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Leptospirosis is a worldwide zoonosis caused by Leptospira interrogans. Laboratory diagnosis of leptospirosis can be done through sample culture. However, microscopic observation of positive Leptospira spp. culture need to be supported by molecular detection to confirm the presence of pathogenic Leptospira spp. This study aimed to compare the effectiveness of two DNA extraction kits, from QIAGEN and Zymo Research, to extract bacterial DNA genome from positive Leptospira spp. culture for downstream molecular detection. This study is an analytical observational study with a cross-sectional design conducted in June-September 2023, and part of the PESTO-RITA 2023 study. Fourteen identified positive Leptospira spp. culture, with Leptospira spp. movement under dark field microscopy, was used for DNA extraction using both extraction kits. QIAGEN kit yielded higher mean DNA concentration (0.900±0.161 vs 0.790±0.167 log10 µg/mL, p>0.05) and better DNA purity (1.854 vs 1.632, p

Forensic short tandem repeats (STR) profiling on touch DNA samples has emerged as a primary method for human identification. The stability and uniqueness of STR combination from the targeted locus in each individual make it a precision marker for human identification. Touch DNA samples can be found in traces of biological material shed from a person. This work aimed to identify the lowest concentration limit required for generating an interpretable DNA profile and the sensitivity of the STR loci applied. Touch DNA samples were collected from donors who were asked to hold a rope for 5 min. A double swab technique was used to lift the touch samples from the rope. These samples are subjected to DNA extraction and quantification. Two STR amplification cycles, 29 and 34 cycles, were used. DNA concentration greatly influences the success of amplifying the target allele at each STR locus to be interpreted into a complete DNA profile, shown by its allele peak. Touch DNA concentration >0.25 ng can produce a complete DNA profile. LCN method successfully amplified touch DNA with a concentration 0.0625–0.25 ng/µL. Limit detection of touch DNA analysis is 0.25 ng/µL. Low-copy DNA can still be analyzed within 0.0625–0.25 ng/µL.

Background. High content of organic compounds that worsen nucleic acid purification in aromatic plants, as well as the use of such toxic substances as phenol and mercaptoethanol in many protocols for plant DNA isolation, make it advisable to take the above disadvantages into account when optimizing the DNA extraction technique for the work with essential oil rose plants. Material and methods. Rose accessions from the Crimean Federal University’s Botanical Garden, and those from the collection held by the Research Institute of Agriculture of Crimea were included in the study. DNA extraction was done according to a modified CTAB protocol. Effectiveness of the technique was assessed using spectrophotometry, horizontal electrophoresis, and ISSR-PCR. Results. DNA preparations extracted with the modified technique were well visualized on the electropherogram and demonstrated high spectrophotometric values. DNA content was twice as high in preparations isolated with an extraction buffer with PVP, compared to a PVP-free buffer. The concentration was also higher in DNA extracts from stems than that from leaves. Purity parameters expressed by the absorption ratios at wavelengths A260/280 and A260/230 were again higher for DNA extracts from stems isolated with an enriched buffer, the A260/230 ratio falling within the normal range only in DNA extracted from stems in the presence of PVP. Besides, DNA extracts were effectively purified from proteins without phenol or mercaptoethanol, due to double rinsing with a chloroform/isoamyl alcohol (24 : 1) mixture. Conclusion. Using rose stem tissues as the research material, adding 2% PVP to the extraction buffer, and twofold rinsing with a chloroform/isoamyl alcohol mixture made it possible to obtain DNA extracts with high concentrations and purity indices within normal ranges suitable for the ISSR analysis of essential oil rose genetic diversity.

Purpose: In some cases of prenatal genetic testing, an ample amount of fetal DNA is needed, to allow for parallel testing (conducting several genetic tests simultaneously). This study investigated the association between amniotic fluid DNA concentration and various factors. We aimed to define the required amount of amniotic fluid to be extracted in amniocentesis, to allow parallel testing throughout gestational weeks. Methods: DNA concentration was analyzed from amniocentesis samples taken during the years 2016–2022. Sex association was also analyzed in postnatal whole blood samples from a separate cohort. Theoretical minimum volume of amniotic fluid needed to ensure enough DNA for chromosomal microarray analysis and exome sequencing was calculated. Results: We focused our analysis on 2573 samples, which were taken during weeks 17–23 and 30–35. DNA concentrations increased from weeks 17 to 21, with relatively stable concentrations thereafter. Significantly higher DNA concentrations were seen in pregnancies of female fetuses. DNA concentrations in postnatal whole blood samples did not show this association. Across most weeks, the volume needed to extract 2 µg of DNA from 95% of the samples was about 34 ml. Conclusion: DNA concentrations in amniotic fluid vary according to gestational age and are higher in pregnancies of female fetuses. This should be considered when determining the volume of fluid extracted and the timing of amniocentesis, with greater volumes needed in earlier stages of pregnancy. [ABSTRACT FROM AUTHOR]

