scholarly journals Optimization of Various ITS rDNA Amplification Protocol of Yeast Isolated from Giant Honey Beehives (Apis dorsata)

2021 ◽  
Vol 3 (2) ◽  
pp. 80-87
Author(s):  
Chumaidatul Choiriyah, S.Si ◽  
Nirmala Fitria Firdhausi ◽  
Esti Tyastirin ◽  
Yuanita Rachmawati ◽  
Moch. Irfan Hadi
Keyword(s):  
Dna Extraction ◽  
Incubation Time ◽  
Dna Amplification ◽  
Extraction Process ◽  
Optimal Time ◽  
Apis Dorsata ◽  
Molecular Approaches ◽  
Amplification Protocol ◽  
Its Gene ◽  
Dna Purity

Indonesia is a country with high variability of microorganisms, including bacteria, yeast, and fungi. Yeast isolates could be isolated from the honeycomb of Apis dorsata. Molecular approaches were used to identify yeast using ribosomal DNA gene sequences, called the ITS gene. The optimum condition for DNA extractions and amplifications are needed for the successfully of molecular identification. Therefore, it is necessary to optimize the DNA extraction and amplification of several protocols to obtain good identification results. This study aimed to compare the effects of DNA extraction with various temperatures and different amplification protocols. LIPI reference DNA extraction protocol with the boiling method and variations in incubation time of 10, 15, and 20 minutes at a temperature of 98° C. Meanwhile, for the amplification of yeast DNA using a variety of different amplification protocols. The results showed the optimal time of incubation was 10 minutes in K1 isolates with DNA purity of 1.896. meanwhile, for isolates K2, K3, and K4 each with a purity of 2.246, 2.335, and 1.748. optimal DNA amplification results were indicated by the presense of DNA bands for each sample K1, K2, K3, and K4, namely 503, 542, 492, and 526 bp. In this study, it can be concluded that the optimal incubation time for the extraction process is 10 minutes. In addition, the optimal amplification protocol was shown in the DNA bands in all sample.

scholarly journals A Disposable DNA Analysis Chip with a Powerless Actuator

2022 ◽  
Author(s):  
Kyungsup Han ◽  
Insup Kim ◽  
Wei Xuan Chan ◽  
Sanglae Kim ◽  
Jeong-Hwan Kim ◽  
...  
Keyword(s):  
Dna Extraction ◽  
Dna Analysis ◽  
Exothermic Reaction ◽  
Microfluidic Channel ◽  
Dna Amplification ◽  
Extraction Process ◽  
Gas Production ◽  
Diagnostic Process ◽  
Hydration Reaction ◽  
Operational Error

Abstract A non-instrumented, single-use, affordable, and fully- yet safely-disposable DNA analysis system for Point Of Care (POC) diagnostic process has been proposed by integrating (1) a hydration-reactive mixture for a portable heating element as a powerless actuator, (2) commercially available optical adhesive films as valves, and (3) an exothermic reaction-based recombinant polymerase amplification (RPA) process for non-instrumented DNA amplification. The operational error tolerance of the adhesive valves was evaluated by gas production and long-lasting ability, and the amplification performance of the RPA device was validated by gel electrophoresis. Finally, a DNA analysis device was fabricated and tested based on a hydration reaction with a DNA extraction microfluidic channel and an exothermic reaction-based RPA device. In the DNA extraction process, dimethyl adipimidate (DMA) solution was used to eliminate some required injection steps from the extraction process. The integrated system's functionality was successfully demonstrated, and the suggested system could become a foundation for the ultimate total solution for POC DNA analysis.

scholarly journals Optimasi Metode Ekstraksi DNA pada Melon (Cucumis melo L.) Berdasarkan Suhu, Lama Inkubasi, dan Kondisi Daun

2021 ◽  
Vol 5 (2) ◽  
pp. 109
Author(s):  
Dewi Retnaningati
Keyword(s):  
Dna Extraction ◽  
Incubation Time ◽  
Economic Value ◽  
Essential Element ◽  
Extraction Technique ◽  
Research Activities ◽  
Determining Factors ◽  
Dna Purity ◽  
Melon Seeds ◽  
The Right

