Comparison of DNA Isolation Methods that Derived from Leaves of a Potential Anti-Cancer Rodent Tuber (Typhonium flagelliforme) Plant

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

Polymerase chain reaction (PCR) provides a reliable detection of pathogenic bacteria in water samples. However, this method can be adversely influenced by the purity of the DNA template. This is a particularly important obstacle when the bacterial DNA is directly extracted from water samples. In this study we compared the suitability of 8 different methods for isolation of bacterial DNA from pure cultures and 10 different methods for isolation of DNA from water samples. The quality of extracted DNA was assessed by PCR amplification of target sequences derived from uid (E. coli and Shigella sp.), tuf (Enterococcus sp.) and hns (Salmonella sp.). Results indicated that there are differences among the methods tested and only a few of them gave satisfactory results. The method based on alkaline lysis of bacterial suspension, which was developed in our laboratory, seemed to be efficient enough for the detection of bacteria from pure cultures. Detection of bacteria directly from water samples was more difficult. The modified method developed by Slusarenko was found as the best of the tested methods.

Orchid Ph. amabilis is one of the plants designated as "Indonesian National Flower" and designated as "Puspa Pesona Indonesia". Orchid Ph. amabilis has a diversity that is not widely known, so that it is necessary to identify it molecularly. DNA isolation is the first step in molecular testing. This study aims to determine the sample weight, incubation time and the appropriate isolation method for DNA isolation from samples of Ph.amabilis orchid roots. In this study, the DNA isolation stage used two experimental methods, namely the CTAB method (Murray and Thompson) and the mini kit. The research method used was a single factor experiment with a combination of sample weight and incubation time consisting of 6 treatments with 3 replications, so 18 experimental units were obtained. The experimental treatment used was from the roots of the orchid Ph. amabilis which has a sample weight of 0.25 g, 0.5 g, and 0.75 g and each sample weight treatment was incubated for 30 minutes and 60 minutes. In addition, DNA isolation using the kit method had a sample weight of 0.25 g, 0.5 g and 0.75 g and an incubation period of 10 minutes. The results showed that the DNA isolation of Ph. amabilis with a sample weight of 0.75g, incubation time of 30 minutes using the CTAB method showed the best treatment in producing DNA with a concentration of 384 ng/µl and a purity of 1.9. This study is expected to provide information on sample weight, incubation time and the appropriate method for DNA isolation of Ph. amabilis orchid roots.

Recycling Isolation of Plant DNA, A Novel Method

<p>A molecular marker of parent and offspring is used to find fast and accurate markers influenced by DNA isolation and amplification. This research aims to find the most suitable DNA isolation and DNA amplification methods. This study used four DNA isolation methods; namely IM01, IM02, IM03, and IM04. DNA amplification used ten protocols (AP01, AP02, AP03, AP04, AP05, AP06, AP07, AP08, AP09, and AP010). The results of the research showed that the most suitable DNA isolation method for salak was IM0, and the most suitable DNA amplification for salak was AP04 that produces the highest value of DNA bands.</p><p><strong> </strong></p><p>Keywords: DNA isolation; DNA amplification; hybrids.</p>

Since Entamoeba histolytica and Entamoeba dispar were formally recognized as two different species at the World Health Organization (WHO)/Pan American Health Organization (PAHO)/United Nations Educational, Scientific and Cultural Organization (UNESCO) meeting in Mexico City in 1997, the specific differentiation of the two morphologically identical species would seem relevant in clinical diagnosis. Several polymerase chain reaction (PCR)-based methods have been described and used successfully, but methods for DNA isolation from cysts in stool samples are time-consuming and problematic due to inhibitory factors in faeces. The use of the slightly modified QIAamp tissue method (Qiagen, Germany) for DNA isolation was evaluated in 657 unpreserved faecal samples from cases of suspected Entamoeba histolytica/Entamoeba dispar infection. In only 1.7% of the cases was PCR hampered by inhibitors present in the faeces. The DNA isolation procedure was found to be rapid, simple and one that could easily be implemented in a routine diagnostic setting. In 98.8% of Entamoeba histolytica/Entamoeba dispar cyst-positive faecal samples, the true identity of the cysts could be determined using PCR specific for Entamoeba histolytica and Entamoeba dispar, respectively.

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.