Background: This study investigates the impact of acute and chronic exposure to gaseous pollutants, specifically sulfur dioxide (SO), nitrogen dioxide (NO), and their combination (SO +NO), on the physiological and genetic integrity of Centropristis striata (sea bass). The research focuses on understanding the adverse effects on gill, liver, and kidney tissues over a 96-hour acute exposure period and during long-term chronic exposure at sub-lethal concentrations. Methods: The study determined lethal concentrations (LC, LC, LC) for SO, NO and their combination. Gill and liver health were evaluated using scoring systems. Total carotenoid and beta -carotene content in liver tissue were quantified. Comet assays were employed to measure DNA damage in gill, liver, and kidney tissues. Results: The findings indicate a dose-dependent increase in mortality rates with escalating concentrations of pollutants. Significant damage to gill and liver tissues was observed, with higher pollutant concentrations resulting in more severe damage. A notable depletion of carotenoid levels in the liver was detected with increasing pollutant exposure. Genotoxic analysis revealed significant DNA damage, particularly in liver tissue, suggesting a mechanistic link between pollutant exposure and genetic injury. Conclusion: The research highlights the detrimental effects of gaseous pollutants on the health and genetic integrity of sea bass. The dose-dependent adverse effects, including increased mortality, tissue damage, and DNA integrity disruption, underscore the necessity of implementing effective pollution control measures to preserve aquatic ecosystems. [ABSTRACT FROM AUTHOR]

Background: Formalin-fixed paraffin-embedded (FFPE) specimen archives are a valuable source of sample material for molecular biological analysis. However, the DNA isolated from FFPE samples is usually low in concentration and fragmented. Thus, it is necessary to optimize the FFPE DNA extraction protocol to obtain the best results. Proteinase K incubation is undoubtedly crucial in DNA extraction procedures, but this step is often not well explained in the manufacturer’s manual. Purpose: This study aimed to find the optimal duration for proteinase K incubation protocols to achieve the highest DNA yields. Methods: Fifteen paraffin blocks of Oral Squamous Cell Carcinoma (OSCC) specimens were obtained, and the cancerous areas were microdissected into smaller cuts for DNA extraction. The samples were randomly divided into three groups (n=5) and subjected to three different proteinase K incubation protocols: one-hour incubation at 56ºC as per the manufacturer’s instructions (Group I), 24-hour incubation at 56ºC (Group II), and 48 hours at room temperature with an additional four hours at 56ºC (Group III). The extracted DNA was then quantified using a Nanodrop spectrophotometer. The recorded data were analyzed using ANOVA-LSD. Results: The highest DNA concentration was found in Group III (107.74 ± 41.92), which was significantly higher compared to Group II (59.46 ± 30.32) and Group I (6.46 ± 1.97) (p

Background: The Polymerase Chain Reaction (PCR) method can identify Mycobacterium tuberculosis in a sputum sample of a patient with TB (TB). One crucial step to ensure accurate PCR results is the DNA extraction process. Objective: The research aims to compare the concentration and purity of DNA from the sputum of TB patients using ultrasound and spin column extraction techniques. Methods: The research uses descriptive study designs with post-only design strategies. The primary data was derived from 18 sputum specimens from TB patients. Concentration measurement and DNA purity testing using a nanodrop spectroscopic photometer. Results: DNA extraction by ultrasound method has an average concentration of 18.9 ± 8.5 ng/L, with a peak of 37.6 ng/ L. The spin column method produces an average of 55.5 ± 27.9 ng/μL; the peak is 105.0 ng/ μL. The purity value of the DNA extract is in the range of 1.8 ± 2.0 with the ultrasound method of 61% and the spin column of 78%. Conclusion: The sonication method has a lower average concentration and a higher percentage of purity than the spin column method, and there are differences in concentrations and purity values between the two methods.

DNA analysis is the core of biotechnology applied in petroleum resources and engineering. Traditionally accurate determination of DNA purity and concentration by spectrometer is the first and critical step for downstream molecular biology research. In this study, three different spectrophotometry methods, BPM, NDTT and NPMTTZ were compared for their performance in determining DNA concentration and purity in 32 oil samples, and molecule methods like quantitative real-time PCR (qPCR) and high-throughput sequence were also performed to help assess the accuracy of the three methods in determining DNA concentration and purity. For ordinary heavy oil (OHO), extra heavy oil (EHO) and super heavy oil (SHO), the characteristics of high viscosity (η), density (ρ) and resin plus asphaltene content will affect the DNA extraction and UV determination. The DNA concentration was decreased as density increased: OHO (11.46 ± 18.34 ng/μL), EHO (6.68 ± 9.67 ng/μL) and SHO (6.20 ± 7.83 ng/μL), and the DNA purity was on the reverse: OHO (1.31 ± 0.27), EHO (1.54 ± 0.20), and SHO (1.83 ± 0.32). The results suggest that spectrophotometry such as BPM and NPMTTZ are qualitatively favorite methods as the quick non-consumable methods in determining DNA concentration and purity of medium oil and heavy oil. [ABSTRACT FROM AUTHOR]

