Efficient DNA Recovery Research Articles

Challenges and Innovations in Forensic Identification: Case Study of Decomposed Remains in a Crocodile's Body for Missing Child Identification

Identifying a missing child from decomposed remains in a crocodile's body poses significant challenges and complexity. Forensic investigations involving human remains within animal’s body can present unique difficulties apart from conventional investigative scenarios. Decomposed remains added an additional layer of difficulty as the digestive process of a crocodile can significantly degrade the body and its features. Forensic anthropology examinations were impeded due to the limited availability of tiny bone samples. In this scenario decomposed bone pieces were the only remaining material for DNA analysis, the choice of an efficient DNA recovery procedure becomes crucial for successful outcome. In the study, prior to the DNA extraction process, a decontamination technique was employed, followed by decalcification method on the bone sample. The resulting, clean and decalcified bone sample was then subjected to extraction using a silica-based method, and the extracted DNA was subsequently typed through STR amplification. The derived DNA profile information from the bone sample pertaining to the child is compared with the profile details of parents for identification purposes.

Comparing preservation substrates under field conditions for efficient DNA recovery in bone

Often bones are the only biological material left for the identification of human remains. As situations may occur where samples need to be stored for an extended period without access to cooling, appropriate storage of the bone samples is necessary for maintaining the integrity of DNA for profiling. To simulate DNA preservation under field conditions, pig rib bones were used to evaluate the effects of bone cleaning, buffer composition, storage temperature, and time on DNA recovery from bone samples. Bones were stored in three different buffers: TENT, solid sodium chloride, and ethanol-EDTA, at 20 °C and 35 °C for 10, 20, and 30 days. Bones were subsequently dried and ground to powder. DNA was extracted and quantified. Results show that temperature and storage time have no significant influence on DNA yield. DNA recovery from bones stored in solid sodium chloride or ethanol-EDTA was significantly higher compared to bones stored in TENT, and grinding of bones was facilitated by the extent of dehydration in solid sodium chloride and ethanol-EDTA compared to TENT. Overall, solid sodium chloride was found to be superior over ethanol-EDTA; when it comes to transportation, dry material such as salt eliminates the risk of leaking; it is non-toxic and in contrast to ethanol not classified as dangerous goods. Based on this study’s results, we recommend NaCl as a storage substrate for forensic samples in cases where no cooling/freezing conditions are available.

A comparison of drying methods on the quality for bryophyte molecular specimens collected in the field.

A major challenge in extracting high-quality DNA from bryophytes is the treatment of bryophyte material in the field. The existing and commonly used treatment methods in the field have several shortcomings. Natural drying methods can lead to DNA breaks. In addition, it is highly cumbersome to carry large quantities of silica gel in the field due to its weight and high risk of contamination among samples. In this study, we explored more convenient drying methods to treat bryophyte specimens and promote more efficient DNA recovery. The quantity and quality of genomic DNA extracted from every bryophyte species using different drying methods, including hot-air drying methods (150°C, 80°C, and 40°C), natural drying method, and silica gel drying method, were measured. Spectrophotometry, electrophoresis, and PCR amplification were performed to assess the effects of different drying methods. The results of total DNA purity, total DNA concentration, PCR success, and OD 260/230 ratios suggested that the hot-air drying (40-80°C) was the best method. The morphological comparison revealed that hot-air drying at 40°C and 80°C exerted no significant adverse effects on plant morphology and taxonomic studies. Thus, this method prevents rapid DNA degradation and silica gel pollution and saves the workforce from carrying large amounts of silica gel to the field. Several inexpensive devices, such as portable hairdryers, fan heaters, and electric blankets, are available that can be easily carried to the field for drying molecular specimens.

Uncovering Diagnostic Value of Mitogenome for Identification of Cryptic Species Fusarium graminearum Sensu Stricto.

