[Objective] To introduce an improved method for DNA extraction from the faeces of red deer. [Method] Based on the traditional method of CTAB lysis, we proposed an improved DNA extraction method according to the charac...[Objective] To introduce an improved method for DNA extraction from the faeces of red deer. [Method] Based on the traditional method of CTAB lysis, we proposed an improved DNA extraction method according to the characteristics of red deer faeces. [Result] This improved method extracted high-quality fecal DNA from Tianshan red deer and amplified the mitochondrial cytochrome b gene. With the muscle and fur DNA of red deer as the control, the sequencing results further con- firmed the reliability of the method. [Conclusion] The method requires no proteinase K in the process of extraction, and the extracted DNA can be used for PCR ampli- fication directly without the purification of DNA purification kit, thus, it is cost-saving.展开更多
[Objective] The aim of this study is to isolate Chlorella vulgaris(chlorella)and extract its genomic DNA.[Method] Both the dilution method and drip method were employed to isolate chlorella from lake water samples;the...[Objective] The aim of this study is to isolate Chlorella vulgaris(chlorella)and extract its genomic DNA.[Method] Both the dilution method and drip method were employed to isolate chlorella from lake water samples;the conditions for culturing chlorella were optimized and its genomic DNA was extracted by improved CTAB method and SDS method.[Result] The proper conditions for chlorella culture were as following:temperature 20-25 ℃,illumination 4.39-5.86 W/m2 and rotational speed 100-150r/min;improved CTAB method was suitable for extracting genomic DNA from chlorella.[Conclusion] The study is helpful to study the chlorella at molecular level and promote the exploitation and utilization of chlorella resources.展开更多
[Objective] This study was to find out a quick,simple,and low-cost method for the extraction of sorghum genomic DNA.[Method] Four plant genomic DNA extraction methods based on CTAB,including liquid nitrogen grinding m...[Objective] This study was to find out a quick,simple,and low-cost method for the extraction of sorghum genomic DNA.[Method] Four plant genomic DNA extraction methods based on CTAB,including liquid nitrogen grinding method（method I）,buffer grinding method（method II）,drying grinding method（method III）and directly grinding method（method IV）,were used to extract the sorghum genomic DNA from leaves;further the quantity and quality of the yielded DNA were detected by gel electrophoresis,SSR-PCR and SRAP-PCR.[Result] These four methods performed no remarkable difference in DNA product.The method I and method II produced DNA with higher purity and better integrity,which,especially from method I,is effective for SRAP-PCR and SSR-PCR.While the DNA extracted via method III and method IV had less integrality and lower purity,and only effective in SSR-PCR.[Conclusion] Enough amount of sorghum genomic DNA to perform tens of PCR could be quickly extracted using all these four methods.The DNA obtained via method I and method II had a broader application spectrum（SRAP,RAPD,ISSR and SSR）than that via method III and method IV which is only proper for PCR targeting small DNA fragments（SSR）.展开更多
[Objective] This study aimed at comparing the four extraction methods of genomic DNA from Clematis fasciculiflora Franch and determining the optimal extraction method for extracting the genomic DNA from Clematis fasci...[Objective] This study aimed at comparing the four extraction methods of genomic DNA from Clematis fasciculiflora Franch and determining the optimal extraction method for extracting the genomic DNA from Clematis fasciculiflora Franch.[Method] Leavies of Clematis fasciculiflora Franch were used as materials for comparing the purity and concentration of extracted DNA and extracting time among the four extraction methods of genomic DNA including improved CTAB method Ⅰ,improved CTAB method Ⅱ,improved CTAB method Ⅲ and improved SDS method.[Result] The four extraction methods could all be successfully used for extracting the genomic DNA from Clematis fasciculiflora Franch.The purity of genomic DNA was the highest using improved CTAB method Ⅰ,with the longest extracting time;while the concentration of genomic DNA was the maximum using the improved SDS method,with the shortest extracting time and relatively low purity;the extracting time of improved CTAB method Ⅲ was the shortest.[Conclusion] This study had established the optimal extraction method for extracting the genomic DNA from Clematis fasciculiflora Franch and supported for the further research using molecular biological methods.展开更多