Escherichia coli is a leading cause of Urinary Tract Infections (UTIs) in Indonesia, with approximately 180,000 cases reported annually. The more cases of UTIs, the more PCR diagnosis is needed with an accurate, fast, simple, and economical DNA isolation method. However, currently, there is no DNA purification stage from protein and RNA contaminants in the boiling DNA isolation method. This study aimed to investigate the impact of incorporating Proteinase-K and RNase into the boiling DNA isolation method on the purity and concentration of E. coli’s DNA during isolation. The boiling method involved heating to 95°C – 100°C bring to cell lysis and release of cellular components, including DNA. Urine samples were artificially contaminated with E. coli at different McFarland standards (0.25, 0.5, and 1). The boiling DNA isolation method was then performed and then analyzed for purity and concentration using a NanoDrop spectrophotometer. This study demonstrated a positive correlation between Proteinase-K and RNase concentrations used in the boiling DNA isolation method and the subsequent increase in DNA purity and concentration. An increase in DNA purity and concentration was obtained even though it was not statistically significant compared to that without ProteinaseK and RNase addition, with p-values of 0.245 for DNA purity and 0.353 for DNA concentration. Further research is recommended with higher Proteinase-K and RNase concentrations in the boiling DNA isolation method to achieve improved purity and concentration of E. coli DNA. Such enhancements could improve PCR amplification and help diagnose E. coli-related UTIs. [ABSTRACT FROM AUTHOR]

DNA extraction from frozen blood clots is challenging. Here, the authors applied QIAGEN Clotspin Baskets and the Gentra Puregene Blood Kit for DNA extraction to cellular fraction of 5.5 ml whole blood without anticoagulating additives. The amount and quality of extracted DNA were assessed via spectrophotometer and gel electrophoresis. Results from array-based genotyping were analyzed. All steps were compared with DNA isolated from anticoagulated blood samples from a separate study. The quality and concentration of DNA extracted from clotted blood were comparable to those of DNA extracted from anticoagulated blood. DNA yield was on average 27 μg per ml clotted blood, with an average purity of 1.87 (A260/A280). Genotyping quality was similar for both DNA sources (call rate: 99.56% from clotted vs 99.49% from anticoagulated blood).

The acquisition of high‐quality biospecimens and the appropriate handling of these materials are indispensable for successful clinical sequencing. We developed a cancer clinical sequencing system targeting 160 cancer genes: PleSSision‐Rapid. Through the PleSSision‐Rapid system, we have analyzed DNA quality evaluated by DIN (DNA integrity number) with 1329 formalin‐fixed paraffin embedded (FFPE) samples including 477 prospectively collected tissues for genomic test (P) and 852 archival samples after routine pathological diagnosis (A1/A2). As a result, the samples with more than DIN 2.1 was 92.0% (439/477) in prospectively collected sample (P), while it was 85.6% (332/388) and 76.7% (356/464) in two types of archival samples (A1/A2). We performed the PleSSision‐Rapid sequence using the samples with over DIN 2.1 and DNA concentration >10 ng/μL with which we were able to construct a DNA library, and the probability of sequence success was almost equivalent during all types of specimen processing, at 90.7% (398/439) in (P), 92.5% (307/332) in (A1) and 90.2% (321/356) in (A2), respectively. Our result indicated the clinical benefit to prepare the prospective collection of FFPE materials for indisputable clinical sequence, and that DIN ≥ 2.1 would be a solid parameter for sample preparation of comprehensive genomic profiling tests. [ABSTRACT FROM AUTHOR]

Deproteinized Bovine Bone Mineral (DBBM) has been widely used as tissue engineering scaffold, because of its not antigenic potential, although non-resorbable. Freeze-Dried Bovine Bone (FDBB) scaffold was developed to overcome the limitations of DBBM but it's still suspected to have antigenic potential. Therefore, to overcome the issues of antigenic potential, decellularization of FDBB was carried out (dc-FDBB). Objective: To analyze antigenic potential of FDBB scaffold with process decellularization. FDBB and dc-FDBB scaffolds compared to DBBM scaffold as gold standard. Ten samples for each group of scaffold FDBB, dc-FDBB, and DBBM. The groups were observed for residual osteoblasts and osteocytes with HE staining using a light microscope with 40 x and 400 x magnification. DNA concentration analysis was carried out on the three types of scaffolds using a spectrophotometer. Statistical analysis was performed using IBM SPSS version 25. Data were statistically analyzed using ANOVA and Post Hoc Games-Howell tests. There were degradation of osteocytes without nucleus and satisfactory condition of extracellular matrix geometrical structure in the FDBB scaffold. There were no osteoblasts and osteocytes with damaged extracellular matrix geometrical structures were seen in the dc-FDBB scaffold. There were no osteoblasts and osteocytes with satisfactory condition of extracellular matrix geometrical structure in the DBBM scaffold. Mean values of DNA concentration: FDBB, dc-FDBB, and DBBM were of (19.75 ng/µL ± 4.80), (16.84 ng/µL ± 6.55), and (8.72 ng/µL ± 0.65) respectively. Comparative p value FDBB and dc-FDBB scaffolds were 0,509 (p>0,05), there were no statistically significant difference between FDBB and dc-FDBB scaffolds. FDBB and dc-FDBB scaffolds can be used because it has low antigenic potential. [ABSTRACT FROM AUTHOR]