Abstract Melon is a fruit commodity that has high economic value and is in demand by the community, so it has the potential to be developed. Therefore it is necessary to study various sciences, one of which is the molecular approach. DNA is an essential element in molecular research. The right extraction technique will determine the quality and quantity of DNA produced. The temperature and incubation time applied in the DNA extraction technique, as well as the quality of the leaves as a source of plant DNA, are among the determining factors for the quality and quantity of extracted DNA, so it is necessary to carry out an assessment and optimization. This study aims to assess the optimal temperature and incubation time in extracting DNA from material (melon leaves) under different conditions. Research activities include planting melon seeds, collecting leaf samples, DNA extraction and quantitative DNA testing. The results showed that the concentration and purity of DNA extracted from cold leaves was higher than that from fresh leaves. The highest DNA concentration was obtained from the 65 ° C incubation treatment for 20 minutes, namely 2707.6 ng / μl, and the highest DNA purity was obtained from the 70 ° C incubation treatment for 10 minutes, namely 1.94 from leaf material that had been cooled overnight at a temperature of -20 ° C . Abstrak Melon merupakan salah satu komoditas buah yang bernilai ekonomi tinggi dan diminati masyarakat, sehingga potensial untuk dikembangkan. Oleh karenanya perlu pengkajian dari berbagai ilmu salah satunya dengan pendekatan molekuler. DNA merupakan unsur yang cukup esensial dalam riset molekuler. Teknik ekstraksi yang tepat sangat menentukan kualitas dan kuantitas DNA yang dihasilkan. Suhu dan lama inkubasi yang diterapkan dalam teknik ekstraksi DNA, serta kualitas daun sebagai sumber DNA tanaman merupakan salah satu faktor penentu kualitas dan kuantitas DNA hasil ekstraksi, sehingga perlu dilakukan pengkajian dan optimasi. Penelitian ini bertujuan untuk mengkaji suhu dan lama inkubasi yang optimal dalam mengekstraksi DNA dari bahan (daun melon) dengan kondisi yang berbeda. Kegiatan penelitian meliputi penanaman benih melon, koleksi sampel daun, ekstraksi DNA dan uji kuantitatif DNA. Hasil penelitian menunjukkan bahwa konsentrasi dan kemurnian DNA hasil ekstraksi dari daun dingin lebih tinggi dibandingkan dari daun segar.  Konsentrasi DNA tertinggi diperoleh dari perlakuan inkubasi 65°C selama 20 menit, yaitu 2707.6 ng/μl, dan kemurnian DNA tertinggi diperoleh dari perlakuan inkubasi 70°C selama 10 menit, yaitu 1.94 dari bahan daun yang sudah didinginkan semalaman pada suhu -20°C.

scholarly journals Study on Aqueous Enzymatic Extraction Process of Common Pistache Oil

2021 ◽  
Vol 251 ◽  
pp. 02058
Author(s):  
Feng Xuehua ◽  
Tao Ali ◽  
Song Zurong ◽  
Gong Panpan
Keyword(s):  
Ph Value ◽  
Orthogonal Experiment ◽  
Extraction Process ◽  
Optimal Time ◽  
Process Conditions ◽  
Optimal Process ◽  
Enzymatic Extraction ◽  
Hydrolysis Time ◽  
Aqueous Enzymatic Extraction ◽  
The Common

The aqueous enzymatic method was applied to extract the common pistache oil and the optimal extraction process conditions were identified. By observing the effect of enzymatic hydrolysis time, pH value, temperature on aqueous enzymatic extraction process and performing the orthogonal experiment based on the single factor test, the optimal process parameters were obtained, namely, the optimal time, temperature, and pH value were respectively 3 h, 50℃, and 7 with a final extraction rate of 25.38 %.

Aqueous Enzymatic Extraction of Rapeseed Oil and Protein from Dehulled Cold-pressed Double-low Rapeseed Cake

2012 ◽  
Vol 8 (3) ◽  
Author(s):  
Yan Xing Niu ◽  
Wenlin Li ◽  
Jun Zhu ◽  
Qingde Huang ◽  
Mulan Jiang ◽  
...  
Keyword(s):  
Incubation Time ◽  
Cell Structure ◽  
Extraction Process ◽  
Enzyme Concentration ◽  
Extraction Yield ◽  
Enzymatic Extraction ◽  
Aqueous Enzymatic Extraction ◽  
Rapeseed Cake ◽  
Cold Press ◽  
Cold Pressed

