fact that variable environment has a significant effect on DNA preservation. During the years 2002-2015 unidentified degraded skeletal remains were accumulated at our institute, National Institute of Forensic Medicine (NIFM), most of them with none or partial DNA profile. As new methods rapidly emerge, we revisited these samples with partial DNA profiles in the hope to add additional alleles and eventually be able to identify these previously unidentifiable samples. We have chosen to use these samples to compare two automated methods: Prepfiler Express BTA (Applied Biosystems) and QIAcube (Quiagen), in hope of acquiring a more complete DNA profile and eventually make new identifications possibly comparing these profiles with missing person database. In both methods, a preparation step is required, after which the samples undergo automatic DNA extraction. The two protocols are based on different extraction methods. Fresh or non-problematic bone samples as the positive control gave the same results in both methods. In the degraded skeletal samples, the results were significantly better using the QIAcube method in our hands, but since degraded samples are highly variable the combination of both methods could be useful to receive better and more reliable profiles.
基金This work was funded by the Special Scientific Foundation of Guangdong Province (No. A305030301)was partly supported by a grant from KLFEE, Ministry of Agriculture (2003-04).
文摘Among many reports investigating microbial diversity from environmental samples with denaturing gradient gel electrophoresis (DGGE), limited attention has been given to the effects of universal primers and DNA extraction on the outcome of DGGE analysis. In this study, these effects were tested with 16S rRNA gene-based DGGE on a bacterial community from farming water samples. The results indicate that the number of discernable bands in the DGGE fingerprint differed with the primer pairs used; the bands produced by 63f/518r, 341f/926r and 933f/1387r primer pairs were obviously fewer than those by 968f/1401r. Also, we found that each DNA extraction method resulted in different community profiles, reflected by the number and intensity of bands in the DGGE fingerprint. Furthermore, the main bands (theoretically representing dominant bacteria) differed with the extraction methods applied. It is therefore believed that the effects of universal primers and DNA extraction should be given more attention and carefully chosen before performing an investigation into a new environment with DGGE.
基金This paper was supported by National Natural Science Foundation of China (No. 30571513) and National High Technology Research and Development Program of China (863 Program) (No. 2002AA241080).
文摘The basic method of DNA extraction (CTAB) was improved as the multi-times STE-CTAB extraction method and used to extract the DNA of birch leaved in this experiment. Results showed that the improved method is suitable not only for genomic DNA extraction of birch but also for that of other plants. The purity of genomic DNA extracted by the multi-times STE-CTAB extraction method is higher than that by one time STE-CTAB method, and it does not need the process of RNase. The factors of influencing ISSR system were explored based on the genomic DNA of birch extracted by the two methods. The optimal conditions for ISSR system were determined as follows: Mg2+ concentration is 1.5–3.0 mmol·L-1, dNTP concentration 0.10–0.25 mmol·L-1, the quantity of Taq polymerase 0.5–2.0 U, template DNA 30–100 ng, and the concentration of primer is 0.2?0.4 μmmol·L-1, and the reaction program was as: initial denaturation for 5 min at 94oC, 30 cycles of denaturation for 30 s at 94oC, annealing for 30 s at 51 oC, extension for 30 s at 72oC, and a final 7 min extension at 72 oC.
文摘A new method CTAB-silica for DNA extraction and purification from the leaves and buds of Castanea mollissima and Ginkgo biloba was tested. The method is based on the silica-based purification protocol developed by Boom et al. (1990). By modifying the protocol, plant genome DNA could be extracted easily from dormant buds, mature leaves, and other parts of plant. Our results showed that the purified DNA was of high purity and could be analyzed by PCR. Furthermore, this CTAB-silica method took much less time for a successful DNA purification process compared to the traditional methods (CTAB and SDS). By our method, the suitable DNA can be extracted and purified from over 10 plant samples by one person in an hour.
