Method For Isolation Of DNA Research Articles

Molecular detection of Toxoplasma gondii in natural surface water bodies in Poland.

The aim of this study was molecular detection of Toxoplasma gondii in 36 natural surface water bodies in Poland, including preliminary genotype identification and determination of co-occurrence of this parasite with other protozoa that have been detected in previous studies. The examined DNA samples were obtained before to detect Cryptosporidium, Giardia and free-living amoebae. Nested polymerase chain reaction (PCR) based on B1 gene and sequencing was performed for both confirmation of parasite presence in water and genotype identification. T. gondii DNA was detected in 19.4% (7/36) water bodies, while in the case of other studies, T. gondii prevalence ranged between 0% and over 56%. These differences may be caused by natural variations in T. gondii occurrence as well as different sample volumes and methods of sample processing or DNA isolation and detection. Two cases of double contamination were reported: T. gondii with Cryptosporidium parvum and T. gondii with potentially pathogenic Acanthamoeba T4 genotype, thus there is a possibility of mixed infection in humans after occasional contact with water. Obtained T. gondii strains were genetically identical or closely similar (99.8%) to RH strain representing genotype I, however, further examinations involving more loci will be conducted to identify the genotype.

Uji Efektivitas Metode Isolasi DNA Genom Kopi Arabika (Coffea arabica L.) Asal Kabupaten Jayawijaya

Genetic substance of DNA has many functions as a basic component of the organism. DNA can be obtained directly through the isolation of DNA. Isolation of genomic DNA Wamena arabica coffee is done by treating the young leaves to get DNA extract. This reasearch is intended to provide scientific contributions in an effort to screen the best methods of DNA isolation, including a modified extraction of Zheng et al method (2005), a modified method of Doyle and Doyle (1990), and method of George and Khan modificaation (2008) or CTAB method. All of methods were tested on Arabica coffee Jayawijaya Wamena. Testing was done by looking at the difference in quality and quantity of products in the form of genomic DNA concentration and DNA thickness, comparing with the marker DNA then electrophoresis on agarose gel. From the results of testing the effectiveness of three types of isolation methods, it was found that the method of George and Khan (2008) or CTAB genomic DNA  produce the best quality than other methods. In terms of quantity, the criteria in the form of DNA concentrations ranging from 100 ng λ-DNA/ml, λ-DNA 50 ng/ml, λ-DNA 10 ng/ml. Concentration of genomic DNA bands visible when the profile is visualized in gel electrophoresis with UV luminescence. This study will be a step and initial information about the genetic composition of a population of arabica coffee which still exists, and will be developed through DNA amplification technique. Key words: coffee, isolation of genomic DNA, CTAB method, profile band pattern

Bovine foetal sex determination-Different DNA extraction and amplification approaches for efficient livestock production.

Foetal sex determination using polymerase chain reaction (PCR) in mammals is based on the amplification of gender-specific foetal DNA sequences circulating in maternal blood. The bovine synepitheliochorial placenta does not allow a direct contact between the trophoblast and the maternal blood, resulting in difficult passage of foetal DNA and, consequently, its very small amounts in maternal bloodstream. Circulating cell-free foetal DNA (ccffDNA) encompasses short nucleotide fragments (300-600bp) in maternal circulation. The aim of this study was to assess this non-invasive method in accurate prenatal sexing in early and late gestational periods in comparison with ultrasound diagnostics. As various DNA isolation and amplification methods were tested, their success in obtaining reliable results was evaluated. Two groups were tested, each consisting of 20 pregnant cows. Blood of a bull and a non-pregnant heifer was the controls. Extraction of foetal DNA was accomplished by three different methods: using tubes with silicone membranes, a single-tube extraction without silicone membranes and phenol-chloroform extraction. Following each extraction method, foetal DNA was amplified using PCR and real-time PCR with both bAML and TSPY primers in a separate reaction. Positive results were obtained only after amplification of foetal DNA extracted with a single-tube extraction kit. In comparison with ultrasound examination results and foetal gender recorded at birth, the sensitivity of the PCR test was 90% in Group I, but the technique failed to detect male foetuses in Group II. The real-time PCR test sensitivity in Group I was 90% and in Group II 91.6%.