The work deals with the issue of standardization and more accurate methodology for the isolation of gluten DNA in gluten-free products of plant origin, which is more demanding due to the more complex structure of plant cells. Three isolation methods were compared, of which the combination of glass and zirconium beads, Proteinase K and a commercially produced isolation kit was confirmed to be the most effective procedure. The given isolation procedure was more effective in one-component gluten-free foods, where the concentration of the obtained DNA ranged from 80.4 ± 0.7 to 99.0 ± 0.0 ng/µL. The subsequent PCR reaction revealed the presence of gluten not only in guaranteed gluten-free products (40%), but also in naturally gluten-free foods (50%). These were mainly gluten-free sponge cakes, gluten-free biscuits "Cranberries", cocoa powder, coffee "3in1", and instant coffee. [ABSTRACT FROM AUTHOR]

Aspects of the growth pattern, reproductive biology, and molecular characteristics of two cichlids, Sarotherodon melanotheron and Coptodon zillii were investigated in Lagos Lagoon. A total of twenty-nine samples were collected from the Lagos Lagoon. Blood samples were obtained for DNA extraction and analysis from 9 specimens of each species. The morphometric data of S. melanotheron varied from 2.5-4.5cm (head length), 13.5-18.0cm (total length) and 35.00-135.00g (body weight), while that of C. zillii were 2.0-4.5cm, 12.0-21.5cm and 39.00-228.00g respectively. The growth pattern of S. melanotheron showed a positive allometric, b= 3.324, while that of C. zillii showed a negative allometric pattern (b= 2.715). The condition factors of S. melanotheron and C. zillii ranged from 4.15-4.55 and 4.12-5.07 respectively. The sex ratio for S. melanotheron (males, 11; females 2) was 1: 0.18, while C. zillii (males, 12; females 4) had a sex ratio of 1:0.33, and these were statistically significant (p< 0.05). From the molecular characterization analysis, the DNA concentration ranged from 374-765 ng/µl and 48-1265 ng/µl, while DNA purity ranged from 1.56-1.82 and 1.49-1.81, for S. melanotheron and C. zilli respectively. These findings form baseline information for conducting further investigations on the taxonomy, ecology, conservation of the genetic purity of these fisheries resources in Lagos Lagoon. [ABSTRACT FROM AUTHOR]

Microinjection of DNA into zygote pronucleus is still one of the most common methods to obtain transgenic animals. Pronuclear injection seems to be a standardized method, however there are problematic constructs that researchers struggle with to obtain transgenic animals. Here we present a modification of the standard transgenesis method, which we suggest to use in those cases in which the standard method results unsuccessful. By increasing DNA concentration by one order of magnitude we obtained 4 founder mice out of 64 born mice. Moreover, we did not notice toxic effect on embryos attributable to the higher DNA concentration employed. According to our results, we suggest that increasing DNA concentration in the standard protocol for pronuclear injection might be viable solution when dealing with problematic constructs. [ABSTRACT FROM AUTHOR]

The work deals with the issue of standardization and more accurate methodology for the isolation of gluten DNA in gluten-free products of plant origin, which is more demanding due to the more complex structure of plant cells. Three isolation methods were compared, of which the combination of glass and zirconium beads, Proteinase K and a commercially produced isolation kit was confirmed to be the most effective procedure. The given isolation procedure was more effective in one-component gluten-free foods, where the concentration of the obtained DNA ranged from 80.4 ± 0.7 to 99.0 ± 0.0 ng/µL. The subsequent PCR reaction revealed the presence of gluten not only in guaranteed gluten-free products (40%), but also in naturally gluten-free foods (50%). These were mainly gluten-free sponge cakes, gluten-free biscuits “Cranberries”, cocoa powder, coffee “3in1”, and instant coffee.