To rapidly obtain high-quality genomic DNA from Chenopodium quinoa Willd, the genomic DAN in different tissues （leaves, stems and roots） of Chenopodi- um quinoa Willd was extracted by modified CTAB method, SDS metho...To rapidly obtain high-quality genomic DNA from Chenopodium quinoa Willd, the genomic DAN in different tissues （leaves, stems and roots） of Chenopodi- um quinoa Willd was extracted by modified CTAB method, SDS method and high- salt Iow-pH method, respectively. The quality and yield of extracted DNA was deter- mined using agarose gel electrophoresis and UV spectrophotometry. At the same time, the PCR-SSR and SSCP molecular detection was also performed. The results showed that the gel test strips, without obvious decomposition, of all the extraction methods were relatively obvious; the genomic DNA yield extracted by modified CTAB method was highest, followed by that by SDS method, and the genomic DNA extracted by high-salt Iow-pH method was lowest： the genomic DNA yields extracted by different methods from Chenopodium quinoa Wiltd leaves were all high- er than those from roots and stems; the quality of Chenopodium quinoa Willd ge- nomic DNA extracted by modified CTAB method and high-salt Iow-pH method was better, and polyphenols, polysaccharides and other impurities were removed more completely. The PCR-SSR and SSCP detection results showed that the genomic DNA extracted by different methods from different tissues of Chenopodium quinoa Willd all could be better amplified, and high-quality strips could be obtained. So the Chenopodium quinoa Willd genomic DNA extracted by the three methods all can be used for subsequent molecular biology research.展开更多
[Objective] The aim was to optimize the mass and rapid method for DNA extraction of Beauvena bassiana. [Method] Boiling water DNA extraction method was improved, DNA extraction liquid was heated by PCR instrument and ...[Objective] The aim was to optimize the mass and rapid method for DNA extraction of Beauvena bassiana. [Method] Boiling water DNA extraction method was improved, DNA extraction liquid was heated by PCR instrument and the extraction process was finished rapidly. [ Resuit] The quality of DNA obtained through mass and rapid extraction of fungal genomic DNA could meet the requirement of RAPD amplification analysis. The clear bands were amplified from 22 tested strains, the number of clear bands were different in the range of 2 -6 and the size of band were mainly concentrated in 450 -800 bp. The DNA extracted by this method also could completely meet the requirement of SCAR amplification. The amplified specific DNA bands used to mark the strain F263 were very clear. [Conclusion] This research provided relatively perfect method for mass and rapid extraction of fungal clenomic DNA.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 suitabl...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 genornic 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 0.104）.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 pmmol·L^-1, and the reaction program was as： initial denaturation for 5 min at 94℃, 30 cycles of denaturation for 30 s at 94℃,annealing for 30 s at 51℃, extension for 30 s at 72℃, and a final 7 min extension at 72℃.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
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
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
In order to thoroughly investigate the diversity of glacier microorganisms, four DNA extraction methods with differem lysis pat- terns were tested and two screened methods （the Bosshard-Bano method and the Zhou metho...In order to thoroughly investigate the diversity of glacier microorganisms, four DNA extraction methods with differem lysis pat- terns were tested and two screened methods （the Bosshard-Bano method and the Zhou method） were optimized for the most ef- fective form of the filter membrane （cut vs. uncut）, the DNA extraction method, and the precipitation method. The two optimized methods were then compared with the commercial Mo-Bio DNA extraction kit, and the results showed that the kit was generally suitable for extraction of microorganism DNA fi＇om glacier surface snow. Procedurally, it was found that a modified Boss- hard-Bano method （i.e., cutting the filter membrane into pieces, using a specific lysis pattern [lysozyme （5 mg/mL）-protease K （ 1 mg/mL）-CTAB （ 1%）-SDS （ 1%）], performing the extraction only once by chloroform-isoamyl alcohol （24： 1）, and conducting DNA precipitation by pure ethanol） was also an effective and less expensive method for extraction of microorganism DNA from glacier surface snow.展开更多
