Effective DNA Extraction Research Articles

Improved Method for DNA Extraction and Purification from Tetrahymena pyriformis.

Tetrahymena pyriformis (protozoa) is intensely investigated as a model organism, offering numerous advantages in comprehensive and multidisciplinary studies using morphologic or molecular methods. Since DNA extraction is a vital step of any molecular experiment, here a new mixed surfactant (Sodium dodecyl sulfate (SDS) 20%/Triton X-100) was adopted for effective DNA extraction from Tetrahymena pyriformis under an easy, fast protocol. The efficiency of this technique was then compared with three widely-used alternative techniques, namely the Chelex 100 matrix, Ammonium pyrrolidine dithiocarbamate (APD) complex and SDS–chloroform methods. DNA extraction was analyzed by pulsed-field gel electrophoresis, spectral measurement, fluorometry (Qubit), restriction enzyme digestion, and polymerase chain reaction. Data analysis revealed that the quantity and quality of the recovered DNA varied depending on the applied DNA extraction method. The new method (SDS 20%/Triton X-100) was the most efficient for extracting DNA from Tetrahymena pyriformis with high integrity and purity, affordable cost, less time, and suitability for molecular applications.

Comparison of Effective DNA Extraction method for Molecular biological study of Mycobacterium leprae

Comparison of Effective DNA Extraction method for Molecular biological study of <i>Mycobacterium leprae</i>

The comparison of DNA extraction techniques in human bone and tooth samples exposed to high heat

In the identification of forensic cases, it is critical to obtain information to identify the victimÂ’s identity from burnt bone and tooth remains. When bone and tooth are burned, both their physical and chemical properties change significantly, which prevents anthropological evaluations and successful DNA profiling for identification purposes. Heat causes difficulties in the identification techniques in which bones and teeth are used, depending on the degree of heat exposure. For this reason, with the changes occurring in bone and tooth during combustion, it is necessary to evaluate the results of observation and analysis, and to be used the information obtained in a certain order. It is very difficult to extract DNA from the samples obtained from fire, explosion, motor vehicle and aircraft accidents and other traumatic events by traditional methods. The only biological material available to determine the typing and the identification of missing persons or unknown remains in different situations, such as mass fatality events, wars or socio-political events, is often only human remains. Effective DNA extraction procedures are critical steps in successful DNA analysis of skeletal remains. Due to variations in DNA and heterogeneity within the bone, unfortunately, the only reliable method for obtaining DNA from overly degraded specimens such as burnt bones and teeth in the literature, is still not available. In the study, present extraction methods are compared and as a result it was suggested that phenol-chloroform and Qiagen DNA Mini Kit could be used in routine with a newly developed modified procedure in order to increase efficiency and efficiency in forensic genetic identification.

Simulation of DNA extraction and separation from salivary fluid by superparamagnetic beads and electromagnetic field in microfluidic platform

The conceptual simulation study of DNA extraction and separation from salivary fluid sample are mainly divided into two parts. The initial study covered microfluidic channel design and effective DNA extraction by using mobilizing superparamagnetic (SPM) beads in COMSOL Multiphysics® software. The subsequent part presented simulation work for DNA separation from SPM beads in electromagnetization field. The main objective of this study in microfluidic platform in presence of electromagnetic field was to achieve higher and uncontaminated yield of DNA as sample preparation step which can be applied for clinical analysis. COMSOL Multiphysics® simulation software was used to study the entire work and presented in this paper with complete study steps. The attached DNA onto SPM beads was separated by specifying computational fluid dynamics in laminar flow, particle tracing module and electromagnetic field by using external electromagnetic coil as magnet array. Most of the SPM beads were captured and held by magnetic field when 100 mT/min was generated by electromagnetic coil. 82.01% of uncontaminated DNA yield from salivary fluid was able to separate from SPM beads after DNA extraction in microfluidic channel. In the absence of external magnetic field, the produced superparamagnetism enable SPM beads to flip in an isotropy provide efficient separation. The mechanical and chemical stability of silica surface of SPM beads provide an excellent chromatography separation medium for DNA sample preparation for further downstream analysis.