Received: 2016-06-25 | Accepted: 2016-07-29 | Available online: 2016-12-22 http://dx.doi.org/10.15414/afz.2016.19.04.144-149 The most suitable method of total DNA extraction still remains the crucial step for many plant species, although there are many different protocols and commercial kits for DNA isolation. In this study, five different extraction protocols were analysed to find out the most appropriate method for DNA extraction from Hedera helix L. This species has numerous medical and pharmaceutical uses and is also characterized by antioxidant effects on human body. In spite of its wide medical utilization, it belongs to those plant species, where the genomic information is very limited. Comparing of different protocols resulted in the yield of extracted DNA that has ranged from 6.3 to 487 ng μl-1. The purity of extracted DNA has ranged from 1.4 up to 2.0 A260/A280. All the extraction methods used in this study were evaluated not only in term of quantity and purity of DNA but also its functionality in the restriction endonuclease digestion and polymerase chain reaction based downstream analysis was performed. Keywords: DNA extraction, CTAB, SDS,commercial kit, Hedera helix L. References ABU-ROMMAN, S. (2011). Comparison of methods for isolating high quality DNA from sage (Salvia officinalis). Journal of Medicinal Plants Research, vol 5, no. 6, pp. 938-941 BALANSARD, G. et al. (1980). Douvicidal and Antifungal Activities of α-Hederin Extracted from Hedera helix leaves. Planta Medica, vol. 39, p. 234. BARRA, M. et al. (2012). Simple and robust DNA extraction mehod for the large-scale analysis of genotypes containing high polyphenolic content, such as landraces of Solanum tuberosum and Zea mays. Plant breeding, genetic and genetic resources, vol. 39, no. 3, pp. 593-601. BEDIR, E. et al. (2000). Triterpene Saponins from the Fruits of Hedera helix. Phytochemistry, vol. 53, pp. 905-909. CLARKE, M.M. et al., (2006). Prevalence of different horticultural taxa of ivy (Hedera spp., Araliaceae) in invading populations. Biological Invasions, vol. 8, pp. 149–157. COTA-SÁNCHEZ, H. J. et al. (2006). Ready-to-Use DNA Extracted with a CTAB Method Adapted for Herbarium Specimens and Mucilaginous Plant Tissue. Plant Molecular Biology Reporter, vol. 24, no. 2, pp.161-167. CRESPIN, F. et al. (1995). Identification of 3O-beta-D-Glucopyranosyl Hederagenin from the Leaves of Hedera helix. Fitoterapia, vol. 66, pp. 47. DABO, S. M. et al. (1993). A method for the isolation of nuclear DNA from cotton (Gossypium) leaves. Analytical Biochemistry, vol. 210, pp. 34-38. DELLAPORTA, S. L. et al. (1983). A Plant DNA Minipreparation: Version II. Plant Mol. Biol. Rep., vol. 1, pp. 19-21. DEMEKE, T. and JENKINS, R. (2010). Influence of DNA extraction methods, PCR inhibitors and quantification methods on Real time PCR assay of biotechnology-derived traits. Analytical and Bioanalytical Chemistry, vol. 396, pp. 1977-1990. DOOSTY, B. et al. (2012). Comparative Analysis and Optimization of Different DNA Extraction Protocols in Satureja khuzistanica. International Journal of Biology, vol. 4, no. 4,pp. 112-116. ELIAS, R. et al. (1991). Triterpenoid saponins from the leaves of Hedera helix. Journal of Natural Products, vol. 54, pp. 98-103. GREEN, A. F. et al. (2011). Phylogeny and Biogeography of Ivies ( Hedera spp., Araliaceae), a Polyploid Complex of Woody Vines. Systematic Botany, vol 36, no. 4, pp. 1114-1127. HUAQIANG, T. et al. (2013). Comparative Analysis of Six DNA Extraction Methods in Cowpea (Vigna unguiculata L.Walp). Journal of Agricultural Science, vol 5, pp. 82-90. IVERSEN, J. (1944). Viscum, Hedera and Ilex as climatic indicators. Geologiska Föreningers Stockholm Förhandlinger, vol. 66, pp. 463–483. JOBES, D. V. et al. (1995) Plant DNA isolation: A Method to Efficiently Remove Polyphenolics, Polysaccharides, and RNA. Taxon, vol. 44, pp. 379-386. KALENDAR, R. et al. 2010. iPBS: a universal method for DNA Fingerprinting and retrotransposon isolation. Theoretical and Applied Genetics, vol. 121, pp. 1419–1430. KRAJEWSKA, A. (1981). Phytochemical Studies of Hedera helix Herbage, Flowers and Fruits. Herba Polonica, vol. 27, pp. 303-309. LADE, B.D. et al., (2014). Efficient genomic DNA extraction protocol from medicinal rich Passiflora foetida containing high level f polysacharide and polyphenol. SpingerPlus, vol. 3, p. 457. LUTSENKO, Y. et al. (2010). Hedera helix as a medicinal plant. Herba Polonica, vol. 56, pp. 83- 96. MAJESTERSAVORNIN, B. et al. (1991). Saponins of the ivy plant. Planta Medica, vol. 57, p. 260- 262.  Â