Polyphenols,teroens,and resinsmake it difficult to obtain high qualitygenomic DNA from rice.Four ex-traction methods were compared inour study,and CTAB precipitationwas the most practical one.Materials used were old l...Polyphenols,teroens,and resinsmake it difficult to obtain high qualitygenomic DNA from rice.Four ex-traction methods were compared inour study,and CTAB precipitationwas the most practical one.Materials used were old leavesfrom Ftransgenic rice in Hainan andfresh leaves from Ftransgenic rice inKunming.Procedures of the four methodswere as follows:1.Basical method:powder 1 gold or new leaves→added 2 ml ex-traction solution(100 mmol/L展开更多
[ Objective ] The objective of this study is to explore a rapid and efficient method of extracting genomic DNA from Artemisia abrotanum. [ Method] Three methods of Cutting Method, Liquid Nitrogen Method and Quartz San...[ Objective ] The objective of this study is to explore a rapid and efficient method of extracting genomic DNA from Artemisia abrotanum. [ Method] Three methods of Cutting Method, Liquid Nitrogen Method and Quartz Sand Method based on SDS method were employed to extract Artemisia abrotanum genomlc DNA from tender leaf at seedling stage, tender spike and old leaf at heading stage. The obtained DNAs were detected by absorbance detection, agarose gel and PCR amplification. [ Result ] Cutting Method performed better than the other two methods compared in purity, extracting cycle and cost, accordingly more suitable for PCR amplification. The results also show that young spike is the best material for extracting genomic DNA from Artemisia Annua.展开更多
[ Objective] The aim was to establish the method of extracting genomic DNA from sheep blood clot on the basis of the improvement of method for extracting genomic DNA from tissues. [Method]The genomic DNA with complete...[ Objective] The aim was to establish the method of extracting genomic DNA from sheep blood clot on the basis of the improvement of method for extracting genomic DNA from tissues. [Method]The genomic DNA with complete primary structure and high purity was obtained from the sheep blood clot after the steps of cutting the sheep blood clot with ophthalmic scissors, cell lysis with tissue DNA extracts and digested by proteinase K, extracting with phenol/chloroform and precipitating with ethanol were performed. [ Result] The concentration of the extracted DNA was 159.90 ±0.70 ng/μl and the ratio of the A260/A280 was 1.80 ＋0.01. The sheep microsatellite locus of BM203 was amplified by using the extracted DNA from the sheep blood clot as template of PCR, and the PCR result was perfect. [Conclusion]This method is simple and feasible, the quantity and quality of the extracted DNA can satisfy the demands for the subsequent researches. It is worth to extending and using for reference.展开更多
The crushing of leaves is the most time and effort-consuming part in DNA extraction which is a fundamental step in the study of molecular biology. In this study, a new rapid and batch-oriented crushing method for DNA ...The crushing of leaves is the most time and effort-consuming part in DNA extraction which is a fundamental step in the study of molecular biology. In this study, a new rapid and batch-oriented crushing method for DNA extraction from maize leaves was developed. In addition, the practicability of the developed method in molecular marker-assisted breeding was verified using SSR molecular maker technology so as to provide a rapid, batch-oriented, low-cost and non-toxic leafcrushing method for a large number of molecular marker tests, improving test efficiency.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 Bo...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