Development of a rapid, sensitive and specific DNA-based method to detect Ralstonia solanacearum in potato for quarantine purposes

Bacterial wilt of potato is considered as one of the most destructive diseases of potato. The use of healthy seed potatoes is the most effective means to control the disease. Ralstonia solanacearum, the causal organism of bacterial wilt of potato, is considered as a quarantine pest in Sri Lanka. Therefore, there is a requirement to detect R. solanacearum in seed potatoes quickly and reliably for quarantine purposes. From this study, it was expected to present a rapid and effective DNA extraction method for PCR mediated detection of R. solanacearum in potato tubers for quarantine purposes. The sensitivity of the developed detection method was assessed. The present study developed a rapid, sensitive and specific DNAbased method for detection of R. solanacearum in potatoes with 102 cfumL-1 sensitivity. The developed detection method is user-friendly as it does not require more complicated and toxic chemical substances; consists of few steps of handling; and generates the result within one day, which makes this method more appropriate for quarantine purposes in Sri Lanka.

Evaluation of Genomic DNA-Extraction Methods for Molecular Analysis of Solanecio biafrae

ABSTRACT Solanecio biafrae (Oliv. & Hiern) C. Jеffrеу, an indigenous vegetable of the humid tropics, is rich in minerals and easy to cultivate, but its vegetative growth is slow, making production fall short of demand. An efficient and effective genomic DNA-extraction method is necessary for a comprehensive molecular analysis to increase leaf yield and further improve proximate and mineral contents of the vegetable. Cetyltrimethylammonium bromide (CTAB), sodium dodecyl sulfate (SDS), and Qiagen kit (QIAGEN) methods were evaluated for extraction of genomic DNA from young leaves suitable for PCR amplification for molecular analyses. Quantity and quality of the DNA were examined spectrophotometerically and with gel electrophoresis. Amplification of the DNA by PCR for molecular analysis was assessed with five inter-simple sequence repeat (ISSR) primers. The mean DNA yield using QIAGEN, CTAB, and SDS methods were 71.0, 35.4, and 38.8 µg∙µL−1∙g−1 of tissue, respectively. The mean 260/280 nm ratios of QIAGEN, CTAB, and SDS were 1.84, 1.83, and 1.79, respectively. Gel electrophoresis indicated presence of DNA free of contaminants in all samples using three methods. A total of 270 distinct bands were produced by five ISSR primers with similar patterns among three DNA-extraction methods, ranging from 200–2900 base pairs. Three methods are capable of extracting adequate genomic DNA suitable for PCR amplification. The QIAGEN method produced the highest DNA yield, which was of the same quality with DNA obtained using CTAB and SDS methods.

Simple DNA extraction of urine samples: Effects of storage temperature and storage time

Urine samples are commonly analysed in cases with suspected illicit drug consumption. In events of alleged sample mishandling, urine sample source identification may be necessary. A simple DNA extraction procedure suitable for STR typing of urine samples was established on the Promega Maxwell® 16 paramagnetic silica bead platform. A small sample volume of 1.7mL was used. Samples were stored at room temperature, 4°C and −20°C for 100days to investigate the influence of storage temperature and time on extracted DNA quantity and success rate of STR typing. Samples stored at room temperature exhibited a faster decline in DNA yield with time and lower typing success rates as compared to those at 4°C and −20°C. This trend can likely be attributed to DNA degradation. In conclusion, this study presents a quick and effective DNA extraction protocol from a small urine volume stored for up to 100days at 4°C and −20°C.

