Optimization Of DNA Extraction Research Articles

Optimization of DNA Extraction and PCR Conditions for Fungal Metagenome Analysis of Atmospheric Particulate Matter

Optimization of DNA Extraction and PCR Conditions for Fungal Metagenome Analysis of Atmospheric Particulate Matter

Optimization of DNA extraction and sampling methods for successful forensic microbiome analyses of the skin and saliva.

Microbiome studies have contributed to many fields, such as healthcare and medicine; however, these studies are relatively limited in forensics. Microbiome analyses can provide information, such as geolocation and ancestry information, when short tandem repeat (STR) profiling fails. In this study, methods for DNA extraction and sampling from the skin and saliva were optimized for the construction of a Korean Forensic Microbiome Database (KFMD). DNA yields were estimated using four DNA extraction kits, including two automated kits (Maxwell® FSC DNA IQ™ Casework Kit and PrepFiler™ Forensic DNA Extraction Kit, updated) and two manual kits (QIAamp DNA Mini Kit and QIAamp DNA Micro Kit) commonly used in forensic DNA profiling laboratories. Next-generation sequencing of the 16S rRNA V4 region was performed to analyze microbial communities in samples. The Bacterial Transport Swab with Liquid Media (NobleBio), two cotton swabs (PoongSung and Puritan), and nylon-flocked swabs (NobleBio and COPAN) were tested for DNA recovery. The PrepFiler and Maxwell kits showed the highest yields of 3.884ng/μL and 23.767ng/μL from the scalp and saliva, respectively. With respect to DNA recovery, nylon-flocked swabs performed better than cotton swabs. The relative abundances of taxa sorted by DNA extraction kits were similar contributions; however, with significant differences in community composition between scalp and saliva samples. Lawsonella and Veillonella were the most abundant genera in the two sample types. Thus, the Maxwell® FSC DNA IQ™ Casework Kit and nylon-flocked swab (NobleBio) were optimal for DNA extraction and collection in microbiome analyses.

Optimization of DNA extraction and amplification of Kikuyu (Pennisetum clandestinum Hochst. ex Chiov) for molecular identification

Kikuyu (Pennisetum clandestinum Hochst. ex Chiov) is an important forage containing high crude protein for livestock. Molecular analysis of kikuyu relies on high yields of pure DNA and suitable PCR conditions. This research aimed to extract DNA from kikuyu based on weight of the sample and amplify the DNA of Burangrang accession using specific primers. 100 grams and 200 grams leaves of 3 accessions of kikuyu from Burangrang, Bukit Tunggul, and Tangkuban Perahu were extracted by Qiagen Mini Kit Plant. Concentration and purity of DNA were analyzed by NanoDrop Spectrophotometer 2000. DNA from Burangrang accession was amplified using six specific primers at different annealing temperatures. The result showed that the yield of DNA ranged 2.2 µg/µl to 21.4 µg/µl and the purity (ratio) were 1.08 to 2.01. Bukit Tunggul and Burangrang accession showed the same interaction pattern on the sample weight for concentration and purity. One hundred grams of leaves from Burangrang accession produce the highest concentration and the best purity of DNA, but no difference between other weight and accession. Reproducible amplifiable products were observed in all PCR reactions except primer K2. These results indicated that optimized protocol is suitable for further work on molecular identification of kikuyu.

Optimization of DNA Extraction from Field-Collected Mammalian Whole Blood on Filter Paper for Trypanosoma cruzi (Chagas Disease) Detection.

