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
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
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
环境DNA宏条形码(Environmental DNA metabarcoding,eDNA metabarcoding)技术在调查鱼类物种丰度和多样性方面具有快速、高效、环境友好等优势,但其敏感性和有效性需进一步评估。本研究选择位于瓯江水系上游一级支流松荫溪发源地的高碧...环境DNA宏条形码(Environmental DNA metabarcoding,eDNA metabarcoding)技术在调查鱼类物种丰度和多样性方面具有快速、高效、环境友好等优势,但其敏感性和有效性需进一步评估。本研究选择位于瓯江水系上游一级支流松荫溪发源地的高碧溪和成屏水库作为调查水域,分别利用传统捕捞方法和eDNA宏条形码技术对2个水域的鱼类物种进行调查。结果表明:捕捞方法在2个水域共发现鱼类24种,88%鱼类物种(21种)在瓯江水系有记录,其中高碧溪采集到鱼类12种,隶属于4目8科11属,成屏水库采集到鱼类16种,隶属于3目4科14属。eDNA宏条形码技术在2个水域共检测到鱼类31种,有84%的物种(26种)在瓯江水系有记录,其中高碧溪共检测出28种,隶属于5目12科25属,成屏水库共检测出27种,隶属于6目11科25属。然而,2种调查方法在溪流中共同发现的鱼类仅1种,在水库中共同发现的鱼类也只有5种。此外,高碧溪Shannon多样性指数、Simpson多样性指数、Margalef丰度指数以及Pielou均匀度指数均高于成屏水库,且2种调查方式得出的结论较为一致。由此可见,eDNA宏条形码技术比传统调查方法能够发现更多物种,具有更高的敏感性,但与实际捕获的鱼类物种组成存在较大差异,因此本研究认为eDNA宏条形码技术目前仅可以作为传统渔业调查方法的参考依据,利用该技术给出的调查结论需持谨慎态度。展开更多
Injectable drugs manufactured in E. coli must be tested for host residual DNA (hr DNA) impurity in ensuring drug purity and safety. Because of low allowable hr DNA as impurity, highly sensitive methods are needed. Dro...Injectable drugs manufactured in E. coli must be tested for host residual DNA (hr DNA) impurity in ensuring drug purity and safety. Because of low allowable hr DNA as impurity, highly sensitive methods are needed. Droplet digital PCR (ddPCR) is a new method where the reaction is partitioned into about 20,000 nanoliter-sized droplets and each droplet acts as individual PCR reaction. After completion of end-point PCR, droplets are analyzed for fluorescence and categorized as positive or negative and DNA quantified using Poisson statistics. Here we describe development of a direct E. coli hr DNA dd PCR method where the drug is directly added to the ddPCR reaction. We show that the ddPCR method has acceptable precision and high accuracy, works with different biologic drugs, and compared to qPCR shows higher tolerance of drug matrices. The method does not require DNA extraction or standard curves for quantification of hr DNA in unknown samples.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
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.
文摘环境DNA宏条形码(Environmental DNA metabarcoding,eDNA metabarcoding)技术在调查鱼类物种丰度和多样性方面具有快速、高效、环境友好等优势,但其敏感性和有效性需进一步评估。本研究选择位于瓯江水系上游一级支流松荫溪发源地的高碧溪和成屏水库作为调查水域,分别利用传统捕捞方法和eDNA宏条形码技术对2个水域的鱼类物种进行调查。结果表明:捕捞方法在2个水域共发现鱼类24种,88%鱼类物种(21种)在瓯江水系有记录,其中高碧溪采集到鱼类12种,隶属于4目8科11属,成屏水库采集到鱼类16种,隶属于3目4科14属。eDNA宏条形码技术在2个水域共检测到鱼类31种,有84%的物种(26种)在瓯江水系有记录,其中高碧溪共检测出28种,隶属于5目12科25属,成屏水库共检测出27种,隶属于6目11科25属。然而,2种调查方法在溪流中共同发现的鱼类仅1种,在水库中共同发现的鱼类也只有5种。此外,高碧溪Shannon多样性指数、Simpson多样性指数、Margalef丰度指数以及Pielou均匀度指数均高于成屏水库,且2种调查方式得出的结论较为一致。由此可见,eDNA宏条形码技术比传统调查方法能够发现更多物种,具有更高的敏感性,但与实际捕获的鱼类物种组成存在较大差异,因此本研究认为eDNA宏条形码技术目前仅可以作为传统渔业调查方法的参考依据,利用该技术给出的调查结论需持谨慎态度。
文摘Injectable drugs manufactured in E. coli must be tested for host residual DNA (hr DNA) impurity in ensuring drug purity and safety. Because of low allowable hr DNA as impurity, highly sensitive methods are needed. Droplet digital PCR (ddPCR) is a new method where the reaction is partitioned into about 20,000 nanoliter-sized droplets and each droplet acts as individual PCR reaction. After completion of end-point PCR, droplets are analyzed for fluorescence and categorized as positive or negative and DNA quantified using Poisson statistics. Here we describe development of a direct E. coli hr DNA dd PCR method where the drug is directly added to the ddPCR reaction. We show that the ddPCR method has acceptable precision and high accuracy, works with different biologic drugs, and compared to qPCR shows higher tolerance of drug matrices. The method does not require DNA extraction or standard curves for quantification of hr DNA in unknown samples.
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.