Evaluation of Three Protocols of DNA Extraction for Detection of Giardia duodenalis in Human Fecal Specimens

Background: Nowadays a number of methods are used for the extraction of genomic DNA from fecal specimens. Identification and selection of an effective DNA extraction method is considered as one of the most important steps in molecular assays of Giardia duodenalis. Objectives: We compared the effects of 3 different DNA extraction techniques in PCR amplification of a specific area of SSU rRNA gene. Methods: A total of 20 fecal samples containing purified cysts were aliquoted in 3 sub-samples. DNA extraction was performed using the Phenol-Chloroform Isoamyl alcohol (PCI), QIAamp DNA stool mini kit, and YTA Stool DNA Isolation mini Kit. The quantity and purity of extracted DNA were compared. The potency of extracted DNA was tested. Results: The results showed that the most concentrated DNA was obtained from phenol/chloroform/Isoamyl alcohol method and the best purity based on comparing the ratio of A260/230 was obtained from QIAamp DNA stool mini kit. In this study the diagnostic sensitivity of QIAamp DNA stool mini kit, phenol/chloroform/Isoamyl alcohol, and YTA Stool DNA Isolation mini Kit methods was 60%, 70%, and 60%, respectively. Conclusions: The application of a proper DNA extraction method leads to obtaining reliable and reproducible results in molecular assays and supports treatment and control strategies of G. duodenalis. In this study, PCR amplification targeting a 350-bp fragment of SSUrRNA gene demonstrated phenol/chloroform/Isoamyl alcohol as the most efficient method.

High-efficiency automated DNA extraction method for degraded old skeletal samples

In case of extreme degradation, bone may be the only suitable material available for successful STR genotyping. However, relatively specialized techniques are required for the extraction of DNA from bone tissues, particularly when the bones have been exposed to adverse environmental conditions and DNA is degraded and present in low concentrations. In this study, to optimize DNA extraction procedure for old and highly degraded skeletal remains, newly developed automated DNA extraction system was compared to the traditional organic DNA extraction methods We adopted Chemagic Magnetic Separation Module I system for automated DNA extraction, with optimized protocols for large volumes (range, 5–15mL) of bone samples. Success rates of genotyping were analyzed by STR genotyping, and mtDNA sequencing analysis. Real-time assays for quantification revealed that human DNA yield from skeletal samples did not show remarkable differences between two methods However, Chemagic MSM I system showed lower concentration of co-extracted PCR inhibitors than the traditional organic extraction methods Furthermore, STR genotyping results showed that Chemagic MSM I system is more effective DNA extraction method for old skeletal remains than organic extraction method. Therefore, here we suggest that the Chemagic MSM I system is efficient automated DNA extraction system for old and highly degraded bone samples and powerful and valuable tool for identification of old skeletal remains.

Evaluation and comparison of FTA card and CTAB DNA extraction methods for non-agricultural taxa.

Premise of the study:An efficient, effective DNA extraction method is necessary for comprehensive analysis of plant genomes. This study analyzed the quality of DNA obtained using paper FTA cards prepared directly in the field when compared to the more traditional cetyltrimethylammonium bromide (CTAB)–based extraction methods from silica-dried samples.Methods:DNA was extracted using FTA cards according to the manufacturer’s protocol. In parallel, CTAB-based extractions were done using the automated AutoGen DNA isolation system. DNA quality for both methods was determined for 15 non-agricultural species collected in situ, by gel separation, spectrophotometry, fluorometry, and successful amplification and sequencing of nuclear and chloroplast gene markers.Results:The FTA card extraction method yielded less concentrated, but also less fragmented samples than the CTAB-based technique. The card-extracted samples provided DNA that could be successfully amplified and sequenced. The FTA cards are also useful because the collected samples do not require refrigeration, extensive laboratory expertise, or as many hazardous chemicals as extractions using the CTAB-based technique.Discussion:The relative success of the FTA card method in our study suggested that this method could be a valuable tool for studies in plant population genetics and conservation biology that may involve screening of hundreds of individual plants. The FTA cards, like the silica gel samples, do not contain plant material capable of propagation, and therefore do not require permits from the U.S. Department of Agriculture (USDA) Animal and Plant Health Inspection Service (APHIS) for transportation.

Inexpensive metagenomic DNA extraction protocol with high quality from marine sediments contaminated by petroleum hydrocarbons.