Blood filter paper strips are cost-effective materials used to store body fluid specimens under challenging field conditions, extending the reach of zoonotic pathogen surveillance and research. We describe an optimized procedure for the extraction of parasite DNA from whole blood (WB) stored on Type I Advantec Nobuto strips from both experimentally spiked and field-collected specimens from canine and skunks, respectively. When comparing two commercial kits for extraction, Qiagen’s DNeasy Blood & Tissue Kit performed best for the detection of parasite DNA by PCR from Trypanosoma cruzi-spiked canine WB samples on Nobuto strips. To further optimize recovery of β-actin from field-collected skunk WB archived on Nobuto strips, we modified the extraction procedures for the Qiagen kit with a 90 °C incubation step and extended incubation post-addition of proteinase K, a method subsequently employed to identify a T. cruzi infection in one of the skunks. Using this optimized extraction method can efficaciously increase the accuracy and precision of future molecular epidemiologic investigations targeting neglected tropical diseases in field-collected WB specimens on filter strips.

Optimization of DNA Extraction from Avian Blood

Our research aimed to optimize a protocol of DNA extraction from avian blood. Three methods of DNA extraction (A, B and C) have been applied to the blood samples, using the protocol of phenol-chloroform isopropanol DNA extraction. Method (A) was set to use 2% SDS. Method (B) was set to use 20% SDS. Method (C) was set to use 20% SDS with modifications in the digestion step, which was accomplished in two different steps instead of one step at normal phenol-chloroform. The modification was very useful to get the lowest value of protein contamination especially, if the samples undergoing a long storage period. DNA concentration and purity were estimated in the samples of each method. The results revealed that gel electrophoresis of method (A) yielded weak bands and very few or no smears, whereas method (B) yielded smeared DNA and method (C) yielded intense DNA band with few or no smear in the gel. The highest average DNA concentration was obtained by method B (137.368ng/ml), followed by the method C (84.102ng/ml), and the lowest DNA concentration was obtained by the method A (76.104ng/ml), but the differences were insignificant. There were significant differences in DNA purity between the methods. The highest purity was achieved by method C (1.9364nm), followed by method B (1.6866), and the lowest purity was by applying method A (1.5676). The study suggests extracting DNA from avian blood by using method (B) or method (C).

Automated, high-throughput DNA extraction protocol for disposable label free, microfluidics integrating DNA biosensor for oil palm pathogen, Ganoderma boninense

Basal Stem Rot and Upper Stem Rot diseases caused by pathogenic fungus Ganoderma boninense continue to be a major plight in the palm oil industry. Despite continuous research in combating the problem, resolution remains stagnant. Here, developed an automated, high-throughput DNA extraction protocol on microfluidics device for a quick, disposable, label-free detection, within 2 h of assessment. Microfluidics was designed using AutoCAD software, fabricated on microscopic glass substrate using negative photoresist (SU-8 2015) and molded with a biopolymer silicone, Polydimethylsiloxane. G. boninense and unknown pathogenic fungus isolated from rotten mushroom were grown and fractions of extracted DNA were pooled and analyzed for comparison along with synthetic ssDNA of G. boninense. Results from LPM and HPM show successful fabrication with ≤0.1 mm variance between the dimensions in the design before and after lithography process. The PDMS microfluidics show no leakage when run with DNA samples. Analyses from I-V measurement, UV–vis, FTIR, and PCR show comparable results between extracted and synthetic ssDNA of G. boninense and a contrast with the unknown pathogenic fungus, indicating a successful DNA extraction protocol via microfluidics for label-free identification of G. boninense. Optimization of DNA extraction can be further devised for applicability on lab-on-a-chip devices.

Optimization of DNA extraction from dental remains.

Efficient DNA extraction procedures is a critical step involved in the process of successful DNA analysis of such samples. Various protocols have been devised for the genomic DNA extraction from human tissues and forensic stains, such as dental tissue that is the skeletal part that better preserves DNA over time. However DNA recovery is low and protocols require labor‐intensive and time‐consuming step prior to isolating genetic material. Herein, we describe an extremely fast procedure of DNA extraction from teeth compared to classical method. Sixteen teeth of 100‐year‐old human remains were divided into two groups of 8 teeth and we compared DNA yield, in term of quantity and quality, starting from two different sample preparation steps. Specifically, teeth of group 1 were treated with a classic technique based on several steps of pulverization and decalcification, while teeth of group 2 were processed following a new procedure to withdraw dental pulp. In the next phase, the samples of both group underwent the same procedure of extraction, quantification and DNA profile analysis. Our findings provide an alternative protocol to obtain a higher amount of good quality DNA in a fast time procedure, helpful for forensic and anthropological studies.