Abstract The oil and protein of dehulled cold-pressed double-low rapeseed cake was extracted by an aqueous enzymatic process. The rapeseed cake was treated by the chosen combined enzymes of Viscozyme L and Alcalase 2.4L (VLA,1:1,w/w). Preliminary experiments and Response Surface Methodology (RSM) were used to study the effects of enzyme concentration, incubation time and water-to-cake ratio on the extraction yield of oil and protein. This is how the desirable conditions were obtained. Transmissive electron microscope photo showed that after cold-pressing the cell structure of rapeseed was partly damaged while dehulling had little effect on the cell structure of rapeseed. In RSM experiments water-to-cake ratio showed significant effects on the extraction of oil and protein (P<0.05),while incubation time only showed significant effects on protein yield (P<0.05).The desirable conditions were as follows: 1.0% concentration (w/w) of VLA; water-to-cake ratio(w/w),6:1; 80 min incubation time. Under this condition, the extraction yield of protein and oil were 82.10% and 71.89%, respectively. Through combining both the cold-press and the aqueous enzymatic processes together, the total oil yield reached 91.6%, which is higher than the normal cold-press process or the aqueous enzymatic extraction process alone.

scholarly journals Improved Protocols of ITS1-Based Metabarcoding and Their Application in the Analysis of Plant-Containing Products

Genes ◽  
2019 ◽  
Vol 10 (2) ◽  
pp. 122 ◽  
Author(s):  
Denis Omelchenko ◽  
Anna Speranskaya ◽  
Andrey Ayginin ◽  
Kamil Khafizov ◽  
Anastasia Krinitsina ◽  
...  
Keyword(s):  
Dna Extraction ◽  
Herbal Medicines ◽  
Herbal Remedies ◽  
Potential Health ◽  
Amplification Success ◽  
Dna Extraction Method ◽  
Dna Metabarcoding ◽  
Food And Beverage ◽  
Dna Purity ◽  
Medicines Quality

Plants are widely used for food and beverage preparation, most often in the form of complex mixtures of dried and ground parts, such as teas, spices or herbal medicines. Quality control of such products is important due to the potential health risks from the presence of unlabelled components or absence of claimed ones. A promising approach to analyse such products is DNA metabarcoding due to its high resolution and sensitivity. However, this method’s application in food analysis requires several methodology optimizations in DNA extraction, amplification and library preparation. In this study, we present such optimizations. The most important methodological outcomes are the following: 1) the DNA extraction method greatly influences amplification success; 2) the main problem for the application of metabarcoding is DNA purity, not integrity or quantity; and 3) the “non-amplifiable” samples can be amplified with polymerases resistant to inhibitors. Using this optimized workflow, we analysed a broad set of plant products (teas, spices and herbal remedies) using two NGS platforms. The analysis revealed the problem of both the presence of extraneous components and the absence of labelled ones. Notably, for teas, no correlation was found between the price and either the absence of labelled components or presence of unlabelled ones; for spices, a negative correlation was found between the price and presence of unlabelled components.

Comparison of commercial DNA extraction kits for isolation and purification of bacterial and eukaryotic DNA from PAH-contaminated soils

2011 ◽  
Vol 57 (8) ◽  
pp. 623-628 ◽  
Author(s):  
Nagissa Mahmoudi ◽  
Greg F. Slater ◽  
Roberta R. Fulthorpe
Keyword(s):  
Dna Extraction ◽  
Contaminated Soils ◽  
Dna Isolation ◽  
Extraction Process ◽  
Organic Carbon Content ◽  
Soil Dna ◽  
Isolation And Purification ◽  
Dna Yield ◽  
Pcr Products ◽  
Eukaryotic Dna