Marine environments are a reservoir of relevant information on dangerous contaminants such as hydrocarbons, as well as microbial communities with probable degradation skills. However, to access microbial diversity, it is necessary to obtain high-quality DNA. An inexpensive, reliable, and effective metagenomic DNA (mgDNA) extraction protocol from marine sediments contaminated with petroleum hydrocarbons was established in this study from modifications to Zhou's protocol. The optimization included pretreatment of sediment with saline solutions for the removal of contaminants, a second precipitation and enzymatic degradation of RNA, followed by purification of mgDNA extracted by electroelution. The results obtained indicated that the modifications applied to 12 sediments with total petroleum hydrocarbon (TPH) concentrations from 22.6-174.3 (µg/g dry sediment) yielded 20.3-321.3 ng/µL mgDNA with A260/A280 and A260/A230 ratios of 1.75 ± 0.08 and 1.19 ± 0.22, respectively. The 16S rRNA amplification confirmed the purity of the mgDNA. The suitability of this mgDNA extraction protocol lies in the fact that all chemical solutions utilized are common in all molecular biology laboratories, and the use of dialysis membrane does not require any sophisticated or expensive equipment, only an electrophoretic chamber.

Metagenomic Characterization of Microbial Communities In Situ Within the Deeper Layers of the Ileum in Crohn's Disease.

Background & AimsMicrobial dysbiosis and aberrant host–microbe interactions in the gut are believed to contribute to the development and progression of Crohn’s disease (CD). Microbiome studies in CD typically have focused on microbiota in feces or superficial mucosal layers of the colon because accessing DNA from deeper layers of the bowel is challenging. In this study, we analyzed the deep tissue microbiome in patients who underwent surgical resection of the small intestine.MethodsParaffin blocks were obtained from 12 CD patients undergoing ileocecal resection, and healthy ileum samples (inflammatory bowel disease–free controls) were obtained from 12 patients undergoing surgery for right-sided colon cancer. Diseased and healthy-appearing ileum was identified using microscopy, and paraffin blocks were macrodissected using a core needle to specifically isolate DNA. Illumina Whole Genome Sequencing was used for microbial sequence identification and subsequent taxonomic classification using the PathSeq tool.ResultsWe observed significant differences between the microbiome of CD samples vs inflammatory bowel disease–free controls, including depletion of Bacteroidetes and Clostridia. Notably, microbial composition at the phyla level did not differ markedly between healthy and diseased areas of CD patients. However, we observed enrichment of potentially pathogenic organisms at the species level.ConclusionsOur study showed dysbiosis within deeper layers of the ileum of CD patients, specifically enrichment of enterotoxigenic Staphylococcus aureus and an environmental Mycobacterium species not described previously. Future studies with larger cohort sizes are warranted to confirm these findings. Studies would benefit from effective microbial DNA extraction methods from paraffin sections and host nucleic acid depletion approaches to increase microbial read coverage.

GENOMIC DNA EXTRACTION METHOD FROM MATURE LEAF OF LAI (Durio kutejensis Becc.)

Lai ( Durio kutejensis Becc.) is an indigeneous germplasm of Kalimantan which has some superiorities compared to its close-relative, durian ( Durio zibethinus Murr.). Genetic exploration of lai is important to support its breeding program. According to rapid development in molecular biology, genetic exploration effort of lai will be easier. One of significant step in any molecular biology activities is DNA isolation to produce high quality DNA for further analysis. Leaves of lai, as other perennial crop, contain of high concen-tration of polysaccharides and polyphenol which will be co-extracted with the DNA. These compounds can interfere enzymes activities in subsequent molecular analysis. The aim of this study was to establish an optimal and effective DNA extraction method to obtain high-quality DNA from mature leaf of lai. An established extraction buffer and its modification were used in this study. The result showed that modification 4 could produce high quality DNA, and was considered to be the most effective DNA extraction method for mature leaf of lai.

