Methodological Studies on Genomic DNA Extraction and Purification from Plant Drug Materials

This paper reports the rationale and methods of DNA extraction and purification from nine species of Compositae and four commercial drugs of corresponding plant Elephantopus scaber. The comparison of three methods: CsCl gradient, CTAB/CsCl gradient and CTAB miniprep extraction by yield, purity and factors affecting PCR was carried out. In conclusion, CTAB miniprep method provides a rapid, effective, economic approach for isolating genomic DNA for Chinese drug identification by genomic fingerprints.

A Rapid DNA Extraction Method for PCR Detection of Arabidopsis thaliana

[Objective] The aim was to introduce a rapid DNA extraction method for PCR detection of Arabidopsis thaliana.[Method] Through the improvement of conventional DNA extraction method,a rapid Arabidopsis thaliana DNA extraction method was obtained.With randomly selected Arabidopsis thaliana transgenic strains and mutants as samples,the method was verified.[Result] After electrophoresis,UV absorption detection,it was found that DNA samples are complete and less pollution,and the result of PCR amplification objective fragment was good which proved DNA is suitable as a template for PCR reaction.After PCR detection,positive plants gene amplified bands were clear,without false-positive,and the test results were satisfactory.[Conclusion] The method is suitable for rapid extraction of Arabidopsis thaliana DNA and PCR detection.

Comparison of Genomic DNA Extraction Methods for Chenopodium quinoa Willd

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.

Comparison on Four Extraction Methods of Genomic DNA from Clematis fasciculiflora Franch

[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.

A Rapid Metagenomic DNA Extraction from Sediments: Potassium Dichromate SDS Method

[Objective] The objective of this study was to report an improved method for rapid DNA extraction from black-order sediments, without any purification step. [Methods] Sediments in eutrophic lake are complex ecosystems and soil microbes involved in anthropogenic nutrient cycling are very important. DNA-based molecular methods offer new tools for characterization of these mixed communities of mi- croorganisms. However, it is very difficult to remove humic substances, heavy met- als that co-existed with genome DNA representing the microbial community directly from these complex systems and can interfere with subsequent genetic analysis. The potassium dichromate solution was firstly used to remove humic substances. [Results] The steps of removing humic substances and DNA extraction were per- formed simultaneously that improved the speed of extraction to approximately 1 hour and the nucleic acids that were obtained with this method did not need to be washed with 70% ethanol and dissolved directly in sterile water for total bacterial 16S rDNA, nosZ gene of denitrifying bacteria, pmoA of methanotrophs, nifH of nitro- gen-fixing bacteria, amoA of ammonia-oxidizing bacteria and ammonia-oxidizing ar- chaea molecular ecology analyses. [Conclusion] This method could provide a plat- form for preparing a fast sediments DNA extraction.

Rapid DNA Extraction Method from Amorphophallas.konjac and ISSR-PCR Amplification

[Objective]The research aimed to study the total DNA extraction methods of Amorphophallas.konjac.[Method]To investigate the optimum DNA extraction method,CTAB,SDS and new modified methods for isolating genomic DNA from Amorphophallas.konjac leaf were studied and the isolated DNAs were examined by ultraviolet absorption test,agarose gel electrophoresis and ISSR-PCR test for comparative analysis.[Result]The results showed that the new modified method was suitable for DNA isolation from Amorphophallas.konjac plants with high purity and yield;quality of DNA could meet ISSR-PCR requirement even extracted once by the new modified method.[Conclusion]The study provided basis for extracting high quality DNA sample,and offered the molecular genetics reference for exploring the genetic diversity of Amorphophallas.konjac.

A New Rapid and Batch-oriented Crushing Method for DNA Extraction from Maize Leaves

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.

DNA Extraction and Molecular Identification of Green Tea

[Objective] The aim of this study was to provide reference for the quality identification of green tea.[Method] Green Tea was used as materials,and its total DNA was extracted through improved CTAB method.And the obtained DNA was used to carry out identification on 10 varieties of green tea through ISSR molecular markers.[Result] The high quality DNA from green tea could be obtained with new method,the DNA yield ranged from 101-498 μg/g tea sample for various green tea samples,and the average yield was 249 μg/g tea sample.The ISSR detection result showed that ISSR markers could effectively differentiate different varieties of green tea.[Conclusion] The result had provided reference for the further study on molecular identification of green tea.