AIM： To rapidly quantify hepatitis B virus （HBV） DNA by real-time PCR using efficient TaqMan probe and extraction methods of virus DNA. METHODS： Three standards were prepared by cloning PCR products which targeted...AIM： To rapidly quantify hepatitis B virus （HBV） DNA by real-time PCR using efficient TaqMan probe and extraction methods of virus DNA. METHODS： Three standards were prepared by cloning PCR products which targeted S, C and X region of HBV genome into pGEM-T vector respectively. A pair of primers and matched TaqMan probe were selected by comparing the copy number and the Ct values of HBV serum samples derived from the three different standard curves using certain serum DNA. Then the efficiency of six HBV DNA extraction methods including guanidinium isothiocyanate, proteinase K, NaI, NaOH lysis, alkaline lysis and simple boiling was analyzed in sample A, B and C by real-time PCR. Meanwhile, 8 clinical HBV serum samples were quantified. RESULTS： The copy number of the same HBV serum sample originated from the standard curve of S, C and X regions was 5.7 × 10^4/mL, 6.3 × 10^2/mL and 1.6 × 10^3/ mL respectively. The relative Ct value was 26.6, 31.8 and 29.5 respectively. Therefore, primers and matched probe from S region were chosen for further optimization of six extraction methods. The copy number of HBV serum samples A, B and C was 3.49 × 10^9/mL, 2.08 × 10^6/mL and 4.40 × 10^7/mL respectively, the relative Ct value was 19.9, 30 and 26.2 in the method of NaOH lysis, which was the efficientest among six methods. Simple boiling showed a slightly lower efficiency than NaOH lysis. Guanidinium isothiocyanate, proteinase K and NaI displayed that the copy number of HBV serum sample A, B and C was around 10^S/mL, meanwhile the Ct value was about 30. Alkaline failed to quantify the copy number of three HBV serum samples. Standard deviation （SD） and coefficient variation （CV） were very low in all 8 clinical HBV serum samples, showing that quantification of HBV DNA in triplicate was reliable and accurate. CONCLUSION： Real-time PCR based on optimized primers and TaqMan probe from S region in combination with NaOH lysis is a simple, rapid and accurate method for quantification of HBV serum DNA.展开更多
[Objective] This study aimed to explore a method for plant genomic DNA extraction, to provide guidance for rapid extraction of genomic DNA from plant tissues. [ Method] Based on the published genomic DNA extraction me...[Objective] This study aimed to explore a method for plant genomic DNA extraction, to provide guidance for rapid extraction of genomic DNA from plant tissues. [ Method] Based on the published genomic DNA extraction methods, operation steps, reagent amount and processing time of SDS extraction method were optimized. [Result] A widely applicable method was established initially for genomic DNA extraction from various varieties of plants and various kinds of plant tissues. Quality of the extracted genomic DNA was relatively good, which meets the requirements for further operation. [ Conclusion] This study provided guidance for rapid plant genomic DNA extraction.展开更多
基金Supported by the National Natural Science Foundation of China(31060152)the Natural Science Foundation of the Xinjiang Uygur Autonomous Region,China(2010211A02)the Key Program for Animal Sciences of Xinjiang University,China~~
文摘[Objective] To introduce an improved method for DNA extraction from the faeces of red deer. [Method] Based on the traditional method of CTAB lysis, we proposed an improved DNA extraction method according to the characteristics of red deer faeces. [Result] This improved method extracted high-quality fecal DNA from Tianshan red deer and amplified the mitochondrial cytochrome b gene. With the muscle and fur DNA of red deer as the control, the sequencing results further con- firmed the reliability of the method. [Conclusion] The method requires no proteinase K in the process of extraction, and the extracted DNA can be used for PCR ampli- fication directly without the purification of DNA purification kit, thus, it is cost-saving.
基金Supported by the High-level Talents Start-up Fund of Shihezi University(07002-500002061401)~~
文摘[Objective] The aim of this study is to isolate Chlorella vulgaris(chlorella)and extract its genomic DNA.[Method] Both the dilution method and drip method were employed to isolate chlorella from lake water samples;the conditions for culturing chlorella were optimized and its genomic DNA was extracted by improved CTAB method and SDS method.[Result] The proper conditions for chlorella culture were as following:temperature 20-25 ℃,illumination 4.39-5.86 W/m2 and rotational speed 100-150r/min;improved CTAB method was suitable for extracting genomic DNA from chlorella.[Conclusion] The study is helpful to study the chlorella at molecular level and promote the exploitation and utilization of chlorella resources.