Fungal complexes are often composed of morphologically nearly indistinguishable species with high genetic similarity. However, despite their close relationship, they can exhibit distinct phenotypic differences in pathogenicity and production of mycotoxins. Many plant pathogenic and toxigenic fungi have been shown to consist of such cryptic species. Identification of cryptic species in economically important pathogens has added value in epidemiologic studies and provides opportunities for better control. Analysis of mitochondrial genomes or mitogenomics opens up dimensions for improved diagnostics of fungi, especially when efficient recovery of DNA is problematic. In comparison to nuclear DNA, mitochondrial DNA (mtDNA) can be amplified with improved efficacy due to its multi-copy nature. However, to date, only a few studies have demonstrated the usefulness of mtDNA for identification of cryptic species within fungal complexes. In this study, we explored the value of mtDNA for identification of one of the most important cereal pathogens Fusarium graminearum sensu stricto (F.g.). We found that homing endonucleases (HEGs), which are widely distributed in mitogenomes of fungi, display small indel polymorphism, proven to be potentially species specific. The resulting small differences in their lengths may facilitate further differentiation of F.g. from the other cryptic species belonging to F. graminearum species complex. We also explored the value of SNP analysis of the mitogenome for typing F.g. The success in identifying F.g. strains was estimated at 96%, making this tool an attractive complement to other techniques for identification of F.g.

Identification of Small Berry Species in Food and Juice Using TaqMan-Based Real-Time PCR

Abstract Background: Public interest is growing for small berries in recent years because they are very delicious, low in energy, and full of bioactive compounds with potential health benefits. Similar to other food products, adulteration of small berry fruit products poses economic and safety problems to consumers. Objective: To protect consumers and regulate the small berry fruit products market, it is necessary to establish a robust method for detecting the authenticity. Methods: In this study, TaqMan-based real-time PCR assay was established for species identification of cranberry, raspberry, and blueberry to ensure authenticity of commercial small berry food products (pulp, dried fruit, fruit juice, jam, and puree). Results: Absolute detection limit was 0.1 pg/μL DNA for raspberries, 1 pg/μL DNA for blueberries, and 10 pg/μL DNA for cranberries. Practical LOD was 0.1% (v/v) for fresh juice. For processed juice, practical LOD was 1% for blueberry and red raspberries, 0.1% for black and yellow raspberries, and 5% for cranberry. Conclusions: The method was shown to be functional and effective to detect the raw material composition of cranberry, raspberry, and blueberry for commercial products. Highlights: TaqMan probe-based real-time PCR methods were designed to identify three small berries (blueberry, raspberry, and cranberry) in berry products. Efficient DNA recovery methods and detection strategy were established to ensure correct and sensitive testing of fresh small berries exhibited a detection limit of 0.1 to 10 pg/μL. The practical minimum detection levels were 0.1 to 5% in fresh and processed juice, including pasteurization and HTHP.

Identification of Small Berry Species in Food and Juice Using TaqMan-Based Real-Time PCR.

Background: Public interest is growing for small berries in recent years because they are very delicious, low in energy, and full of bioactive compounds with potential health benefits. Similar to other food products, adulteration of small berry fruit products poses economic and safety problems to consumers. Objective: To protect consumers and regulate the small berry fruit products market, it is necessary to establish a robust method for detecting the authenticity. Methods: In this study, TaqMan-based real-time PCR assay was established for species identification of cranberry, raspberry, and blueberry to ensure authenticity of commercial small berry food products (pulp, dried fruit, fruit juice, jam, and puree). Results: Absolute detection limit was 0.1 pg/μL DNA for raspberries, 1 pg/μL DNA for blueberries, and 10 pg/μL DNA for cranberries. Practical LOD was 0.1% (v/v) for fresh juice. For processed juice, practical LOD was 1% for blueberry and red raspberries, 0.1% for black and yellow raspberries, and 5% for cranberry. Conclusions: The method was shown to be functional and effective to detect the raw material composition of cranberry, raspberry, and blueberry for commercial products. Highlights: TaqMan probe-based real-time PCR methods were designed to identify three small berries (blueberry, raspberry, and cranberry) in berry products. Efficient DNA recovery methods and detection strategy were established to ensure correct and sensitive testing of fresh small berries exhibited a detection limit of 0.1 to 10 pg/μL. The practical minimum detection levels were 0.1 to 5% in fresh and processed juice, including pasteurization and HTHP.