Molecular characterization of the microbial populations of soils and sediments contaminated with polycyclic aromatic hydrocarbons (PAHs) is often a first step in assessing intrinsic biodegradation potential. However, soils are problematic for molecular analysis owing to the presence of organic matter, such as humic acids. Furthermore, the presence of contaminants, such as PAHs, can cause further challenges to DNA extraction, quantification, and amplification. The goal of our study was to compare the effectiveness of four commercial soil DNA extraction kits (UltraClean Soil DNA Isolation kit, PowerSoil DNA Isolation kit, PowerMax Soil DNA Isolation kit, and FastDNA SPIN kit) to extract pure, high-quality bacterial and eukaryotic DNA from PAH-contaminated soils. Six different contaminated soils were used to determine if there were any biases among the kits due to soil properties or level of contamination. Extracted DNA was used as a template for bacterial 16S rDNA and eukaryotic 18S rDNA amplifications, and PCR products were subsequently analyzed using denaturing gel gradient electrophoresis (DGGE). We found that the FastDNA SPIN kit provided significantly higher DNA yields for all soils; however, it also resulted in the highest levels of humic acid contamination. Soil texture and organic carbon content of the soil did not affect the DNA yield of any kit. Moreover, a liquid–liquid extraction of the DNA extracts found no residual PAHs, indicating that all kits were effective at removing contaminants in the extraction process. Although the PowerSoil DNA Isolation kit gave relatively low DNA yields, it provided the highest quality DNA based on successful amplification of both bacterial and eukaryotic DNA for all six soils. DGGE fingerprints among the kits were dramatically different for both bacterial and eukaryotic DNA. The PowerSoil DNA Isolation kit revealed multiple bands for each soil and provided the most consistent DGGE profiles among replicates for both bacterial and eukaryotic DNA.

scholarly journals Analysis of residual sludge stored in UASB of a WWT in Petrolina-PE-Brazil

2020 ◽  
Vol 24 ◽  
pp. 10
Author(s):  
Erick De Aquino Santos ◽  
Keyla Vitória Marques Xavier ◽  
Marcella Vianna Cabral Paiva ◽  
Miriam Cleide Cavalcante de Amorim ◽  
Michely Correia Diniz
Keyword(s):  
Dna Extraction ◽  
Waste Water Treatment ◽  
Extraction Process ◽  
Upflow Anaerobic Sludge Blanket ◽  
Anaerobic Sludge ◽  
Physical Analysis ◽  
High Concentration ◽  
Residual Sludge ◽  
Anaerobic Sludge Blanket ◽  
Solids Analysis

Anaerobic digestion is a process that occurs through microorganisms in an anoxic condition and aims to digest organic matter resulting mainly in biogas. This process is common in wastewater treatment WWTs (Waste Water Treatment), which usually occur in bioreactors. In Brazil the most widespread is the UASB (Upflow Anaerobic Sludge Blanket) reactor due to its temperature conditions, which found in the country an ideal parameter. Archeas make up the microbiota responsible for digestion acting in the final stage of methanogenesis. The studies of these organisms are mainly through metagenomics, because laboratory cultivation is difficult. Therefore, the research aimed to study the physical and molecular parameters of the sludge. Four UASB reactors from WWT Center in Petrolina – Pernambuco- Brazil were evaluated. For the DNA extraction process the adapted protocol was applied, the physical analysis of the solids obeyed the determinations of APHA (2005). DNA extraction was achieved with the modified protocol and demonstrated a high concentration of DNA present in the samples, being the 4 most abundant reactor. Physical quantifications of the solids analysis showed that the values found are in compliance with current standards.

scholarly journals Evaluation and Application of an Efficient Plant DNA Extraction Protocol in Laboratory and Field Testing

2020 ◽  
Author(s):  
Qi Wang ◽  
Xiaoxia Shen ◽  
Tian Qiu ◽  
Wei Wu ◽  
Zhian Wang ◽  
...  
Keyword(s):  
Dna Extraction ◽  
Molecular Identification ◽  
Filter Paper ◽  
Biological Samples ◽  
Gc Content ◽  
Dna Amplification ◽  
Nucleic Acid Amplification ◽  
Lamp Method ◽  
Paper Strip ◽  
Cellulose Filter

Abstract Background CTAB has been considered as the standard protocol for DNA extraction. But the complex and time-consuming procedures can’t meet the needs of rapid molecular identification. The method of using cellulose filter paper strips to transfer the DNA in the plant tissue lysate to the nucleic acid amplification system shortening the DNA extraction time to 30 s. However, cellulose filter paper strips have some shortcomings that cannot be put into widespread use. And the data supporting the rapidly purification of DNA by cellulose filter paper is not sufficient.ResultsIn the study, the published filter paper strip was modified by sticking the filter paper on the PVC sheet. This modified method is named EZ-D, for easy DNA extraction. Compared with the original method, the DNA extracted by EZ-D is more efficient in PCR amplification. We also came up with a new DNA extraction buffer, which exhibited higher DNA extraction efficiency. When compared with classic CTAB, EZ-D also showed great advantages for higher efficiency, easier protocol and lower cost. PCR analyses showed that DNA extracted from several types of plants by EZ-D were appropriate for specific identification of biological samples. PCR using DNA extracted by EZ-D was sensitive enough to detect 0.1 ng/μL. Evaluation of the EZ-D showed that the DNA extracts can be successfully amplified by PCR reaction for the DNA fragments up to 3000 bp in length and up to 80% in GC content. EZ-D was successfully used for DNA extraction from a variety of plant species and plant tissues. Moreover, when EZ-D was combined with the loop-mediated isothermal amplification (LAMP) method, DNA identification of biological samples could be achieved in an equipment-free way. Conclusion Combined with DNA amplification technology, EZ-D protocol is a rapid, specific and sensitive method for molecular identification of plant samples. In addition, EZ-D method is the first application of cellulose filter paper in the identification of genetically modified crops and traditional Chinese medicine ultra-fine powder. In terms of practicability, EZ-D has realized the popularization of cellulose filter paper for rapid DNA purification in laboratories and markets.