Aim. To study the effect of the shelf life of frozen whole blood samples in a biobank on the amount of released deoxyribonucleic acid (DNA).Material and methods. The study included whole blood samples placed in tubes with the anticoagulant EDTA (ethylenediaminetetraacetic acid at a concentration of 1,8 mg/ml) from participants in the epidemiological study ESSE-RF-1 and ESSE-RF-2 and cohort studies conducted at the National Medical Research Center for Therapy and Preventive Medicine. The samples were stored in the biobank of the National Medical Research Center for Therapy and Preventive Medicine at temperature from -22О C to -32О C. The shelf life from blood collection to DNA extraction ranged from several weeks to 11 years. DNA was extracted using QIAamp DNA Blood Mini Kit (250) and 96 Blood Kit (Qiagen, Germany). Statistical analysis was performed using the R 3.6.1 software. To analyze the association of blood storage time with the logarithm of DNA concentration, a linear regression was used.Results. The analysis included data on the DNA concentration of 5405 samples. Multivariate regression showed that the blood shelf life was significantly associated with a decrease in concentration by 3,92% (3,16-4,68) for each year of storage (p

Abstract A modified genomic DNA extraction method named the combination of lysozyme and ultrasonic lysis (CLU) method was used to analyze the fish intestinal microflora. In this method, the physical disruption and chemical lysis steps were combined, and some parameters in the key steps were adjusted. In addition, the results obtained by this method were compared with the results obtained by the Zirmil-beating cell disruption method and the QIAamp Fast DNA Stool Mini Kit. The OD260/OD280 ratio and concentration of the DNA extracted using the CLU method were 2.02 and 282.8 µg/µL, respectively; when the incubation temperatures for lysozyme and RNase were adjusted to 37 °C, those values were 2.08 and 309.8 µg/µL, respectively. On the agarose gel, a major high-intensity, discrete band of more than 10 kb was found for the CLU method. However, the smearing intensity of degraded DNA was lower when the incubation temperatures were 60 °C for lysozyme and 30 °C for RNase than when incubation temperatures of 37 °C for lysozyme and 37 °C for RNase were used. The V3 variable region of the prokaryotic 16S rDNA was amplified, and an approximately 600-bp fragment was observed when the DNA extracted using the CLU method was used as a template. The CLU method is simple and cost effective, and it yields high-quality, unsheared, high-molecular-weight DNA, which is comparable to that obtained with a commercially available kit. The extracted DNA has potential for applications in critical molecular biology techniques.

The aim of this study was to detect the effect of active compounds in fruit, leave and stem of Conocarpus (which extracted by methanol alcohol) on growth of Staphylococcus aureus, Enterococcus, E.coli, Klebsiella pneumonia, Proteus merabilis and Pseudomonas aeruginosa. Also, this study aims to detect the effect of active compounds separation from the methanol extract (phenol, alkaloid, and terpene) on bacterial growth, and study the effect of phenol and terpene which gave antibacterial activity as anti-biofilm. This study showed the methanolic extract gave higher effect on Staphylococcus aureus and inhibition zone diameter was 19mm, while the least effect was on Klebsiella pneumonia and inhibition zone diameter was 9mm. Also, the recent study showed the phenolic compounds were more activity than terpene on bacterial growth, while the alkaloid compounds were absent in methanol extract for all part, the phenolic compounds in of leave were higher effect than phenolic compounds in fruit and stem on bacterial growth. The ability of bacteria to the formation of biofilm and biofilm inhibitors by phenolic and terpene was investigated by phenotypic method. The results of the antibiotic sensitivity test showed that the bacterial isolates were highly resistant to the antibiotic; therefore, it is considered multidrug-resistance (MDR). Also, this study showed present variation in effect the phenolic and terpene on bacterial DNA. [ABSTRACT FROM AUTHOR]

For bacteria sampling studies, various collection methods have been used to identify bacteria. To obtain accurate information about bacteria, high quality samples should be obtained. In order to obtain a high quality sample, a stable and large number of DNA copies must be collected. This study compared the efficiency of different methods of bacterial gDNA extraction and bacteria collection according to swabbing solution volumes and types. The efficiency of bacterial genomic DNA extraction was compared using a AccuPrep® Genomic DNA Extraction kit, a QIAamp® DNA Mini kit, and a MOBIO® DNeasy PowerSoil kit. The DNA Mini kit was shown to extract the highest amount of gDNA, and sub-experiments were conducted using this kit. Phosphate-buffered saline and phosphate-buffered saline with 0.1% Tween 20 were used as collection solutions of various volumes (0, 40, 50, 60, 70, 80, 90, 100, 110, and 120 μL) using cotton swabs. Bacteria collection efficiency was highest when 70 μL PBS was used. The target strains collected in this experiment were Staphylococcus aureus and Escherichia coli, and these were quantified using the number of colony-forming units, DNA concentrations, and the number of DNA copies. These results can be used to efficiently bacterial collection for experiments in various fields. [ABSTRACT FROM AUTHOR]