文摘Molecular microbiological methods, such as competetive PCR, real-time PCR, denaturing gradient gel electrophoresis (DGGE) and large-scale parallel-pyrosequencing, require the extraction of sufficient quantity of high quality DNA from microbiologically and chemically complex matrices. Due to difficulties in the field to standardize/select the optimum DNA preservation-extraction methods in view of laboratories differences, this article attempts to present a straight-forward mathematical framework for comparing some of the most commonly used methods. To this end, as a case study, the problem of selecting an optimum sample preservation-DNA extraction strategy for obtaining total bacterial DNA from swine feces was considered. Two sample preservation methods (liquid nitrogen and RNAlater?) and seven extraction techniques were paired and compared under six quantitative DNA analysis criteria: yield of extraction, purity of extracted DNA (A260/280 and A 260/230 ratios), duration of extraction, degradation degree of DNA, and cost. From a practical point of view, it is unlikely that a single sample preservation-DNA extraction strategy can be optimum for all selected criteria. Hence, a systematic multi-criteria decision-making (MCDM) approach was used to compare the methods. As a result, the ZR Fecal DNA MiniPrepTM DNA extraction kit for samples preserved either with liquid nitrogen or RNAlater? were identified as potential optimum solutions for obtaining total bacterial DNA from swine feces. Considering the need for practicality for in situ applications, we would recommend liquid nitrogen as sample preservation method, along with the ZR Fecal DNA MiniPrepTM kit. Total bacterial DNA obtained by this strategy can be suitable for downstream PCR-based DNA analyses of swine feces.
文摘There is an increased interest in the extraction of nucleic acids from various environmental samples since culture-independent molecular techniques contribute to deepen and broaden the understanding of a greater portion of uncultivable microorganisms. Due to difficulties to select the optimum DNA extraction method in view of downstream molecular analyses, this article presents a straightforward mathematical framework for comparing some of the most commonly used methods. Four commercial DNA extraction kits and two physical-chemical methods (bead-beating and freeze-thaw) were compared for the extraction of DNA under several quantitative DNA analysis criteria: yield of extraction, purity of extracted DNA (A260/280 and A260/230 ratios), degradation degree of DNA, easiness of PCR amplification, duration of extraction, and cost per extraction. From a practical point of view, it is unlikely that a single DNA extraction strategy can be optimum for all selected criteria. Hence, a systematic Technique for Order Preference by Similarity to Ideal Solution (TOPSIS) was employed to compare the methods. The PowerSoil? DNA Isolation Kit was systematically defined as the best performing method for extracting DNA from soil samples. More specifically, for soil:manure and soil:manure:biochar mixtures, the PowerSoil?DNA Isolation Kit method performed best, while for neat soil samples its alternative version gained the first rank.
基金Supported by Ministry of Education of China (Grant No. 01029)
文摘There have been many arguments on the classification of Eriocaulon Linn. by morphology so far, and little is known about the use of molecular marker for genetic for genetic diversity of Eriocaulon plants. To apply the technique of molecular marker to the research of genetic diversity of Eriocaulon plants, the study of the extraction method of DNA from the Eriocaulon plants and the RAPD system are essential for researchers. In this paper, the extraction of genome DNA from the silica-gel-dried leaves of several species of Eriocaulon distributed in China was studied, and the best RAPD analysis technique condition of Eriocaulon plants was analyzed.