Simple DNA extraction method for SSR-PCR analysis from different tissues ofTapiscia sinensis, an endangered plant species

A simple and rapid method to process different tissues of Tapiscia sinensis for effective plant DNA extraction were established in the study. DNA quality and yield obtained from bark, leaves and seeds were evaluated by Eppendorf BioPhotometer and SSR-PCR analysis. The results indicated that extracted genomic DNA based on the established method was sufficient for SSR-PCR profiling of different tree tissues. Hence, the sample treatment and extraction method can be used for SSR-PCR applications in tree genetic and molecular analysis research.

A hybrid DNA extraction method for the qualitative and quantitative assessment of bacterial communities from poultry production samples.

The efficacy of DNA extraction protocols can be highly dependent upon both the type of sample being investigated and the types of downstream analyses performed. Considering that the use of new bacterial community analysis techniques (e.g., microbiomics, metagenomics) is becoming more prevalent in the agricultural and environmental sciences and many environmental samples within these disciplines can be physiochemically and microbiologically unique (e.g., fecal and litter/bedding samples from the poultry production spectrum), appropriate and effective DNA extraction methods need to be carefully chosen. Therefore, a novel semi-automated hybrid DNA extraction method was developed specifically for use with environmental poultry production samples. This method is a combination of the two major types of DNA extraction: mechanical and enzymatic. A two-step intense mechanical homogenization step (using bead-beating specifically formulated for environmental samples) was added to the beginning of the "gold standard" enzymatic DNA extraction method for fecal samples to enhance the removal of bacteria and DNA from the sample matrix and improve the recovery of Gram-positive bacterial community members. Once the enzymatic extraction portion of the hybrid method was initiated, the remaining purification process was automated using a robotic workstation to increase sample throughput and decrease sample processing error. In comparison to the strict mechanical and enzymatic DNA extraction methods, this novel hybrid method provided the best overall combined performance when considering quantitative (using 16S rRNA qPCR) and qualitative (using microbiomics) estimates of the total bacterial communities when processing poultry feces and litter samples.

A Simple and Rapid Leaf Genomic DNA Extraction Method for Pearl Millet (Pennisetum glaucum L. R. Br)

Pearl millet is widely grown as a multi-purpose cereal grain crop for feed, fodder, fuel and mulch predominantly in the semi-arid tropics. It is highly adapted to drought, representing an essential component of the food security and livelihood of many million poor farmers. DNA extraction is unusually difficult in some plants due to the presence of secondary metabolites that interfere with DNA isolation making it very laborious and time consuming. An effective genomic DNA extraction should be simple, cost effective, with good yield and high purity. The protocol used in this study involved the extraction of genomic DNA from fresh leaves using the Sodium dodecyl sulphate (SDS) method with slight modifications including absence of use of liquid nitrogen which is difficult and expensive to obtain in the developing World. Genomic DNA obtained from the ten pearl millet samples using this procedure was good.

Effective DNA extraction method to improve detection of Mycobacterium avium subsp. paratuberculosis in bovine feces

Effective DNA extraction method to improve detection of Mycobacterium avium subsp. paratuberculosis in bovine feces

Evaluation of MolYsis™ Complete5 DNA extraction method for detecting Staphylococcus aureus DNA from whole blood in a sepsis model using PCR/pyrosequencing

Bacterial bloodstream infections (BSI) and ensuing sepsis are important causes of morbidity and mortality. Early diagnosis and rapid treatment with appropriate antibiotics are vital for improving outcome. Nucleic acid amplification of bacteria directly from whole blood has the potential of providing a faster means of diagnosing BSI than automated blood culture. However, effective DNA extraction of commonly low levels of bacterial target from whole blood is critical for this approach to be successful. This study compared the Molzyme MolYsis™ Complete5 DNA extraction method to a previously described organic bead-based method for use with whole blood. A well-characterized Staphylococcus aureus-induced pneumonia model of sepsis in canines was used to provide clinically relevant whole blood samples. DNA extracts were assessed for purity and concentration and analyzed for bacterial rRNA gene targets using PCR and sequence-based identification. Both extraction methods yielded relatively pure DNA with median A260/280 absorbance ratios of 1.71 (MolYsis™) and 1.97 (bead-based). The organic bead-based extraction method yielded significantly higher average DNA concentrations (P<0.05) at each time point throughout the experiment, closely correlating with changes observed in white blood cell (WBC) concentrations during this same time period, while DNA concentrations of the MolYsis™ extracts closely mirrored quantitative blood culture results. Overall, S. aureus DNA was detected from whole blood samples in 70.7% (58/82) of MolYsis™ DNA extracts, and in 59.8% (49/82) of organic bead-based extracts, with peak detection rates seen at 48h for both MolYsis™ (87.0%) and organic bead-based (82.6%) methods. In summary, the MolYsis™ Complete5 DNA extraction kit proved to be the more effective method for isolating bacterial DNA directly from extracts made from whole blood.