Optimization of DNA Extraction from Individual Sand Flies for PCR Amplification.

Numerous protocols have been published for extracting DNA from phlebotomines. Nevertheless, their small size is generally an issue in terms of yield, efficiency, and purity, for large-scale individual sand fly DNA extractions when using traditional methods. Even though this can be circumvented with commercial kits, these are generally cost-prohibitive for developing countries. We encountered these limitations when analyzing field-collected Lutzomyia spp. by polymerase chain reaction (PCR) and, for this reason, we evaluated various modifications on a previously published protocol, the most significant of which was a different lysis buffer that contained Ca2+ (buffer TESCa). This ion protects proteinase K against autolysis, increases its thermal stability, and could have a regulatory function for its substrate-binding site. Individual sand fly DNA extraction success was confirmed by amplification reactions using internal control primers that amplify a fragment of the cacophony gene. To the best of our knowledge, this is the first time a lysis buffer containing Ca2+ has been reported for the extraction of DNA from sand flies.

Optimization of DNA extraction and PCR protocols for plants with high Phenolics: Bael, Mango, Pomegranate as examples

The genetic studies of tropical tree species containing high amount of phenols are greatly hampered by the inability to extract sufficient quantities of high quality DNA and the pres­ence of PCR inhibitors in extracted DNA samples. While there are some DNA extraction and PCR protocols for such species, no consistency in results. Hence, optimization of such protocols is a prerequisite for conducting research studies on tropical flora. This study reports the optimization of cheaper DNA extraction and PCR procedures for plants having high amount of phenol­ic compounds and PCR inhibitors. Three fruit crop species, Bael (Aegle marmelos L. Correa), Pomegranate (Punica granatum L.) and Mango (Mangifera indica L.), with distinctly diverse sec­ondary metabolic profiles, were used as examples. The CTAB DNA extraction method, with some modifications, was compared with two commercially available DNA extraction kits namely; Promega Wizard® Genomic DNA purification kit and QIAGEN DNeasy® Plant Mini kit. DNA from three to five genotypes from each species was extracted from each method and the quality and quantity were assessed. Spermidine was added to the PCR mix at the rate of 0.8 μM to block the PCR inhibitors and the DNA samples were amplified using universal plant barcoding primer pair rbcL, and SSR or ISSR primers. The modified CTAB method resulted significantly higher quantity of quality DNA in all sam­ples compared to two commercial kits. Henceforth DNA extract­ed from CTAB method, and the two commercial kits were used to precede PCR. However, expected bands were not generated in regular PCR. Interestingly, the inclusion of spermidine amplified the relevant band/s in relatively easy PCR reactions such as rbcL, as well as trickier reactions such as SSR and ISSR. These results suggest that cheaper alternative procedures used in this research study could be used successfully for the range of applications in plants with array of secondary metabolic profiles.

Optimising DNA extraction from a critically endangered marine alga

Prior to conducting genetic studies on critically endangered species, it is desirable to optimise DNA extraction to limit destructive sampling and reduce impacts on populations. We optimized DNA extraction for the critically endangered marine alga, Nereia lophocladia. Only gentle cryogenic homogenization using a mortar and pestle, followed by brief incubation in lysis buffer, produced high molecular weight DNA. However, this DNA still required post hoc cleanup to be of sufficient quantity for PCR and next generation sequencing. Importantly, sufficient DNA can be obtained from as little as 25 mg of algal material while still allowing contingencies for losses associated with cleanup. Optimisation of DNA extraction for this critically endangered species is paving the way for genomic studies to inform management strategies and conservation.