scholarly journals Validation of Half-reaction Volumes of the Promega PowerPlex® Forensic Amplification Kits (PowerPlex® 18D Systems, PowerPlex ® 21System, PowerPlex® Fusion System and PowerPlex® Y23 System) in STR Analysis

2020 ◽  
Vol 2 (2) ◽  
pp. 164-171
Author(s):  
Hanan K. Mahmood ◽  
Nadia F. Salman ◽  
Dhurgham H. Hasan ◽  
Khaleefah M. Salih ◽  
Maryam A. Sadiq ◽  
...  
Keyword(s):  
Dna Analysis ◽  
Dna Amplification ◽  
Sensitivity Study ◽  
Fusion System ◽  
Reaction Volume ◽  
Forensic Dna Analysis ◽  
Str Analysis ◽  
Amplification Protocol ◽  
Reaction Amplification ◽  
Reaction Volumes

DNA amplification is known to be the most expensive step during forensic DNA analysis. This study evaluated the half-reaction amplification protocol (12.5 µL PCR product) using DNA amplification kits from Promega PowerPlex® (PowerPlex® 18D System, PowerPlex ®21System, PowerPlex® Fusion System and PowerPlex® Y23 System), which might aid in reducing sample analysis cost by half and allow the analysis of more samples. A sensitivity study (15 samples) along with testing of various blood stain samples (n=100) that were submitted to the Medico-Legal Directorate laboratory for DNA testing was accomplished to compare the DNA profiles resulting from half-reaction volume procedure to those with full-reaction volume procedure, using three differed methods along with standard protocol to evaluate the effect of half reaction volume with some variables. Results demonstrated the use of half-reaction amplification protocol preceded by washing step for all aforementioned DNA amplification kits gave a robust and reliable amplification result that aid to increase the number of samples analyzed and decreased the test cost for each kit without compromising the quality of 3DNA profiles obtained.

scholarly journals A Protocol for Rapid and Inexpensive Genotyping of Mutant Mice from Dried Blood Spots: An Update

2021 ◽  
Author(s):  
Antonella Romano ◽  
Candida Zuchegna ◽  
Giuseppa Zannini ◽  
Roberta Grillo ◽  
Samantha Messina ◽  
...  
Keyword(s):  
Dna Extraction ◽  
Genomic Dna ◽  
Fragment Length Polymorphism ◽  
Dna Amplification ◽  
Dried Blood Spots ◽  
Nucleotide Polymorphisms ◽  
Single Nucleotide ◽  
Pcr Rflp ◽  
Polymerase Chain ◽  
Sensitivity Specificity

Abstract Background: Dried blood spot (DBS) testing is a well-known method of bio-sampling by which blood samples are blotted and dried on filter paper. The dried samples can then be analyzed by several techniques such as DNA amplification and HPLC. We have developed a homemade DBS method followed by an alternative protocol for genomic DNA extraction from a drop of blood adsorbed on paper support. This protocol consists of two separate steps: (1) organic DNA extraction from the DBS, followed by (2) DNA amplification by polymerase chain reaction (PCR). The PCR-restriction fragment length polymorphism (PCR-RFLP) is an advantageous and simple approach to detect single nucleotide polymorphisms (SNPs). Results: We have evaluated the efficiency of our method for the extraction of genomic DNA from DBS by testing its performance in genotyping mouse models of obesity and herein discuss the sensitivity, specificity and feasibility of this novel procedure. Conclusions: Our protocol is easy to perform, fast and inexpensive and allows the isolation of pure DNA from a miniscule amount of sample.

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