Culture purification and DNA extraction procedures suitable for next-generation sequencing of euglenids

In the present study, five different DNA extraction procedures were examined to determine their effectiveness for extracting DNA suitable for NGS applications. This included two silica-membrane spin column kits, phenol:chloroform, and two CTAB-based methods. Spectrophotometric and fluorimetric measurements as well as standard gel electrophoresis were used as criteria for evaluating the quantity and quality of the isolated DNA prior to the sequencing. Herein, the method of establishing and maintaining axenic Euglena cultures is also presented. The modified CTAB-based method proved to be highly efficient. In terms of DNA quantity and purity (according to the absorbance ratios), the chosen method resulted in DNA of high molecular weight and quality, which fulfills the library construction requirements. Genomic DNA of Euglena hiemalis (CCAP 1224/35) and E. longa (CCAP 1204-17a) isolated using the suggested protocol had been successfully sequenced on the Illumina HiSeq platform. A modified, rapid CTAB-based method of total DNA isolation from Euglena has been described. In terms of the DNA quantity and quality, the protocol devised involving the washing step with DMSO:acetonitrile proved superior to the commonly used, commercially manufactured kits and isolation with phenol:chloroform. The method is also less labor-intensive and time-consuming than the traditional CTAB-based protocol.

Isolation of DNA from Mouse Tails without Extraction by Organic Solvents.

This protocol describes a variation of a simple method for isolation of DNA from mouse tails that does not require extraction with phenol:chloroform. This variant is useful when processing very large numbers of samples.

Comparison of detergent and CTAB method for isolation of DNA from Salak ( Salacca zalacca (Gaert.) Voss. ‘Pondoh’)

This study conducted in Laboratory of Molecular Biology, Balai Besar Bioteknologi dan Sumberdaya Genetik Pertanian (BB- Biogen) Bogor. The aims of this study are to determine and comparing the quantity, quality and the efficiency of DNA isolation result using detergent method and CTAB method. The parameters observed in this study are the value of DNA concentration, purity, and visualization result using gel electrophoresis. The samples are the leaves of Salak ‘Pondoh’ (Salacca zalacca (Gaert.) Voss.). Detergent method is a method which was developed by Faculty of Biology UGM, it has simple method and relatively affordable cost. Meanwhile, CTAB method is one of the commonly used methods of DNA isolation protocol with relatively expensive cost. Detergent method used detergent in the cell wall separation and protein removal in the sample. The CTAB method used Cetyltrimethyl ammonium bromide (CTAB) for cell membrane separation in the sample. The research methods included DNA isolation with detergent and CTAB methods, PCR analysis and electrophoresis. Data analysis was done quantitatively using spectrophotometric method and qualitative used electrophoresis method. The result of the study showed that DNA isolation using CTAB method showed higher purity compared with detergent method with the purity values ranging from 1,3- 1,4 . Meanwhile, the concentration of DNA in the detergent method was higher than that of CTAB with the highest concentration of 1730 µg/ml. There is no difference between the quality of genomic DNA isolated by CTAB and detergent methods.

Determining the optimal method for DNA isolation from fruit jams

DNA extraction is a crucial step in PCR analysis especially when analysing food samples that can be degraded and can potentially contain PCR-inhibiting substances. In this study, we compared the suitability of three DNA extraction methods – two kits: DNeasy<sup>®</sup> Plant Mini Kit and NucleoSpin<sup>®</sup> Food, and the CTAB method – for DNA extraction from commercial fruit jams. Fourteen jams with different contents of fruit, sugar and other additives were extracted in triplicate using the above-mentioned methods directly and after a washing step. The concentration and optical density were analysed using UV spectrophotometry and the amplifiability of the obtained DNA was evaluated using a PCR assay targeting a sequence coding for chloroplast tRNA-Leu. Samples isolated using the NucleoSpin<sup>®</sup> Food kit contained non-amplifiable DNA in eight cases, and samples isolated using the CTAB method could not be quantified. The DNeasy<sup>®</sup> Plant Mini Kit thus proved to be the most suitable method, since well-amplifiable DNA was obtained for all the analysed samples.