Superparamagnetic-bead Based Method: An Effective DNA Extraction from Dried Blood Spots (DBS) for Diagnostic PCR.

Storing blood as dried spots on filter paper is a trustworthy approach used in genetic screening issues which justifies the necessity for a reliable DNA extraction method. The present work aims to investigate the effectiveness of superparamagnetic-bead based method in extracting DNA from dried blood spots (DBS). Sixteen venous blood samples collected in K3-EDTA tubes (400μl of whole blood) were used for the spotting (4 circles each 100μl) on Ahlstrom 226 grad filter papers, for extraction and comparison. To ensure effectiveness, the extracted DNA was checked for quantity using the Quant-iT™ dsDNA Broad-Range Assay Kit and for quality by polymerase chain reaction (PCR) amplification of 344 bp segment of the HBB gene. Hybridization assays based on the dynamic allele specific hybridization (DASH) technique for two hemoglobin beta (HBB) mutations in genomic DNA extracted from DBS of ß-thalassemia patients were also performed to ensure the quality of extraction. The results revealed a compatible effectiveness of the superparamagnetic-bead based method in extracting DNA from DBS particularly when incubating the DBS with lysis buffers BL+BLM overnight. A mean concentration of 21ng/ μl was obtained with lysis buffers BL+BLM overnight incubation compared to 5.2 ng/μl for 2 h incubation with lysis buffers BL+BLM and 4.7 ng/μl when extraction performed using the lysis buffer BLM alone. Moreover, PCR amplification of 344 bp segment of the HBB showed a good quality of the extracted DNA. It was concluded that the superparamagnetic-bead based method is a reliable and effective method for DNA extraction from DBS and can be adopted for genetic diagnostic purposes.

Forensic DNA Typing of Old Skeletal Remains Using AmpFlSTR®Identifiler® PCR Amplification Kit

Background: In this study DNA typing of old skeletal remains was improved through AmpFlSTR®Identifiler®PCR amplification kit using different approaches. Methodology: DNA extraction was carried out by silica columns based total demineralization extraction method.DNA quantification was carried out by Real Time PCR. DNA amplification was carried out by using AmpFlSTR®Identifiler® PCR amplification kit with modified conditions. Capillary electrophoresis and Data analysis were carried out by Genetic Analyzer 3130 (ABI) and GeneMapper ID software version 3.2. Results: DNA was detected in 17 out of 24 skeletal remains. Among them, in 7 samples DNA was in the range of 1-10 pg/µL, in 4 samples it was in the range of 22-69 pg/µL and in 6 samples the DNA was in the range of >100 pg/ µL. The CT value (<30) of 40 cycles indicated that the PCR inhibitors were removed during DNA extraction method. Promising results were obtained by increasing the number of PCR cycles from standard 28 to 33 instead of 32 in PCR reaction. Finally it was observed that consensus approach produced reliable and reproducible DNA profiles from old skeletal remains. Conclusions: Forensic DNA typing of old skeletal remains, through a multiplex AmpFlSTR®Identifiler® PCR amplification kit, is improved by using: a highly effective DNA extraction method, modified and optimized PCR conditions, increasing sensitivity of PCR amplification and consensus approach