Determination of the metagenome has become an important component of forensic identification, which requires efficient environmental sampling techniques. Therefore, in this study, we compared the efficiency of sample collection using swabbing with cotton swabs and three types of medical swabs (S7, S22, S24) along with three different solutions: phosphate-buffered saline (PBS), 1% Tween 20 + 1% glycerol in PBS (TG), and GS commercial solution (Noble Bio, Hwaseong, Republic of Korea). Combinations of the three solutions with the three types of swabs were tested at different volumes (cotton swab, S7: 0, 30, 50, 70 μL; S22, S24: 0, 70, 100, 130 μL). Escherichia coli and Staphylococcus aureus were selected as representative environmental microbial samples, and the number of colony-forming units (CFUs), DNA concentration, and DNA copy numbers were compared across groups. The sampling process had a clear effect on the efficiency of extraction, which allowed for determination of a more efficient sample sampling method. In particular, cotton swabs showed 2–10-fold greater CFUs of both species than the medical swabs, and resulted in significantly greater amounts of extracted DNA. TG was found to be the most efficient solution for bacterial DNA extraction, with higher CFUs and DNA obtained than with the other three solutions at all volumes tested. This study highlights the need for a standardized sampling method that can be applied to all environmental samples, especially for microbial quantification, and provides valuable reference data for the efficient collection of environmental samples for metagenomic analyses in microbial-based forensic assessments. • Sampling methods of bacterial DNA from artificial dried samples were compared. • Conventional cotton swabs showed better efficiency than medical swabs. • TG (1% Tween 20 + 1% glycerol in PBS) resulted in the most efficient recovery. • Efficient and standardized sampling can promote metagenomics in forensics analyses. [ABSTRACT FROM AUTHOR]

Agarose gel electrophoresis is performed routinely by molecular biologists as both an analytical and a preparative method for characterization of nucleic acids. Gel analysis of highly dilute DNA solutions is challenging because of the limited sensitivity of detection available with conventional methods. In this study a new approach is described for concentrating samples directly within gels called SURE (su ccessive re loading) electrophoresis. The approach involves loading of dilute samples multiple times into a single well, with each loading followed by a brief pulse of electrical current before the next sample is loaded. The procedure generates single bands created by molecular stacking that exhibit strongly enhanced signal intensities and minimal band broadening. Using optimized voltages and time intervals as many as 20 successive loadings could be performed and up to 800 μL could be loaded into a single well. Gel extraction and fluorescent quantitation demonstrated that approximately 97 % of the DNA from each loading was incorporated into the resultant band. Highly dilute DNA samples (<0.0007 ng per microliter) could be readily detected after six loadings. The method produced good results with either TAE or TBE as electrophoresis buffers, using loading dyes with or without SDS, and in both minigels and large gels. [Display omitted] • Conventional methods for DNA analysis using gel electrophoresis have limited sample loading and detection capabilities. • A new approach involving successive reloading produces strong signal amplification with minimal band broadening. • Up to twenty successive loadings are possible using optimized voltages and time intervals. • Band quality is high using either of the two most common electrophoresis buffers, TAE or TBE. • Previously undetectable DNAs in highly dilute samples are readily detectable with the new method. [ABSTRACT FROM AUTHOR]

The purity and concentration of DNA information from some sample are important to measure the degree of contamination. Anopheles barbirostris mosquitoes samples from Kalukutinggu Village was extracted to obtain the genome DNA using PureLink Genomic DNA mini Kits. The purity and concentration of DNA Anopheles barbirostris was measured by using nano spectrophotometer. The results showed that DNA concentration was 17,33 – 34,79 ug/ml, and their purity were 1,90 average. The purity and concentration of the DNA from the samples showed were not contaminated and eligible for the next step, that is DNA amplification

Graphical abstract Highlights • The waste changed the activity of microorganisms and DNA concentration. • The wastes applied most often induced positive changes in all parameters. • The most favourable changes were induced by sewage sludge. • Used indicators are useful in assessing soil remediation processes. Abstract The investigations were carried out in the form of a field reclamation experiment established in the former "Jeziórko" Sulphur Mine (Poland). For soil reclamation, various combinations of sewage sludge, mineral wool, post-flotation lime and mineral fertilisation were introduced in a single application to the degraded soil. The following parameters were determined in the reclaimed soil in spring and autumn of the 2nd and 3rd year after waste application: number of oligotrophic, cellulolytic and lipolytic bacteria, number of filamentous, cellulolytic and lipolytic fungi, respiratory activity, intensification of cellulose mineralisation, lipase activity and DNA concentration. The applied wastes had a stimulating effect on the growth of the analysed bacterial groups. The growth of fungi was stimulated only in the soil treated with sewage sludge, whereas the values of these parameters in other treatments were usually reduced. The addition of wastes contributed to the intensification of respiration processes and cellulose mineralisation. Lipase activity and DNA concentration fluctuated depending on the type of waste and the time period of investigations; these parameters exhibited the highest values in the soil treated with sewage sludge. In general, the increase in microbiological and biochemical activities was clearly evident in the soil treated with sewage sludge applied separately or in combination with other wastes. The use of sewage sludge together with post-floatation lime, rather than sewage sludge alone, and the application of mineral wool as a 5-cm pad, rather than a mixture of mineral wool and soil, proved to be more beneficial for most of the analysed parameters. The current study is important in the context of safe and effective application of waste for reclamation purposes. In addition, the work raises the innovative aspect of using DNA concentration as an indicator of degraded soil status. [ABSTRACT FROM AUTHOR]