基金Supported by Key Technology R&D Program of Tianjin(10ZCKFNC00100)National Key Technology R&D Program(2007BAD42B03)~~
文摘[Objective] This study was to find out a quick,simple,and low-cost method for the extraction of sorghum genomic DNA.[Method] Four plant genomic DNA extraction methods based on CTAB,including liquid nitrogen grinding method（method I）,buffer grinding method（method II）,drying grinding method（method III）and directly grinding method（method IV）,were used to extract the sorghum genomic DNA from leaves;further the quantity and quality of the yielded DNA were detected by gel electrophoresis,SSR-PCR and SRAP-PCR.[Result] These four methods performed no remarkable difference in DNA product.The method I and method II produced DNA with higher purity and better integrity,which,especially from method I,is effective for SRAP-PCR and SSR-PCR.While the DNA extracted via method III and method IV had less integrality and lower purity,and only effective in SSR-PCR.[Conclusion] Enough amount of sorghum genomic DNA to perform tens of PCR could be quickly extracted using all these four methods.The DNA obtained via method I and method II had a broader application spectrum（SRAP,RAPD,ISSR and SSR）than that via method III and method IV which is only proper for PCR targeting small DNA fragments（SSR）.
基金Supported by Applied Basic Research Project of Yunnan Province(2010ZC089)the948Project of National Forestry Bureau(2008-4-11)+1 种基金Sharing Platform Project of Provincial and Ministerial Key Subject,Key Laboratory and School Laboratory of Provincial Colleges and Universities in Yunnan ProvinceScience and Technology Innovation Fund of Southwest Forestry University~~
文摘[Objective] This study aimed at comparing the four extraction methods of genomic DNA from Clematis fasciculiflora Franch and determining the optimal extraction method for extracting the genomic DNA from Clematis fasciculiflora Franch.[Method] Leavies of Clematis fasciculiflora Franch were used as materials for comparing the purity and concentration of extracted DNA and extracting time among the four extraction methods of genomic DNA including improved CTAB method Ⅰ,improved CTAB method Ⅱ,improved CTAB method Ⅲ and improved SDS method.[Result] The four extraction methods could all be successfully used for extracting the genomic DNA from Clematis fasciculiflora Franch.The purity of genomic DNA was the highest using improved CTAB method Ⅰ,with the longest extracting time;while the concentration of genomic DNA was the maximum using the improved SDS method,with the shortest extracting time and relatively low purity;the extracting time of improved CTAB method Ⅲ was the shortest.[Conclusion] This study had established the optimal extraction method for extracting the genomic DNA from Clematis fasciculiflora Franch and supported for the further research using molecular biological methods.
基金Supported by National Natural Science Foundation of China(31301372)Key Project of Science and Technology Plan of Zhejiang Province(2011C12030)Innovation Training Project of Zhejiang Agriculture and Forestry University(201301004)~~
文摘To rapidly obtain high-quality genomic DNA from Chenopodium quinoa Willd, the genomic DAN in different tissues （leaves, stems and roots） of Chenopodi- um quinoa Willd was extracted by modified CTAB method, SDS method and high- salt Iow-pH method, respectively. The quality and yield of extracted DNA was deter- mined using agarose gel electrophoresis and UV spectrophotometry. At the same time, the PCR-SSR and SSCP molecular detection was also performed. The results showed that the gel test strips, without obvious decomposition, of all the extraction methods were relatively obvious; the genomic DNA yield extracted by modified CTAB method was highest, followed by that by SDS method, and the genomic DNA extracted by high-salt Iow-pH method was lowest： the genomic DNA yields extracted by different methods from Chenopodium quinoa Wiltd leaves were all high- er than those from roots and stems; the quality of Chenopodium quinoa Willd ge- nomic DNA extracted by modified CTAB method and high-salt Iow-pH method was better, and polyphenols, polysaccharides and other impurities were removed more completely. The PCR-SSR and SSCP detection results showed that the genomic DNA extracted by different methods from different tissues of Chenopodium quinoa Willd all could be better amplified, and high-quality strips could be obtained. So the Chenopodium quinoa Willd genomic DNA extracted by the three methods all can be used for subsequent molecular biology research.
