Pyrimido[1,2-a]-purin-10(3H)-one, M(1)G, is less prone to artifact than base oxidation

Pyrimido[1,2-a]-purin-10(3H)-one (M(1)G) is a secondary DNA damage product arising from primary reactive oxygen species (ROS) damage to membrane lipids or deoxyribose. The present study investigated conditions that might lead to artifactual formation or loss of M(1)G during DNA isolation. The addition of antioxidants, DNA isolation at low temperature or non-phenol extraction methods had no statistically significant effect on the number of M(1)G adducts measured in either control or positive control tissue samples. The number of M(1)G adducts in nuclear DNA isolated from brain, liver, kidney, pancreas, lung and heart of control male rats were 0.8, 1.1, 1.1, 1.1, 1.8 and 4.2 M(1)G/10(8) nt, respectively. In rat liver tissue, the mitochondrial DNA contained a 2-fold greater number of M(1)G adducts compared with nuclear DNA. Overall, the results from this study demonstrated that measuring M(1)G is a reliable way to assess oxidative DNA damage because the number of M(1)G adducts is significantly affected by the amount of ROS production, but not by DNA isolation procedures. In addition, this study confirmed that the background number of M(1)G adducts reported in genomic DNA could have been overestimated by one to three orders of magnitude in previous reports

Evaluation of DNA Extraction Methods of Mule Dung

DNA isolation is a critical step in microbial community analysis of animal dung. DNA isolation from mule dung is challenging due to microbial diversity, composition and chemical nature of mule dung. Therefore, selection of an appropriate DNA isolation method is important to analyse the complete microbial diversity. In the current study, we evaluated the DNA isolation from mule dung samples (n=11) using QiAmp Mini stool kit as per manufacturer’s procedure with modifications. The results suggest that modifications in proprietary column based method improved the DNA quality and quantity suitable for mule dung microbial community analyses

Efficient DNA isolation from moroccan arar tree [Tetraclinis articulata (Vahl) Masters] leaves and optimization of the rapd-pcr molecular technique

Efficient DNA isolation from Moroccan Arar tree [Tetraclinis articulata (Vahl) Masters] leaves and optimization of the RAPD-PCR molecular technique. Molecular genetic analysis of Arar tree [Tetraclinis articulata (Vahl) Masters] is often limited by the availability of fresh tissue and an efficient and reliable protocol for high quality genomic DNA extraction. In this study, two DNA extraction protocols were specifically developed for extracting high quality genomic DNA from Arar tree leaves: modified QIAgen DNA Kit and protocol developed by Ouenzar et al. (1998). DNA yield and purity were monitored by gel electrophoresis and by determining absorbance at UV (A260/A280 and A260/A230). Both ratios were between 1.7 and 2.0, indicating that the presence of contaminating metabolites was minimal. The DNA yield obtained ranged between 20 to 40 μg/g of plant materiel. The Ouenzar and collaborators protocol gave higher yield but was more time consuming compared to QIAgen Kit. However, both techniques gave DNA of good quality that is amenable to RAPD-PCR reactions. Additionally, restriction digestion and PCR analyses of the obtained DNA showed its compatibility with downstream applications. Randomly Amplified Polymorphic DNA profiling from the isolated DNA was optimized to produce scorable and clear amplicons. The presented protocols allow easy and high quality DNA isolation for genetic diversity studies within Arar tree

A rapid and easy method for the DNA extraction from Cryptococcus neoformans

DNA isolation from C. neoformans is difficult due to a thick and resistant capsule. We have optimized a new and rapid DNA isolation method for Cryptococcus using a short urea treatment followed by a rapid method using a chelex resin suspension. This procedure is simpler than previously reported methods

Congenital diaphragmatic hernia : the importance of genetic and environmental factors

For the studies described in this thesis we used a study protocol 'Environmental and Genetic factors in Congenital Diaphragmatic Hernia and Esophageal Atresia', approved by the Institutional Review Board, in collaboration with the parent support groups, 'Stichting Hernia Diafragrnatica' and 'Vereniging Ouders Kinderen Slokdarmafsluiting'. During admission of the patient in our hospital and via meetings of the parent support groups, patients and their parents were included. After informed consent by the parents we took blood samples from the parents for DNA-isolation and storage, and also blood from the mother for PCB-analysis. From the patients we took blood samples, if possible combined with a regular blood sample, for karyotyping and for a cell-line and DNA-isolation, or cheekswaps for DNA-isolation