文摘Extraction of DNA from fresh tissues is routine in studies of tropical forest species, but DNA extraction from wood is considered as difficult due to its highly degraded nature and adequate quality of genomic DNA extraction is essential for molecular studies. Very few studies have validated the potential for isolating DNA from dried wood (Heartwood and Sapwood). Wood genomic DNA extraction is difficult from mature timber (Teak (Tectona grandis f;verbanaceae), Black Rosewood (Dalbergia latifolia f;Fabaceae) Ben Teak (Lagerstroemia lanceolata f;Lytheraceae) tissues due to presence of high quantity of secondary metabolites polyphenols, tannins and terpenoids and protein inhibitors. Mostly in laboratories DNA extraction kits are available but by using kits, DNA yield is very low and it is quite expensive too. We have standardized and validated the DNA extraction through manual protocol which is applicable for Bark, Sapwood and Heartwood samples of tree species which contains huge amount of inflexible tissues and fibers. The quality of the DNA was tested by spectrophotometer, gel electrophoresis and PCR (ISSR and SSR) amplification. An avrage DNA yield for heartwood ranges from 0.186 - 0.166 μg/μL and sapwood was ranges from 0.26 - 0.244 μg/μL. Modification of CTAB method was by addition of polyvinylpyrrolidone (PVP) appx 0.25%, variation in Rnase concentration, proteinase treatment with different concentration and incubation time. In order to evaluate the standardized wood genomic DNA extraction protocol, we compared it with the mature leaf and core samples (heartwood and sapwood) of the same timber species. The outcome was also quantified and proved by means of polymerase chain reaction analysis by using ISSR and SSR microsatellite markers conducted with isolated pure DNAs. This modified protocol made increased yield and purity of wood total genomic DNA and facilitate the important application of forensic timber species effort.
基金Supported by the Jilin Science & Technology Development Plan,China(No.201201060)the Scientific Research Foundation of Jilin Province,China(No.20100942)+1 种基金the Fund of Developing and Reforming Community of Jilin Province,China(No.2010-1928)the Health Scientific Research Foundation of Jilin Province,China(Nos.2009z081,2010Z083)
文摘For first-line non-small-cell lung cancer(NSCLC) therapy,detecting mutation status of the epidermal growth factor receptor(EGFR) gene constitutes a prudent test to identify patients who are most likely to benefit from EGFR-tyrosine kinase inhibitor(TKI) therapy.Now,the material for detecting EGFR gene mutation status mainly comes from formalin-fixed and paraffin-embedded(FFPE) tissues.DNA extraction from FFPE and the amplification of EGFR gene by polymerase chain reaction(PCR) are two key steps for detecting EGFR gene mutation.We showed a simple method of DNA extraction from FFPE tissues for the effective amplification of EGFR gene.Extracting DNA from the FFPE tissues of NSCLC patients with 1% Triton X-100(pH=10.0) was performed by heating at 95 °C for 30 min.Meanwhile,a commercial kit was used to extract DNA from the same FFPE tissues of NSCLC patients for comparison.DNA extracted products were used as template for amplifying the exons 18,19,20 and 21 of EGFR by PCR for different amplified fragments.Results show that DNA fragment size extracted from FFPE tissues with 1% Triton X was about 250―500 base pairs(bp).However,DNA fragment size extracted from FFPE tissues via commercial kit was about from several hundreds to several thousands bp.The DNA yield extracted from FFPE tissues with 1% Triton X was larger than that via commercial kit.For about 500 bp fragment,four exons of EGFR could not be amplified more efficiently from extracted DNA with 1% Triton X than with commercial kit.However,for about 200 bp fragment.This simple and non-laborious protocol could successfully be used to extract DNA from FFPE tissue for the amplification of EGFR gene by PCR,further screening of EGFR gene mutation and facilitating the molecular analysis of a large number of FFPE tissues from NSCLC patients.
文摘Approximately 200 mg of fin tissue from each specimen was cut into pieces and treated with a gradient of ethanol (65%, 70%, 75%, 80%, 85%, 90%, 95% and 100%) and then DNA was extracted from formalin fish fin tissues. Agarose gel visualization of the DNA confirmed DNA extracted. MtDNA cyt b gene banding pattern of samples with agarose gel was clearly visible and could be used to be sequenced. In terms of DNA purity, the 260/280 ratio of 87.5% samples ranged between 1.8 and 2.0, indicating that DNA of the majority of the samples was pure. The developed DNA extraction procedure from formalin fish fin tissues by pretreatment with a gradient of ethanol system will be a useful tool to study the genetic structure and phylogenesis of endangered fish, by specimens preserved formalin-fixed in museum and herbarium.
文摘A simple protocol was established for DNA extraction using etiolated rice seedlings, whereby rice DNA was directly extracted in 0.5 mol/L NaOH solution in a single eppendorf tube. Results of comparative PCR analyses and electrophoresis showed that the DNA extracted using this method was as good and useful as that using standard CTAB method.