基金Supported by Anhui Natural Science Foundation(090411004)General Administration of Quality Supervision,Inspection and Quarantine of the People's Republic of China Project 2006IK110)Japanese Science Promotion Society Project(P06578)~~
文摘[Objective] The aim was to optimize the mass and rapid method for DNA extraction of Beauvena bassiana. [Method] Boiling water DNA extraction method was improved, DNA extraction liquid was heated by PCR instrument and the extraction process was finished rapidly. [ Resuit] The quality of DNA obtained through mass and rapid extraction of fungal genomic DNA could meet the requirement of RAPD amplification analysis. The clear bands were amplified from 22 tested strains, the number of clear bands were different in the range of 2 -6 and the size of band were mainly concentrated in 450 -800 bp. The DNA extracted by this method also could completely meet the requirement of SCAR amplification. The amplified specific DNA bands used to mark the strain F263 were very clear. [Conclusion] This research provided relatively perfect method for mass and rapid extraction of fungal clenomic DNA.
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 genornic 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 0.104）.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 pmmol·L^-1, and the reaction program was as： initial denaturation for 5 min at 94℃, 30 cycles of denaturation for 30 s at 94℃,annealing for 30 s at 51℃, extension for 30 s at 72℃, and a final 7 min extension at 72℃.
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.
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.
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supported by the National Natural Science Foundation of China (Nos. 40825017, 40576001 and 31100369)
文摘In order to thoroughly investigate the diversity of glacier microorganisms, four DNA extraction methods with differem lysis pat- terns were tested and two screened methods （the Bosshard-Bano method and the Zhou method） were optimized for the most ef- fective form of the filter membrane （cut vs. uncut）, the DNA extraction method, and the precipitation method. The two optimized methods were then compared with the commercial Mo-Bio DNA extraction kit, and the results showed that the kit was generally suitable for extraction of microorganism DNA fi＇om glacier surface snow. Procedurally, it was found that a modified Boss- hard-Bano method （i.e., cutting the filter membrane into pieces, using a specific lysis pattern [lysozyme （5 mg/mL）-protease K （ 1 mg/mL）-CTAB （ 1%）-SDS （ 1%）], performing the extraction only once by chloroform-isoamyl alcohol （24： 1）, and conducting DNA precipitation by pure ethanol） was also an effective and less expensive method for extraction of microorganism DNA from glacier surface snow.
文摘Polyphenols,teroens,and resinsmake it difficult to obtain high qualitygenomic DNA from rice.Four ex-traction methods were compared inour study,and CTAB precipitationwas the most practical one.Materials used were old leavesfrom Ftransgenic rice in Hainan andfresh leaves from Ftransgenic rice inKunming.Procedures of the four methodswere as follows:1.Basical method:powder 1 gold or new leaves→added 2 ml ex-traction solution(100 mmol/L
基金Project of Key Laboratory of Biological Resource Protection and Utilization in Hubei Province(2007025)Open Fund of Hubei Key Laboratory of Biotechnology in Traditional Chinese Medicine in Hubei University(20060201)Project of Hubei Institute for Nationalities~~
文摘[ Objective ] The objective of this study is to explore a rapid and efficient method of extracting genomic DNA from Artemisia abrotanum. [ Method] Three methods of Cutting Method, Liquid Nitrogen Method and Quartz Sand Method based on SDS method were employed to extract Artemisia abrotanum genomlc DNA from tender leaf at seedling stage, tender spike and old leaf at heading stage. The obtained DNAs were detected by absorbance detection, agarose gel and PCR amplification. [ Result ] Cutting Method performed better than the other two methods compared in purity, extracting cycle and cost, accordingly more suitable for PCR amplification. The results also show that young spike is the best material for extracting genomic DNA from Artemisia Annua.
基金Supported by Natural Science Foundation of Shanxi Province(2007011081 )Returning Brains Project in Shanxi Province(2007066 )Agricultural Science and Technology Achievement Transformation Fund Project(2008GB2A300032)~~
文摘[ Objective] The aim was to establish the method of extracting genomic DNA from sheep blood clot on the basis of the improvement of method for extracting genomic DNA from tissues. [Method]The genomic DNA with complete primary structure and high purity was obtained from the sheep blood clot after the steps of cutting the sheep blood clot with ophthalmic scissors, cell lysis with tissue DNA extracts and digested by proteinase K, extracting with phenol/chloroform and precipitating with ethanol were performed. [ Result] The concentration of the extracted DNA was 159.90 ±0.70 ng/μl and the ratio of the A260/A280 was 1.80 ＋0.01. The sheep microsatellite locus of BM203 was amplified by using the extracted DNA from the sheep blood clot as template of PCR, and the PCR result was perfect. [Conclusion]This method is simple and feasible, the quantity and quality of the extracted DNA can satisfy the demands for the subsequent researches. It is worth to extending and using for reference.