Using alternative buffer for DNA genomic isolation in forest trees

DNA optimization procedures can be carried out on the type of buffer used during extraction or physical handling techniques in separating genomic DNA from other compounds. The research using Three types of buffer: 1. CTAB, 2. Detergents containing Alkyl Benzene Sulfonate (ABS) surfactants and Three Detergents containing Sodium Laureth Sulphate (SLS) surfactants. This study aims to obtain an optimal DNA isolation method to produce genomic DNA of good quality and sufficient quantity so that it can be used for genetic diversity analysis in forest trees and to determine the optimal alternative buffer for CTAB to facilitate DNA isolation in remote areas, which generally hard to get CTAB. The parameters measured in this study were the presence of DNA and DNA concentration. The results showed that DNA isolation of 12 species of forest trees was successfully carried out using CTAB buffer and Detergent containing ABS surfactants by visualizing the genomic DNA bands from the results of the electrophoresis and Nanodrop spectrophotometer meanwhile Detergent containing SLS surfactants buffer was not successful in DNA isolation. The highest DNA quantity (DNA concentration) was found in 19 samples using CTAB buffer and the detergent containing ABS surfactants buffer with a concentration of 1403,8 - 3412,7 ng/µl. The conclusion of this study was CTAB buffer and the Detergent containing ABS surfactants can also be used as an alternative to a simple buffer for DNA isolation experiments

Comparative Study of Sperm DNA Isolation Method for Forensic Analysis

Forensic is a multi-discipline science that is used to obtain evidence of various criminal cases, such as rape. DNA analysis on sperm specimen is needed to identify the rapist. However, the success of this analysis depends on the DNA isolation method used. Several methods of DNA isolation from human sperm have been developed, but no method has been proven effective for the forensic analysis need. This study aimed to determine the effective sperm DNA isolation method for forensic analysis. In this study, the DNA of sperm specimens was isolated using three methods: Boiling Water, modified TRIzol, and Chelex-100. The DNA isolation result was visualized using agarose gel electrophoresis method. The concentration and purity of isolated DNA were measured using a Nanodrop by comparing the absorbance of DNA at λ 260 nm and protein at λ 280 nm. The effectiveness of the sperm DNA isolation method was determined based on the concentration and purity of DNA, the specimen volume, the implementation time, and the costs involved. The result showed that the successful methods for isolating sperm DNA were TRIzol and Chelex-100. The quantity of DNA isolated using the modified TRIzol method was 1,5 times higher than Chelex-100 but equired 120 times more specimen volume than Chelex-100. From 25 µl sperm specimens, the concentration of DNA isolated using the Chelex-100 method was 612.6 ng/µl with a purity of about 1.7. Therefore, it can be concluded that the Chelex-100 is the most effective method for isolating sperm DNA for forensic analysis.Forensic is a multi-discipline science that is used to obtain evidence of various criminal cases, such as rape. DNA analysis on sperm specimen is needed to identify the rapist. However, the success of this analysis depends on the DNA isolation method used. Several methods of DNA isolation from human sperm have been developed, but no method has been proven effective for the forensic analysis need. This study aimed to determine the effective sperm DNA isolation method for forensic analysis. In this study, the DNA of sperm specimens was isolated using three methods: Boiling Water, modified TRIzol, and Chelex-100. The DNA isolation result was visualized using agarose gel electrophoresis method. The concentration and purity of isolated DNA were measured using a Nanodrop by comparing the absorbance of DNA at λ 260 nm and protein at λ 280 nm. The effectiveness of the sperm DNA isolation method was determined based on the concentration and purity of DNA, the specimen volume, the implementation time, and the costs involved. The result showed that the successful methods for isolating sperm DNA were TRIzol and Chelex-100. The quantity of DNA isolated using the modified TRIzol method was 1,5 times higher than Chelex-100 but equired 120 times more specimen volume than Chelex-100. From 25 µl sperm specimens, the concentration of DNA isolated using the Chelex-100 method was 612.6 ng/µl with a purity of about 1.7. Therefore, it can be concluded that the Chelex-100 is the most effective method for isolating sperm DNA for forensic analysis

Comparison of methods for isolation of bacterial and fungal DNA from human blood

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

Kualitas DNA dari bakso yang beredar di pasaran Kabupaten Bojonegoro

Meatballs are one of the most popular processed meat products in Indonesia. As processed food produced by livestock, meatballs are susceptible to alduteration. The DNA-based detection method is the most widely used and the susceptibility depend on the DNA quality. The aim of this research was to investigate the DNA isolation procedure of commercial meatballs. The study was conducted by taking 36 samples of meatballs from meatball stalls in Bojonegoro Regency. Reference meatballs as a positive control were prepared in the laboratory. DNA isolation was carried out on the collected sample meatballs, reference meatballs, and 10 species of fresh meat for comparison. The concentration and purity of DNA were measured using a spectrophotometer and visualization of the isolation results was performed using agarose gel electrophoresis and UV transilluminator. The results of DNA isolation were compared descriptively qualitatively between samples of meatballs on the market, reference meatballs, and fresh meat. The results of DNA isolation showed that the meatball samples from the market were successfully isolated by showing the concentration values ​​and visualization with agarose gel electrophoresis. The results of electrophoresis and the value of the DNA purity index showed that the DNA was not pure