文摘Use of a single seed is very useful for genetic studies on Vitis vinifera. However, molecular markers require a fair amount of high purity DNA. Grapevine contains high concentrations of polysaccharides, polyphenols, tannins and other secondary metabolites. These compounds may hamper the DNA isolation processes and subsequent analysis. In this study we have compared two DNA isolation methods: the NucleoSpin Plant II method and a modified protocol from Doyle and Doyle. The average value of 260/280 nm absorption ratio, which is used to assess the purity of DNA and RNA was 1.8 (accepted as “pure” DNA) and 0.9 (presence of protein or other contaminants) for the first and second method, respectively. Using the NucleoSpin protocol, from a single seed (20 - 35 mg) we obtained an average yield of extracted DNA of 24.8 ± 5.2 to 38.4 ± 11.5 ng·mg-1 dry weight. Among the two protocols examined, the NucleoSpin method was more efficient and gave better quality of DNA values compared to those from the modified Doyle and Doyle procedures.
基金Project(51104189)supported by the National Natural Science Foundation of ChinaProject(2010CB630901)supported by the National Basic Research Program of China+1 种基金Project(1343-77341)supported by the Graduate Education Innovative Program of Central South University,ChinaProject(DOE-ER64125)supported by Department of Energy,Office of Science under the Environmental Remediation Science Program of the United States
文摘Our previously described environmental DNA extraction method has been widely used in environmental microbial community analysis.However,residual humic substances may remain with obtained environmental DNA,which interferes downstream molecular analyses.To remedy this situation,two DNA extraction buffers (PIPES and Tris-HCl) and four purification strategies including our new modified low melting point gel purification method and three commercial kits from QIAEX,Omega and Promega were evaluated with diverse soil samples.The PIPES buffer (pH 6.5) is found to be more effective for removing the humic substances,but it leads to lower DNA yield and causes more severe DNA shearing than using the Tris-HCl buffer (pH 8.0).Gel purification and the Promega purification kit achieve much higher DNA recoveries than QIAEX or Omega kit,and higher purity of DNA is obtained by gel purification than by the Promega kit with both DNA extraction buffers mentioned above.Considering all results together,two alternative methods for DNA extraction and purification are proposed:one uses Tris-HCl buffer extraction and gel purification as the primary approach when the amount of soil or biomass is not a major concern,and the other uses PIPES buffer extraction and the Promega kit purification when severe DNA shearing and/or limited biomass occurs.Purified DNA samples by both methods are amenable for use as templates for whole community genome amplifications and PCR amplifications of bacterial 16S rRNA genes.It is demonstrated that these two alternative methods could be applied to a wide variety of environmental samples.
基金Supported by Dean Youth Innovation Fund of Anhui Academy of Agricultural Sciences"ITS Sequence and Evolutionary Analysis of Local Mulberry Germplasm Resources in Anhui Province"(15B0634)Hefei Comprehensive Test Station of National Sericulture Technology System"Special Project for Construction of China Agricultural Industry Research System"(CARS-22-SYZ09)+1 种基金Special Talent Development Fund of Anhui Academy of Agricultural Sciences"Breeding of High Quality Ecological Mulberry Varieties and Integration and Demonstration of Matching Cultivation Technology"(17F0610)Discipline Construction of Anhui Academy of Agricultural Sciences"Research of Silkworm Gender Tag Technology"(16A0618)
文摘[Objective] The paper aimed to get a efficient,stable and low-priced DNA extraction method of mulberry leaves. [Method] With tender leaves of mulberry varieties in Anhui Province as materials,genomic DNA was extracted with CTAB method,and further identified by ultraviolet spectrophotometer,agarose gel electrophoresis and PCR methods. [Result] The A260/A280 ratio of extracted DNA was 1. 80-1. 90,and the yield was 0. 187-0. 275 μg/mg. With extracted DNA as the template,clear target bands were obtained by PCR. [Conclusion]Extracted DNA was good in quality,which could satisfy the needs of follow-up molecular biological operation.