A silica sands-based method for faithful analysis of microbial communities and DNA isolation from a wide range of species

ABSTRACTA silica sands-based method has been developed to isolate high quality genomic DNAs from cells of animals, plants and microorganisms, such as Hemisalanx prognathus, Spinacia oleracea, Pichia pastoris, Bacillus licheniformis and Escherichia coli. To the best of our knowledge, no DNA isolation method has so wide application until now. In addition, this method and a commercially available kit were compared in analysis of microbial communities using high-throughput 16s rDNA sequencing. As a result, the silica sands-based method was found to be even more efficient in isolating genomic DNA from gram-positive bacteria than the kit, indicating that it would become a very valuable choice to faithfully reflect the composition of microbial communities.

Two efficient methods for isolation of high-quality genomic DNA from entomopathogenic fungi

Conventional and commercial methods for isolation of nucleic acids are available for fungal samples including entomopathogenic fungi (EPF). However, there is not a unique optimal method for all organisms. The cell wall structure and the wide range of secondary metabolites of EPF can broadly interfere with the efficiency of the DNA extraction protocol. This study compares three commercial protocols: DNeasy® Plant Mini Kit (Qiagen), Wizard® Genomic DNA Purification Kit (Promega), and Axygen™ Multisource Genomic DNA Miniprep Kit (Axygen) and three conventional methods based on different buffers: SDS, CTAB/PVPP, and CTAB/β-mercaptoethanol versus three cell lysis procedures: liquid nitrogen homogenization and two bead-beating materials (i.e., tungsten-carbide and stainless-steel) for four representative species of EPF (i.e., Beauveria bassiana, Hirsutella citriformis, Isaria javanica, and Metarhizium anisopliae). Liquid nitrogen homogenization combined with DNeasy® Plant Mini Kit (i.e., QN) or SDS buffer (i.e., SN) significantly improved the yield with a good purity (~1.8) and high integrity (>20,000 bp) of genomic DNA in contrast with other methods, also, these results were better when compared with the two bead-beating materials. The purified DNA was evaluated by PCR-based techniques: amplification of translation elongation factor 1-α (TEF) and two highly sensitive molecular markers (i.e., ISSR and AFLP) with reliable and reproducible results. Despite a variation in yield, purity, and integrity of extracted DNA across the four species of EPF with the different DNA extraction methods, the SN and QN protocols maintained a high-quality of DNA which is required for downstream molecular applications.

Development of a Membrane-Based Method for Isolation of Genomic DNA from Human Blood.

High quality and sufficient quantity of genomic DNA (gDNA) are the primary requisites of several molecular biologic applications, including clinical studies related to genetics, genomics, gene polymorphism, and DNA fingerprinting. Whole blood is the primary source of gDNA in most of the clinical investigations. Currently, commercial kits are primarily used to achieve these goals. However, the use of kits is limited by the cost and involvement of several centrifugal steps. Other methods reported are either laborious or do not produce high quality or quantity of gDNA or both. Here, we present the data on the development of a centrifugation-free, cost-effective, and user-friendly method for the isolation of human gDNA from the buffy coat of human blood that involves limited numbers of steps with about 15 min of hands-on time per sample.