Simultaneous detection of bovine and porcine DNA in pharmaceutical gelatin capsules by duplex PCR assay for Halal authentication

BackgroundIn the pharmaceutical industry, hard- and soft-shelled capsules are typically made from gelatin, commonly derived from bovine and porcine sources. To ensure that pharmaceutical products comply with halal regulations in Muslim countries (no porcine products allowed), development of a valid, reliable, quick, and most importantly, cost-effective tests are of utmost importance.MethodsWe developed a species-specific duplex polymerase chain reaction (PCR) assay targeting 149 bp porcine and 271 bp bovine mitochondrial DNA (mtDNA) to simultaneously detect both porcine and bovine DNA (in one reaction at the same time) in gelatin. Some additional simplex PCR tests (targeting 126 bp bovine and 212 bp porcine mtDNA) and real-time PCR using a commercially available kit (for identification of porcine DNA) were used to verify the selectivity and sensitivity of our duplex PCR. After optimization of DNA extraction and PCR methods, hard/soft pharmaceutical gelatin capsules (containing drug) were tested for the presence of porcine and/or bovine DNA.ResultsDuplex PCR detected the presence of as little as 0.1% porcine DNA, which was more accurate than the commercially available kit. Of all gelatin capsules tested (n = 24), 50% contained porcine DNA (pure porcine gelatin alone or in combination with bovine gelatin).ConclusionsDuplex PCR presents an easy-to-follow, quick, low-cost and reliable method to simultaneously detect porcine and bovine DNAs (>100 bp) in minute amounts in highly processed gelatin-containing pharmaceutical products (with a 0.1% sensitivity for porcine DNA) which may be used for halal authentication.Graphical abstractSimultaneous detection of porcine and bovine DNA in gelatin capsules by duplex PCR

Optimization of DNA extraction for advancing coral microbiota investigations

BackgroundDNA-based sequencing approaches are commonly used to identify microorganisms and their genes and document trends in microbial community diversity in environmental samples. However, extraction of microbial DNA from complex environmental samples like corals can be technically challenging, and extraction methods may impart biases on microbial community structure. MethodsWe designed a two-phase study in order to propose a comprehensive and efficient method for DNA extraction from microbial cells present in corals and investigate if extraction method influences microbial community composition. During phase I, total DNA was extracted from seven coral species in a replicated experimental design using four different MO BIO Laboratories, Inc., DNA Isolation kits: PowerSoil®, PowerPlant® Pro, PowerBiofilm®, and UltraClean® Tissue & Cells (with three homogenization permutations). Technical performance of the treatments was evaluated using DNA yield and amplification efficiency of small subunit ribosomal RNA (SSU ribosomal RNA (rRNA)) genes. During phase II, potential extraction biases were examined via microbial community analysis of SSU rRNA gene sequences amplified from the most successful DNA extraction treatments.ResultsIn phase I of the study, the PowerSoil® and PowerPlant® Pro extracts contained low DNA concentrations, amplified poorly, and were not investigated further. Extracts from PowerBiofilm® and UltraClean® Tissue and Cells permutations were further investigated in phase II, and analysis of sequences demonstrated that overall microbial community composition was dictated by coral species and not extraction treatment. Finer pairwise comparisons of sequences obtained from Orbicella faveolata, Orbicella annularis, and Acropora humilis corals revealed subtle differences in community composition between the treatments; PowerBiofilm®-associated sequences generally had higher microbial richness and the highest coverage of dominant microbial groups in comparison to the UltraClean® Tissue and Cells treatments, a result likely arising from using a combination of different beads during homogenization.ConclusionsBoth the PowerBiofilm® and UltraClean® Tissue and Cells treatments are appropriate for large-scale analyses of coral microbiota. However, studies interested in detecting cryptic microbial members may benefit from using the PowerBiofilm® DNA treatment because of the likely enhanced lysis efficiency of microbial cells attributed to using a variety of beads during homogenization. Consideration of the methodology involved with microbial DNA extraction is particularly important for studies investigating complex host-associated microbiota.