An Improved Method for DNA Extraction from the Faeces of Red Deer

[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.

A Mass and Rapid Method for DNA Extraction of Beauveria bassiana

[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.

Studies on Genomic DNA Extraction and Establishment of AFLP Reaction System in Chinese Cabbage

[Objective] The obtained clear AFLP fingerprint of Chinese cabbage provided basis for studies on the molecular markers of Chinese cabbage cultivars and the phylogenetic relationship among Chinese cabbage cultivars. [Method] With the test materials of leaves of Chinese cabbages, the high-quality total DNA from leaves of Chinese cabbages was extracted by the modified CTAB method. DNA restriction-ligase reaction, pre-amplification and selective amplification were optimized, and the AFLP silver-staining reaction system for Chinese cabbage was established. [Result] The quality of DNA template influenced restriction enzyme digestion and the subsequent ligase amplification reaction, while the modified CTAB extraction method could be used in AFLP analysis of Chinese cabbage to obtain a clear AFLP fingerprint. The optimum conditions for restriction enzyme digestion of genomic DNA from Chinese cabbage were as follows: 150 g DNA template, 12.5 μl reaction volume, 1.25 U Eco R Ⅰ, 1.25 U Mse Ⅰ and 5×Reaction Buffer with 4 h at 37 ℃. The ligation reaction with 2.5 h at 20 ℃ was the optimum condition. Six pairs of primers including E-AAC/M-CAG, E-AAG/M-CAC, E-ACA/M-CTG, E-ACT/M-CAC, E-ACT/M-CTT and E-ACT/M-CTC all had its own stable and clear patterns. [Conclusion] With abundant bands and high polymorphism, AFLP selective amplification is an efficient molecular marker for genomic polymorphism of Chinese cabbage.

Effects of DNA extraction and universal primers on 16S rRNA gene-based DGGE analysis of a bacterial community from fish farming water

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.

CTAB-silica Method for DNA Extraction and Purification from Castanea mollissima and Ginkgo biloba

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.

A Simplified Rice DNA Extraction Protocol for PCR Analysis

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.

Effects of sampling strategies and DNA extraction methods on eDNA metabarcoding: A case study of estuarine fish diversity monitoring

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.

Modified pretreatment method for total microbial DNA extraction from contaminated river sediment

Extraction of high-quality microbial DNA from contaminated environmental samples is an essential step in microbial ecological study. Based on previously published methods for soil and sediment samples, a modified pretreatrnent method was developed for extracting microbial DNA from heavily contaminated river sediment samples via selection of optimal pretreatment parameters （i.e., reagent solution, reaction duration, and temperature）. The pretreatment procedure involves wash ing the river sediment sample for three times with a solution containing 0.1 mol.L-1 ethylene diamine tetra- acetic acid （EDTA）, 0.1 mol- L-1 Tris （pH 8.0）, 1.5 mol. L1 NaC1, 0.1 mol. L-1 NaH2PO4, and Na2HPO4 at 65~C with 180r.min-1 for 15min to remove humic materials and heavy metals prior to the employment of standard DNA extraction procedures. We compared the results of standard procedure DNA extraction following pretreatrnent, without pretreatment, and with using a commercial PowerSoilTM DNA Isolation Kit. The results indicated that the pretreatment significantly improved the DNA quality based on DNA yield, DNA fragment length, and determination of prokaryotic diversity. Prokaryotic diversity exhibited in the DNA with the pretreatment was also considerably higher than that extracted with the Power- SoilTM DNA Isolation Kit only. The pretreatment method worked well even with a small amount of sediment sample （0.25 g or even lower）. The method provides a novel, simple, cost-effective tool for DNA extraction for microbial community analysis in environmental monitoring and remediation processes.

A Simple,Efficient and Rapid Method for Good Quality DNA Extraction from Rice Grains

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.

DNA Extraction from Formalin-fixed and Paraffin-embedded Tissues by Triton X-100 for Effective Amplification of EGFR Gene by Polymerase Chain Reaction

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.

Standardization of DNA Extraction Method from Mature Dried Leaves and ISSR-PCR Conditions for Melia dubia Cav. —A Fast Growing Multipurpose Tree Species

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 MgCl2, 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.

DNA Extraction from Eriocaulon Plants and Construction of RAPD System

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.