Robust DNA Isolation and High-throughput Sequencing Library Construction for Herbarium Specimens

Herbaria are an invaluable source of plant material that can be used in a variety of biological studies. The use of herbarium specimens is associated with a number of challenges including sample preservation quality, degraded DNA, and destructive sampling of rare specimens. In order to more effectively use herbarium material in large sequencing projects, a dependable and scalable method of DNA isolation and library preparation is needed. This paper demonstrates a robust, beginning-to-end protocol for DNA isolation and high-throughput library construction from herbarium specimens that does not require modification for individual samples. This protocol is tailored for low quality dried plant material and takes advantage of existing methods by optimizing tissue grinding, modifying library size selection, and introducing an optional reamplification step for low yield libraries. Reamplification of low yield DNA libraries can rescue samples derived from irreplaceable and potentially valuable herbarium specimens, negating the need for additional destructive sampling and without introducing discernible sequencing bias for common phylogenetic applications. The protocol has been tested on hundreds of grass species, but is expected to be adaptable for use in other plant lineages after verification. This protocol can be limited by extremely degraded DNA, where fragments do not exist in the desired size range, and by secondary metabolites present in some plant material that inhibit clean DNA isolation. Overall, this protocol introduces a fast and comprehensive method that allows for DNA isolation and library preparation of 24 samples in less than 13 h, with only 8 h of active hands-on time with minimal modifications.

Robust DNA Isolation and High-throughput Sequencing Library Construction for Herbarium Specimens.

Herbaria are an invaluable source of plant material that can be used in a variety of biological studies. The use of herbarium specimens is associated with a number of challenges including sample preservation quality, degraded DNA, and destructive sampling of rare specimens. In order to more effectively use herbarium material in large sequencing projects, a dependable and scalable method of DNA isolation and library preparation is needed. This paper demonstrates a robust, beginning-to-end protocol for DNA isolation and high-throughput library construction from herbarium specimens that does not require modification for individual samples. This protocol is tailored for low quality dried plant material and takes advantage of existing methods by optimizing tissue grinding, modifying library size selection, and introducing an optional reamplification step for low yield libraries. Reamplification of low yield DNA libraries can rescue samples derived from irreplaceable and potentially valuable herbarium specimens, negating the need for additional destructive sampling and without introducing discernible sequencing bias for common phylogenetic applications. The protocol has been tested on hundreds of grass species, but is expected to be adaptable for use in other plant lineages after verification. This protocol can be limited by extremely degraded DNA, where fragments do not exist in the desired size range, and by secondary metabolites present in some plant material that inhibit clean DNA isolation. Overall, this protocol introduces a fast and comprehensive method that allows for DNA isolation and library preparation of 24 samples in less than 13 h, with only 8 h of active hands-on time with minimal modifications.

One-Tube Isolation of DNA from Mouse Tails.

This protocol describes a variation of a simple method for isolation of DNA from mouse tails that uses commercially available gel-barrier tubes to eliminate the tedious transfer of samples during serial extractions with organic solvents. This variant is useful when processing very large numbers of samples.

An Alternative Method for Extracting Plasmodium DNA from EDTA Whole Blood for Malaria Diagnosis.

Molecular techniques have been introduced for malaria diagnosis because they offer greater sensitivity and specificity than microscopic examinations. Therefore, DNA isolation methods have been developed for easy preparation and cost effectiveness. The present study described a simple protocol for Plasmodium DNA isolation from EDTA-whole blood. This study demonstrated that after heating infected blood samples with Tris–EDTA buffer and proteinase K solution, without isolation and purification steps, the supernatant can be used as a DNA template for amplification by PCR. The sensitivity of the extracted DNA of Plasmodium falciparum and Plasmodium vivax was separately analyzed by both PCR and semi-nested PCR (Sn-PCR). The results revealed that for PCR the limit of detection was 40 parasites/μl for P. falciparum and 35.2 parasites/μl for P. vivax, whereas for Sn-PCR the limit of detection was 1.6 parasites/μl for P. falciparum and 1.4 parasites/μl for P. vivax. This new method was then verified by DNA extraction of whole blood from 11 asymptomatic Myanmar migrant workers and analyzed by Sn-PCR. The results revealed that DNA can be extracted from all samples, and there were 2 positive samples for Plasmodium (P. falciparum and P. vivax). Therefore, the protocol can be an alternative method for DNA extraction in laboratories with limited resources and a lack of trained technicians for malaria diagnosis. In addition, this protocol can be applied for subclinical cases, and this will be helpful for epidemiology and control.