Optimization of DNA extraction in the spiny spider crab Maja brachydactyla: determining the best extraction method, sample conservation and starting tissue

Methodological studies are an important previous step in molecular research. We optimize a method for DNA extraction from eggs and a noninvasive extraction method for adult tissues of Maja brachydactyla. Our results show that lyophilized eggs in B developmental stage are the most suitable for DNA extraction. Regarding adult tissues, we propose setae as an alternative tissue for DNA extraction, since prevents mutilation of specimens, which avoids infections and fitness reduction. That is especially relevant in studies with endangered species of crustaceans.

Extraction of high quality genomic DNA from Madhuca longifolia (J. Koenig ex l.) J.F. macbr.

Madhuca longifolia commonly known as the Mahua tree is a medium to large sized deciduous tree distributed in India, Nepal and Sri Lanka. It is found growing wild and cultivated. The tree is considered as a kalpavriksha by the tribal who are forest dwellers and keenly conserve this tree. The flowers and fruits are edible and the flowers are fermented to prepare mahua drink which is an integral part of their cultural heritage. The seeds are good source of edible fats and used to extract oil. Several pharmacological activities are reported using various parts of the tree. Very little information exists on the molecular aspects of Madhuca longifolia which requires high quality DNA. Optimization of DNA extraction using CTAB and SDS buffer system was standardized. A protocol for extraction of high quality DNA from tree species which are rich in polyphenols is hereby discussed.

Optimization of DNA extraction and PCR protocols for phylogenetic analysis in Schinopsis spp. and related Anacardiaceae.

The Anacardiaceae is an important and worldwide distributed family of ecological and socio-economic relevance. Notwithstanding that, molecular studies in this family are scarce and problematic because of the particularly high concentration of secondary metabolites—i.e. tannins and oleoresins—that are present in almost all tissues of the many members of the group, which complicate the purification and amplification of the DNA. The objective of this work was to improve an available DNA isolation method for Schinopsis spp. and other related Anacardiaceae, as well as the PCR protocols for DNA amplification of the chloroplast trnL-F, rps16 and ndhF and nuclear ITS–ETS fragments. The modifications proposed allowed the extraction of 70–120 µg of non-degraded genomic DNA per gram of dry tissue that resulted useful for PCR amplification. PCR reactions produced the expected fragments that could be directly sequenced. Sequence analyses of amplicons showed similarity with the corresponding Schinopsis accessions available at GenBank. The methodology presented here can be routinely applied for molecular studies of the group aimed to clarify not only aspects on the molecular biology but also the taxonomy and phylogeny of this fascinating group of vascular plants.Electronic supplementary materialThe online version of this article (doi:10.1186/s40064-016-2118-4) contains supplementary material, which is available to authorized users.

Optimization of DNA Extraction and PCR Conditions for Genetic Diversity Study in Artemisia Herba- Alba from Algeria

Artemisia herba alba is an aromatic species very rich in secondary metabolites that can be used in traditional medicine.

Catostylus tagi: partial rDNA sequencing and characterisation of nematocyte structures using two improvements in jellyfish sample preparation.