A plasma modified cellulose-chitosan porous membrane allows efficient DNA binding and provides antibacterial properties: A step towards developing a new DNA collecting card

In forensic DNA analyses, biological specimens are collected and stored for subsequent recovery and analysis of DNA. A cost-effective and efficient DNA recovery approach is therefore a need. This study aims to produce a plasma modified cellulose-chitosan membrane (pCE-CS) that efficiently binds and retains DNA as a potential DNA collecting card. The pCE-CS membrane was produced by a phase separation of ionic liquid dissolving CE and CS in water with subsequent surface-modification by a two-step exposure of argon plasma and nitrogen gas. Through plasma modification, the pCE-CS membrane demonstrated better DNA retention after a washing process and higher rate of DNA recovery as compared with the original CE-CS membrane and the commercial FTA card. In addition, the pCE-CS membrane exhibited anti-bacterial properties against both Escherichia coli and Staphylococcus aureus. The results of this work suggest a potential function of the pCE-CS membrane as a DNA collecting card with a high recovery rate of captured DNA.

DNA recovery from a single bacterial cell using charge-reversible magnetic nanoparticles

Highly efficient DNA recovery from a single bacterial cell was performed by means of imidazole-modified magnetic nanoparticles (Imi-MNPs). The modification by imidazole was confirmed by Fourier transform infrared spectroscopy. The Imi-MNPs were highly efficient at DNA extraction owing to the charge-reversible properties of Imi-MNPs, whereby DNA is attached to the particles at low pH and eluted at high pH because of electrostatic interactions. The DNA recovery ratio was determined by real-time PCR, and it revealed that complete recovery was guaranteed at ≥103 genome copies of Bacillus subtilis. Extraction of DNA from single bacterial cells was followed by PCR amplification of 16S rDNA and capillary electrophoresis. We achieved detection of single bacterial cells with a detection rate of 80%. We believe that our DNA recovery strategy may serve as a powerful tool for efficient DNA extraction and should be useful for quality control of cosmetics, foods, and pharmaceutical products.

Characterization of virus-like particles associated with the human faecal and caecal microbiota

This work represents an investigation into the presence, abundance and diversity of virus-like particles (VLPs) associated with human faecal and caecal samples. Various methodologies for the recovery of VLPs from faeces were tested and optimized, including successful down-stream processing of such samples for the purpose of an in-depth electron microscopic analysis, pulsed-field gel electrophoresis and efficient DNA recovery. The applicability of the developed VLP characterization method beyond the use of faecal samples was then verified using samples obtained from human caecal fluid.

Ultrasensitive isolation, identification and quantification of DNA-protein adducts by ELISA-based RADAR assay.

Enzymes that form transient DNA–protein covalent complexes are targets for several potent classes of drugs used to treat infectious disease and cancer, making it important to establish robust and rapid procedures for analysis of these complexes. We report a method for isolation of DNA–protein adducts and their identification and quantification, using techniques compatible with high-throughput screening. This method is based on the RADAR assay for DNA adducts that we previously developed (Kiianitsa and Maizels (2013) A rapid and sensitive assay for DNA–protein covalent complexes in living cells. Nucleic Acids Res., 41:e104), but incorporates three key new steps of broad applicability. (i) Silica-assisted ethanol/isopropanol precipitation ensures reproducible and efficient recovery of DNA and DNA–protein adducts at low centrifugal forces, enabling cell culture and DNA precipitation to be carried out in a single microtiter plate. (ii) Rigorous purification of DNA–protein adducts by a procedure that eliminates free proteins and free nucleic acids, generating samples suitable for detection of novel protein adducts (e.g. by mass spectroscopy). (iii) Identification and quantification of DNA–protein adducts by direct ELISA assay. The ELISA-based RADAR assay can detect Top1–DNA and Top2a–DNA adducts in human cells, and gyrase–DNA adducts in Escherichia coli. This approach will be useful for discovery and characterization of new drugs to treat infectious disease and cancer, and for development of companion diagnostics assays for individualized medicine.

Optimization of a method for preserving nucleic acids in tissue lysates from archival formalin-fixed tissue samples

Abstract Biomarker analysis of patient biopsies is essential for identifying biomarkers that have predictive or prognostic value. Such analyses not only have the potential for helping us understand the molecular mechanisms underlying response to therapy, but also in personalizing healthcare based on biomarker profiles in patient sub-populations. Patient biopsies are commonly preserved by formalin-fixing and paraffin-embedding (FFPE). Ability to evaluate nucleic-acid-based biomarkers in FFPE tissue is limited by the fact that formalin-fixing degrades the quality of nucleic acids. RNA and miRNA are further degraded in FFPE blocks as time passes. Hence, there is the basic need to find methods for stabilizing nucleic acids in FFPE tissues so that nucleic-acid-based biomarkers can be evaluated in archival tissue samples. We have evaluated various methods of tissue lysate preparation to identify ones that are optimal for stabilizing nucleic acids in storage, and for efficient recovery of DNA, RNA and miRNA. We also evaluated the quality and quantity of DNA and RNA recovered from these lysates across time. We will present a method that preserves quality of nucleic acids across time, and is compatible with protocols for DNA, RNA and miRNA isolation. This method is currently used in our laboratories to prepare tissue lysates from clinical trial samples and has enabled the development of molecular diagnostic assays.