DNA extraction from hydatid cyst protoscolices: Comparison of five different methods.

Aim:The current study aimed to find out a simple, practical and high throughput DNA isolation method for extraction of DNA from hydatid cyst samples.Materials and Methods:Cattle and sheep isolate of hydatid cysts were obtained from the slaughterhouse, and hydatid fluid and protoscolices were collected in a sterile condition. Protoscolices were washed, 3 times with phosphate buffered saline, and DNA was extracted by different methods including manual extraction with freeze/thawing and phenol-chloroform, Triton X-100 extraction, and by a commercial kit (YTA, Yekta Tajhiz Azma, Iran) with three different modifications in the kit’s manufacturer instructions. The obtained DNA from the different methods was evaluated by Nanodrop in terms of the yield of DNA and carbohydrates or protein contaminations. To compare the quality of the extracted DNA, two pieces of the mitochondrial genome of Echinococcus granulosus, cox1, and nad1, were polymerase chain reaction (PCR)-amplified, using each of the DNA prepared by different methods. Electrophoresis of PCR products was carried out on the agarose gel.Results:The DNA extracted by manual method, using phenol/chloroform, had the highest yield, yet with the highest level of protein and carbohydrate contamination. The DNA extracted using two-step incubations, initially at 60°C for 2 h and then overnight at 37°C, was the most purified DNA with the lowest rate of contamination.Conclusion:Findings of the study demonstrated that modification in the currently available commercially DNA extraction kit resulted in the development of a high throughput DNA isolation method. This method can be recommended for the extraction of DNA from hydatid cysts, especially the cattle isolate where the extraction of DNA in these samples are usually problematic.

Blood Collection and Cell-Free DNA Isolation Methods Influence the Sensitivity of Liquid Biopsy Analysis for Colorectal Cancer Detection.

During colorectal cancer (CRC) development tumor-derived cell-free DNA (cfDNA) can be released into the bloodstream. Many different cfDNA isolation methods and specific blood collection tubes preventing the release of genomic DNA and stabilizing cfDNA with preservative reagents became available. These factors may affect greatly on the further liquid biopsy analyses. Our aim was to test different blood collection tubes and cfDNA isolation methods to determine whether these factors influence the cfDNA amount and the promoter methylation of four previously described hypermethylated biomarkers. Three manual isolation methods (High Pure Viral Nucleic Acid Large Volume Kit; Epi proColon 2.0 Kit; Quick-cfDNA™ Serum & Plasma Kit) and automated sample preparation systems (InviGenius and InviGenius PLUS) were examined. Furthermore, K3EDTA Vacuette tubes and Streck Cell-Free DNA BCT® tubes were compared. After cfDNA isolation and bisulfite conversion of samples, the methylation level of SFRP1, SFRP2, SDC2, and PRIMA1 were defined with MethyLight assays. We have ascertained that there are differences between the cfDNA amounts depending on the isolation methods. Higher cfDNA yield was observed using InviGenius system than column-based manual isolation method; however, InviGenius PLUS has produced lower cfDNA amounts. No remarkable variance could be found between K3EDTA and Streck tubes; slightly higher cfDNA quantity was detected in 60% of plasma samples using Streck tubes. In point of methylation level and frequency, manual column-based isolation produced more consistent results. Automated cfDNA extraction systems are easy-to-use and high-throughput; however, further improvements in the isolation protocols might lead to the increase of the sensitivity of furthermethylation analysis.