BackgroundMore than 200 Scyphozoa species have been described, but few have been properly studied regarding their chemical and genetic characteristics. Catostylus tagi, an edible Scyphozoa and the sole European Catostylidae, occurs in summer at Tagus and Sado estuaries. Neither a systematic comparison between the two Catostylus communities nor a chemical approach on their nematocytes had been carried out yet.MethodsIn order to achieve these purposes, optimisation of DNA extraction and of histochemical staining procedures were developed. Catostylus specimens from Tagus and Sado estuaries were compared by ribosomal 18S, 28S, and ITS1 partial sequencing. The morphochemistry of nematocytes was studied by optical and electronic microscopy.ResultsMacroscopic and molecular results indicated that both communities belong to the same species, C. tagi. The hematoxylin and eosin staining allowed the visualisation of nematocyst genesis and indicated a basic character for the macromolecules on the shaft of euryteles and on the tubule of isorhizae and birhopaloids. By Masson’s trichrome procedure, the basic properties of the tubules were confirmed and a collagenous profile for the toxins was suggested. Results of the alcian blue staining showed that the outer membrane of nematocyte may consist of macromolecules with acidic polysaccharides, consistent with NOWA and nematogalectin glycoproteins detected in Hydra, but also with poly-gamma-glutamate complex, chitin-like polysaccharides and hyaluronic acids. Through the von Kossa assays, calcium was detected; its position suggested interactions with polysaccharides of the membrane, with proteins of the contractile system or with both.ConclusionsThe optimisation of sample preparation for DNA extraction may facilitate further studies on little known jellyfish species. The improvement of the smear procedure simplified the use of stained reactions in zooplankton. Moreover, it was shown that good slide images might be acquired manually. The development of specific reactions, with traditional dyes and others, can give important contributions to clarify the chemical nature of the components of nematocytes. The characterisation of nematocyst toxins by staining tests is a goal to achieve.

Review of processing and analytical methods for Francisella tularensis in soil and water

The etiological agent of tularemia, Francisella tularensis, is a resilient organism within the environment and can be acquired in many ways (infectious aerosols and dust, contaminated food and water, infected carcasses, and arthropod bites). However, isolating F. tularensis from environmental samples can be challenging due to its nutritionally fastidious and slow-growing nature. In order to determine the current state of the science regarding available processing and analytical methods for detection and recovery of F. tularensis from water and soil matrices, a review of the literature was conducted. During the review, analysis via culture, immunoassays, and genomic identification were the methods most commonly found for F. tularensis detection within environmental samples. Other methods included combined culture and genomic analysis for rapid quantification of viable microorganisms and use of one assay to identify multiple pathogens from a single sample. Gaps in the literature that were identified during this review suggest that further work to integrate culture and genomic identification would advance our ability to detect and to assess the viability of Francisella spp. The optimization of DNA extraction, whole genome amplification with inhibition-resistant polymerases, and multiagent microarray detection would also advance biothreat detection.

Plant genotyping using fluorescently tagged inter-simple sequence repeats (ISSRs): basic principles and methodology.

Inter-simple sequence repeat PCR (ISSR-PCR) is a fast, inexpensive genotyping technique based on length variation in the regions between microsatellites. The method requires no species-specific prior knowledge of microsatellite location or composition. Very small amounts of DNA are required, making this method ideal for organisms of conservation concern, or where the quantity of DNA is extremely limited due to organism size. ISSR-PCR can be highly reproducible but requires careful attention to detail. Optimization of DNA extraction, fragment amplification, and normalization of fragment peak heights during fluorescent detection are critical steps to minimizing the downstream time spent verifying and scoring the data.

DNA extraction protocols for molecular studies of Asiatic black bears

Abstract Optimization of DNA extraction and amplification techniques increases the reliability of genetic studies used to estimate population size and assess genetic diversity, particularly for non-invasively collected samples. We evaluated the effectiveness of 4 DNA extraction procedures—the Roboscreen and Qiagen kit methods and the Organic and Chelex methods, as applied to invasively and non-invasively collected samples from Asiatic black bears (Ursus thibetanus). We assessed effectiveness based on quantity and purity of the extracts by method, and their ability to amplify genomic DNA via polymerase chain reaction (PCR) amplification of a single simple sequence repeat (SSR) marker. For each method, the greatest average DNA quantity and purity index (86.25%) was from blood, while muscle showed greatest DNA quantity and purity index using the Qiagen DNA extraction method. The Roboscreen kit and Chelex method provided a 57% and 58% PCR success rate, respectively, for hairs, demonstrating the value of such ...