The study aimed at optimization of DNA isolation from blood of representatives of four microbial groups causing sepsis, i.e., Gram negative: Escherichia coli, Gram positive: Staphylococcus aureus, yeast: Candida albicans, and filamentous fungus: Aspergillus fumigatus. Additionally, the five commercial kits for microbial DNA isolation from the blood were tested. The developed procedure of DNA isolation consisted of three consecutive steps, i.e., mechanical disruption, chemical lysis, and thermal lysis. Afterward, DNA was isolated from the previously prepared samples (erythrocyte lysis) with the use of five commercial kits for DNA isolation. They were compared paying heed to detection limit, concentration, DNA purity, and heme concentration in samples. The isolation of DNA without preliminary erythrocyte lysis resulted in far higher heme concentration than when lysis was applied. In the variant with erythrocyte lysis, two of the commercial kits were most effective in purifying the DNA extract from heme. Designed procedure allowed obtaining microbial DNA from all four groups of pathogens under study in the amount sufficient to conduct the rtPCR reaction, which aimed at detecting them in the blood.

Optimization of DNA isolation and amplification protocol for Gracilaria and Sargassum species of Tamil Nadu coast

Objective: The identification of meat species in meat products is important for protection of human health, economic reasons, religious factors and for controlling the compliance with food regulations. For this purpose, DNA must be obtained in good quality and quantity. The aim of this study was to compare different DNA isolation methods from different meat products. Materials and methods: Comparison among different DNA isolation methods was done. DNA was isolated from different meat products ( e.g ., sucuk, salami, sausage, braised meet, meatball and pastrami). The methods included phenol/chloroform, DNA isolation kit, Cetyl Trimethyl Ammonium Bromide (CTAB) and Dodecyle Trimethyl Ammonium Bromide (DTAB). Results: Although DNA was obtained from all of these methods, the phenol/chloroform and DNA isolation kit methods were found to be the most effective methods for obtaining high quantity DNA. RNA contamination was determined to be common in DTAB method. High quantity of DNA and RNA contamination in terms of quality was detected in CTAB method. Ruminant specific 16S rRNA primer was used to amplify genomic DNA by polymerase chain reaction and all samples were amplified except for some samples of DTAB. Conclusion: DNA isolation kit, another best method, is recommended due to quality and quantity of DNA for researchers who do not want that phenol/chloroform method have toxic substances. This study is also the first study in which DTAB method is used for DNA extraction from meat products. http://doi.org/10.5455/javar.2016.c175

ABSTRACTWe developed a method for isolation of DNA of cellulolytic microbies from soil, so that DNA sample was free from soil organic matter (SOM). The method consists of three procedures: propagation of cellulolytic microbes, separation of microbial cells from SOM, and DNA extraction using cetyl trimethyl-ammonium bromide (CTAB). Differential centrifugation with Ficoll-PM400 successfully separated the cells from soil particles and SOM. From 1 g of soil, 0.12-0.51 μg of microbial DNA with an average size of 15-20 kbp was isolated. Denaturing Gradient Gel Electrophoresis showed that DNA from a variety of cellulolytic microbes was recovered.