Comparison of CTAB Method and Wizard Genomic DNA Purification System Kit from Promega on DNA Isolation of Local Varities of Rice of South Sumatera

Research in the molecular field requires DNA with a high degree of purity. Local rice varieties of South Sumatra have different leaf texture so that in isolation process to obtain DNA with high purity level required the right method. This study aims to compare the quality and quantity of purity of local rice insulation of local rice varieties of South Sumatra with different methods of CTAB and Wizard Genomic DNA Purification System Kit from Promega. The research was conducted from August 2015 to December 2015 at the Laboratory of Microbiology and Genetics and Biotechnology Laboratory, Department of Biology, Faculty of Mathematics and Natural Sciences, Sriwijaya University. The method used is DNA isolation method Wizard Genomic DNA Purification System Kit from promega and CTAB. Based on research that has been done result of DNA visualization showed that DNA isolate CTAB method still have smears while DNA isolate method of Wizard Genomic DNA Purification System Kit from promega not. The average DNA isolate concentration of Wizard Genomic DNA Purification System Kit method is A260 / 280 = 1.853 μg / ml. The average DNA isolate concentration of CTAB method is A260 / 280 = 1,705 μg / ml. Isolation of Rice DNA of local variety of South Sumatera using DNA method of Genomic DNA Purification System Kit from promega has higher quality and quantity compared to CTAB method.

A Simple and Rapid DNA Isolation Method from Dry Pea Seeds Suitable for PCR Analyses

Pea (Pisum sativum L.) is an important legume grown and consumed extensively worldwide. As a rich source of proteins, carbohydrates and vitamins, peas are important in human nutrition. In this study, we compared four different DNA isolation methods from four pea cultivars (F95-927, Specter, Windham and Nicoleta), with some modifications to the original extraction protocols: one SDS extraction buffer without PVP and one SDS extraction buffer with 1% PVP; one CTAB extraction buffer without PVP and one with 1% (1.5%) PVP. For all four extraction methods we used the same quantity of plant material (0.05 g) and an equal quantity of extraction buffer, so the comparison between methods to be very accurate. To establish which is the most efficient extraction method, after DNA isolation and purification, we submitted our samples to PCR analyzes with two markers: ISSR marker 17899A and SSR marker AA175. In my study, based on spectrophotometric measurements and PCR results, I concluded that both CTAB extraction methods were not suitable for DNA extraction from dry pea seeds because they did not show amplification products. The most appropriate DNA extraction method was SDS1 which provided a good quality DNA.

Planarian High Molecular Weight DNA Isolation bySpooling.

High-quality large molecular weight genomic DNA is important for genomic studies. Most commercial available genomic DNA purification kits have failed to generate high molecular weight DNA of sufficient quality from planarians. Here, we describe a simple and efficient genomic DNA isolation method, which has worked for several different planarian species, including Schmidtea mediterranea. This phenol-chloroform based method can be used to obtain genomic DNA of up to 150kb and can be used for bacterial artificial chromosome (BAC) library construction, next-generation sequencing and PCR cloning.

DNA Isolation and Optimization of ISSR-PCR Reaction System in Oryza sativa L.

Inter simple sequence repeats (ISSRs) have been utilized widely for molecular markers in analyzing the genetic diversity and phylogenetic and regions in the genome flanked by microsatellite sequences. PCR amplification of these regions using a single primer yields multiple amplification products that can be used as a dominant multilocus marker system for the study of genetic variation in various organisms. For this study provides, DNA isolation, adjusting in six factors (Buffer, MgCl2, dNTPs, ISSR primers, Template DNA and Taq polymerase ) at six levels, and optimization of PCR temperature for the ISSR reaction was 60-45 °C, primers screening on indica rice ( Oryza sativa ). In this research, simple method of DNA isolation by using seedling. The objective of the present investigation was to assess the optimizations and quantification. Has been shown that stalk enhanced the maximum value of genomic. The results show that 100 ISSR primers were examined as well as, 56 ISSR primers was productively amplified. Optimum components for PCR reactions were 5.0 μl of 5X PCR Buffer, 1.5 μl of 25mM MgCl2, 1 μl of 10 mM dNTP, 1 μl of 10 Μm ISSR primers, 2 μl Template DNA, and 0.1 μl of 5 units/ml Taq polymerase. Based on this study, has brought out some information on the relationship between these ISSR primers will be applied further for molecular profiling as well as response evaluation in rice varieties