基金Supported by Special Fund for Agro-scientific Research in the Public Interest of China(201303008)Science and Technology Plan Project of Guangdong Province(2012B020301006)Key Breeding Project for Special Maize of Department of Agriculture of Guangdong Province(B3071328)~~
文摘The crushing of leaves is the most time and effort-consuming part in DNA extraction which is a fundamental step in the study of molecular biology. In this study, a new rapid and batch-oriented crushing method for DNA extraction from maize leaves was developed. In addition, the practicability of the developed method in molecular marker-assisted breeding was verified using SSR molecular maker technology so as to provide a rapid, batch-oriented, low-cost and non-toxic leafcrushing method for a large number of molecular marker tests, improving test efficiency.
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.
基金Supported by the National Natural Science Foundation of China(No. 30371328), the Key Project of Natural Science Foundationof Shandong Province (No. Z2002C01), and the Key Project ofShandong Academy of Medical Sciences (No. 2005007)
文摘AIM： To rapidly quantify hepatitis B virus （HBV） DNA by real-time PCR using efficient TaqMan probe and extraction methods of virus DNA. METHODS： Three standards were prepared by cloning PCR products which targeted S, C and X region of HBV genome into pGEM-T vector respectively. A pair of primers and matched TaqMan probe were selected by comparing the copy number and the Ct values of HBV serum samples derived from the three different standard curves using certain serum DNA. Then the efficiency of six HBV DNA extraction methods including guanidinium isothiocyanate, proteinase K, NaI, NaOH lysis, alkaline lysis and simple boiling was analyzed in sample A, B and C by real-time PCR. Meanwhile, 8 clinical HBV serum samples were quantified. RESULTS： The copy number of the same HBV serum sample originated from the standard curve of S, C and X regions was 5.7 × 10^4/mL, 6.3 × 10^2/mL and 1.6 × 10^3/ mL respectively. The relative Ct value was 26.6, 31.8 and 29.5 respectively. Therefore, primers and matched probe from S region were chosen for further optimization of six extraction methods. The copy number of HBV serum samples A, B and C was 3.49 × 10^9/mL, 2.08 × 10^6/mL and 4.40 × 10^7/mL respectively, the relative Ct value was 19.9, 30 and 26.2 in the method of NaOH lysis, which was the efficientest among six methods. Simple boiling showed a slightly lower efficiency than NaOH lysis. Guanidinium isothiocyanate, proteinase K and NaI displayed that the copy number of HBV serum sample A, B and C was around 10^S/mL, meanwhile the Ct value was about 30. Alkaline failed to quantify the copy number of three HBV serum samples. Standard deviation （SD） and coefficient variation （CV） were very low in all 8 clinical HBV serum samples, showing that quantification of HBV DNA in triplicate was reliable and accurate. CONCLUSION： Real-time PCR based on optimized primers and TaqMan probe from S region in combination with NaOH lysis is a simple, rapid and accurate method for quantification of HBV serum DNA.
基金Supported by National Natural Science Foundation of China(31060042)Applied Basic Research Project of Yunnan Province(2009ZC079M)
文摘[Objective] This study aimed to explore a method for plant genomic DNA extraction, to provide guidance for rapid extraction of genomic DNA from plant tissues. [ Method] Based on the published genomic DNA extraction methods, operation steps, reagent amount and processing time of SDS extraction method were optimized. [Result] A widely applicable method was established initially for genomic DNA extraction from various varieties of plants and various kinds of plant tissues. Quality of the extracted genomic DNA was relatively good, which meets the requirements for further operation. [ Conclusion] This study provided guidance for rapid plant genomic DNA extraction.