The isolation of high quantity and intact DNA is of a great significance for molecular identification of higher fungi. The aim of this study was to compare two DNA extraction methods for isolation of DNA from medicinal mushrooms of Agaricomycetes class. A modified CTAB method and a modified SDS method were compared by the yield and purity of the extracted DNA, its fragmentation state and suitability for amplification. The results demonstrated high efficiency of both methods in regard to DNA yield (14.18 -144.28 ng DNA/mg biomass with CTAB method and 15.03 -108.34 ng DNA/mg biomass with SDS method). The CTAB method provided DNA extracts with higher purity (A260/A280 ranged from 1.83 to 1.99) in comparison with the SDS method (A260/A280 = 1.53 -1.86). The modified CTAB method produced amplifiable DNA from all mushroom isolates, while the SDS method demonstrated suitability for amplification only in 50% of the samples. Therefore, the modified CTAB method could be the method of choice for DNA extraction from medicinal mushrooms. The analyzed isolates were subjected to molecular identification by ITS1-5.8S-ITS2 rRNA gene sequence analysis and were identified as Ganoderma resinaceum, Trametes versicolor, Fomitopsis pinicola and Inonotus hispidus.

Genotyping with a dried blood spot method: A useful technique for application in pharmacogenetics

Objective: This study compared the effects of different DNA extraction methods (CTAB, phenol-chloroform, silica columns, magnetic beads) on the DNA yield and purity obtained from maize (Zea mays L.), bean (P. vulgaris L.), oak (Q. robur L.) and scots pine (P. sylvestris L.). Materials and Methods: In this study, young apical leaves (the most distal fully expanded leaves) of maize, bean, oak and scots pine were used. For each species, 10 leaf tissue samples were collected from young apical leaves during the vegetation season of the year 2024 from plantations located at different sites within the Bayramiç district of Çanakkale province. Leaf tissue samples were collected during the vegetation season of the year 2024, specifically during the early phenological stages of each species. For maize and bean, samples were obtained in the early vegetative growth phase, when the apical leaves were fully expanded but still young and metabolically active. For oak and Scots pine, sampling was conducted during the early spring, shortly after bud burst, when newly emerged apical leaves were in the initial phase of leaf expansion. Young apical leaves were preferred as they contain fewer secondary metabolites and phenolic compounds, which can interfere with DNA extraction and downstream molecular applications. This sampling strategy ensured the collection of high-quality tissue suitable for genomic DNA isolation. DNA isolation was performed using four different methods: CTAB, phenol-chloroform, silica-based columns, and magnetic beads. These methods were compared in terms of their suitability for genetic analyses specific to each plant species. Results: Using the phenol-chloroform method, a notably high DNA yield was obtained from P. sylvestris L. samples (371.75 ng/µL). Similarly, Q. robur L. also exhibited high yield (352.00 ng/µL), suggesting that these species are particularly compatible with this method. The CTAB method also yielded successful results. P. sylvestris L. achieved a high DNA yield (323.75 ng/µL), indicating that this species' genetic material is compatible with the CTAB method. A DNA yield of 308.00 ng/µL was observed for Q. robur L., which may necessitate additional steps for protein removal. The silica column method yielded DNA samples of a lower quantity than the other three methods. For P. sylvestris L. and Q. robur L., yields of (230.75 ng/µL) and (222.25 ng/µL), respectively, were recorded. Lower yields were observed in P. vulgaris L. (171.75 ng/µL) compared to Zea mays L. (211.75 ng/µL). The diminished DNA recovery may be attributable to the constrained binding capacity of silica columns or inadequate initial material. In the magnetic beads method, DNA yields for P. sylvestris L. (206.25 ng/µL), Q. robur L. (201.75 ng/µL), Zea mays L. (173.50 ng/µL) and P. vulgaris L. (151.75 ng/µL) were the lowest among all methods. Conclusion: This study compares DNA isolation methods including CTAB, phenol-chloroform, silica columns, and magnetic beads for Zea mays L., P. vulgaris L., Q. robur L., and P. sylvestris L. The findings reveal that the differences in DNA yields and purity depend on the plant species and tissue characteristics. The phenol-chloroform method provided the highest DNA yields and purity across all plant species. This method excels in obtaining DNA free from proteins, polysaccharides, and phenolic compounds, ensuring high molecular integrity. The results demonstrate that Q. robur L. and P. sylvestris L., which are rich in lignin and phenolic compounds, had the highest yields and preserved molecular integrity. This supports the use of the phenol-chloroform method in molecular analyses requiring high-quality DNA.