Over the recent years, isolation of high quantities of pure, intact, double-stranded, highly concentrated and not contaminated genomic DNA is prerequisite for successful and reliable large scale genetic and epigenetic analysis and also high quantities of pure DNA are required for these types of studies. The DNA extraction methods developed for human somatic cells are not effective for sperm because of the high rating of nuclear compaction and DNA integrity in sperm cells. In the present study, an economic, reliable and simple method of DNA extraction procedures, were examined to ascertain their relative effectiveness for extracting DNA from human sperm. The quality and quantity of the extracted DNA were assessed by spectrophotometric measurements, methylation-specific PCR (MSP) amplification, and gel electrophoresis. This method was applied successfully from sperm samples (N = 50, age = 33.9 ± 7.8 years) with DNAs absorbency ratios of A260/A280 (1.82 ± 0.03) and A260/230 (1.95 ± 0.16). Gel electrophoresis revealed non-degraded and suitable quality genomic DNA for epigenetic and genetic analysis. The purity and quality of this protocol was closer to the optimum value sand no contamination during manual extraction was observed. It concluded that this described method might have a sufficient quality for subsequent molecular analysis and general genetic and epigenetic methods. [ABSTRACT FROM AUTHOR]

Molekul DNA menunjukkan polarisasi yang kuat sehingga memungkinkan baik gerak elektroforesis berdasarkan muatan negatifnya maupun gerak dielektroforesis berdasarkan induksi polarisasi. Perancangan alat menggunakan kombinasi prinsip elektroforesis dan dielektroforesis dilengkapi perangkat lunak untuk mengukur konsentrasinya sangat diperlukan. Utamanya mengingat uji kualitatif DNA berbasis visualisasi pada gel elektroforesis bersifat sangat subyektif dan kurang terukur. Pengukuran konsentrasi DNA menggunakan spektrofotometer UV/VIS sangat tergantung oleh ketersediaannya di laboratorium. Penelitian bertujuan untuk mendesain piranti untuk mengukur konsentrasi DNA berdasarkan visualisasinya pada gel elektroforesis menggunakan perangkat lunak berbasis MatLab. Pengukuran konsentrasi DNA didasarkan visualisasinya pada gel elektroforesis lalu dibandingkan dengan hasil penghitungan spektrofotometer UV/VIS. Hasil penelitian menggunakan piranti tersebut memperlihatkan visualisasi DNA yang lebih optimal. Hasil pengukuran jumlah DNA menggunakan spektrofotometer memiliki kecenderungan yang sama dengan hasil pengukuran menggunakan perangkat lunak berbasis MatLab meskipun terdapat perbedaan nilai kuantitatif.ABSTRACTMolecules of deoxyribo nucleic acid (DNA) show a strong polarization allowing for both motions of the dielectrophoresis induced by polarization and electrophoresis based on its negative charge. Considering high subjective and less quantifiable result of the visualization based qualitative test of DNA on gel electrophoresis, designing the tool using a combination of the principles of electrophoresis and dielectrophoresis completed with a software for optimization of DNA visualization and to measure the concentration of small and large–sized DNA fragment is very needed. Accuracy of measurement of DNA concentration using a spectrophotometer UV /VIS is depend on its availability in the laboratory. The aim of this study was to design device for optimization of DNA visualization and measuring the concentration in the gel electrophoresis using MatLab- based software. Experiment using this software measured the concentration of DNA based on its visualization and compared it with calculation obtained from spectrophotometer UV/VIS. The research results showed that the amount of DNA analysed using a spectrophotometer tend to similar with the measurement results using the MatLab-based software although there was differences in quantitative values.

Stretching and manipulating DNA efficiently is significant for exploring the properties and applications of single DNA molecules. Here, the influence of concentrations of buffer and DNA on properties of stretched DNA molecules in the molecular evaporation combing (MEC) is investigated systematically with the single molecule fluorescence imaging microscopy and the high-precision drop shape analyzing technology. The stretched degree and uniformity of combed DNA molecules decrease as the buffer concentration are increased from 7 to 20 mM. When the buffer concentration changes from 12 to 15 mM, the stretched DNA molecules are apt to form a ringlike pattern. During the MEC process, there exist two kinds of evaporation modes, i.e., the constant contact angle mode and the constant contact radius mode. The former only takes effect in the lower concentration of buffer and DNA, enabling the uniform stretching. While the latter plays the leading role in the higher concentration, promoting the formation of the ringlike pattern of DNA molecules. [ABSTRACT FROM AUTHOR]

Enterohemorrhagic Escherichia coli (EHEC) serotype O157:H7 (O157) is one of the most notorious human pathogens, causing severe disease in humans worldwide. O157 specifically colonizes the large intestine of mammals after passing through the small intestine, and this process is influenced by differential signals between the two regions. Small regulatory RNAs (sRNAs) are able to sense and respond to environmental changes and regulate diverse physiological processes in pathogenic bacteria. Although some sRNAs of O157 have been extensively investigated, whether these molecules can sense differences between the small and large intestine and influence the preferential colonization in the large intestine by O157 remains unknown. In this study, we identified a new sRNA, Esr055, in O157 which senses the low DNA concentration in the large intestine and contributes to the preferential colonization of the bacteria in this region. The number of O157 wild-type that adhered to the colon is 30.18 times higher than the number that adhered to the ileum of mice, while the number of the 1Esr055 mutant that adhered to the colon decreased to 13.27 times higher than the number adhered to the ileum. Furthermore, we found that the expression of Esr055 is directly activated by the regulator, DeoR, and its expression is positively affected by DNA, which is significantly more abundant in the ileum than in the colon of mice. Additionally, combining the results of informatics predictions and transcriptomic analysis, we found that several virulence genes are up-regulated in the ΔEsr055 mutant and five candidate genes (z0568, z0974, z1356, z1926, and z5187) may be its direct targets. [ABSTRACT FROM AUTHOR]

Background: UK Biobank is a large prospective cohort study in the UK established by the Medical Research Council (MRC) and the Wellcome Trust to enable approved researchers to investigate the role of genetic factors, environmental exposures and lifestyle in the causes of major diseases of late and middle age. A wide range of phenotypic data has been collected at recruitment and has recently been enhanced by the UK Biobank Genotyping Project. All UK Biobank participants (500,000) have been genotyped on either the UK Biobank Axiom® Array or the Affymetrix UK BiLEVE Axiom® Array and the workflow for preparing samples for genotyping is described. The genetic data is hoped to provide further insight into the genetics of disease. All data, including the genetic data, is available for access to approved researchers. Data for two methods of DNA quantification (ultraviolet-visible spectroscopy [UV/Vis]) measured on the Trinean DropSenseTM 96 and PicoGreen®) were compared by two laboratories (UK Biobank and Affymetrix). Results: The sample processing workflow established at UK Biobank, for genotyping on the custom Affymetrix Axiom® array, resulted in high quality DNA (average DNA concentration 38.13 ng/μL, average 260/280 absorbance 1.91). The DNA generated high quality genotype data (average call rate 99.48% and pass rate 99.45%). The DNA concentration measured on the Trinean DropSenseTM 96 at UK Biobank correlated well with DNA concentration measured by PicoGreen® at Affymetrix (r = 0.85). Conclusions: The UK Biobank Genotyping Project demonstrated that the high throughput DNA extraction protocol described generates high quality DNA suitable for genotyping on the Affymetrix Axiom array. The correlation between DNA concentration derived from UV/Vis and PicoGreen® quantification methods suggests, in large-scale genetic studies involving two laboratories, it may be possible to remove the DNA quantification step in one laboratory without affecting downstream analyses. This would result in reductions in cost and time to complete the project, allowing generation of genetic data faster and cheaper. [ABSTRACT FROM AUTHOR]

A protocol for molecular characterization of soil microbial communities of tropical ecosystems (Tropical Dry Forest, TDF; Tropical Dry Scrub, TDS and Tropical Montane Forest, TMF) in southern Ecuador, was optimized. These ecosystems were selected due to their great biological interest, its high biodiversity and its vulnerability. Total genomic DNA from soil samples was isolated and then a polymerase chain reaction (PCR) was performed in order to amplify conserved regions of fungal and bacterial genome (using universal primers; ITS F-1 - 5.8S, EUB338 - EUB 518; fungal and bacteria; respectively). We evaluated the effect of some parameters to reach the maximum efficiency of PCR. The results suggest that the DNA starting concentration was the variable with the highest impact on the reaction efficiency. We therefore identified the optimal dilutions per soil type: 1:2, 1:5, 1:20 of the starting DNA preparation for TDS, TDF, and TMF respectively. Furthermore, the efficiency was increased when we determined the optimal concentration of MgCl2 and optimal annealing temperature for every soil type. The obtained average DNA concentration varies between ecosystems, being higher in the samples from TMF. This reflects the presence of higher microbial biomass in soils of this ecosystem compared to TDF and TDS. We, conclude that extrapolating parameters from one soil type to another which affect the PCR efficiency may result in errors. Our results suggest that it is necessary to standardize various parameters, taking into account the soil type and the conserved region of the genome to be amplified. Besides, we could verify that DNA concentration could be a consistent variable to estimate or compare the microbial biomass in different soil types. [ABSTRACT FROM AUTHOR]