COMPARATIVE ANALYSIS OF METHODS FOR THE PURIFICATION OF DNA FROM LOW‐BIOMASS SAMPLES BASED ON TOTAL YIELD AND CONSERVED MICROBIAL DIVERSITY

ABSTRACT Despite advances in the specificity and sensitivity of molecular biological technologies, the efficient recovery of DNA from low‐biomass samples remains extremely challenging. Optimal methods to purify biomolecules from such environments should (1) achieve the greatest total yield and (2) reflect the true microbial diversity of the sample. These attributes were assessed from five DNA purification regimes: a standard‐manual procedure, MoBio Ultraclean and Promega Wizard kits, and an automated Axcyte AutoLyser method with and without bead‐beating agitation. A homogenous mixture of known concentrations of nine distinct bacterial lineages isolated from low‐biomass environments was prepared and suitable aliquots of subsamples were processed in parallel. DNA products from each of these methods were then subjected to polymerase chain reaction (PCR), quantitative PCR and 16S rRNA clone‐library analysis. The Axcyte AutoLyser outperformed all other purification regimes examined. This automated method consistently both yielded the highest concentration of PCR‐amplifiable DNA, and reported species composition most consistent with the starting solution. PRACTICAL APPLICATIONSThis communication carefully examines the effectiveness of common DNA purification regimes as well as an automated method. Comparative analyses convincingly demonstrate that the different methods not only result in different recovery of genomic DNA, but more importantly, different estimations of microbial diversity in the sample. This report will hopefully inspire investigators from various industries (pharmaceutical, ecological, medical, semiconductor, etc.) who find themselves in the initial phases of large‐scale studies to devote a significant effort into optimizing sample extraction protocols to achieve the most accurate information.

Rapid recovery of DNA from agarose gel slices by coupling electroelution with monolithic SPE

An amino silica monolithic column prepared by in situ polymerization of tetraethoxysilane and N-(beta-aminoethyl)-gamma-aminopropyltriethoxysilane was firstly applied to recover DNA from agarose gel slices by coupling electroelution with monolithic SPE. DNA was electroeluted from the agarose gel slices onto the amino silica monolithic column. The DNA adsorbed on this monolithic column was then recovered using sodium phosphate solution at pH 10. The whole recovery procedure could be completed within 10 min because the use of amino silica monolithic column accelerated the DNA capture and facilitated the DNA release. Electroelution conditions, such as buffer pH, buffer concentration and applied voltage, were online optimized. The average yield for herring sperm DNA, pBR 322 DNA and lambda DNA recovered from 1.0% w/v agarose gel slices were 55+/-4, 50+/-6 and 42+/-7% (n=3), respectively. The polymerase chain reaction performance of pGM plasmid recovered from agarose gel slices demonstrated that the method could provide high-quality DNA for downstream processes. The combination of electroelution with monolithic SPE allows a rapid, simple and efficient DNA recovery method. This technique is especially useful for applications that need to purify small starting amounts of DNA.

Highly magnetizable superparamagnetic iron oxide nanoparticles embedded mesoporous silica spheres and their application for efficient recovery of DNA from agarose gel

Mesoporous silica spheres embedded with the superparamagnetic iron oxide nanoparticles were prepared by a new sol–gel/emulsion approach. The resulting superparamagnetic silica spheres with a saturation magnetization value of 30 emu/g could be enriched completely within 10 s under the application of a 0.4 T magnet. Transmission electron microscopy (TEM) and scanning electron microscopy (SEM) imaging indicated a core-shell structure of the spheres. Adsorption and elution experiments of salmon sperm DNA showed an excellent ability of the superparamagnetic mesoporous silica spheres to adsorb DNA. Electrophoresis experiments show that the superparamagnetic mesoporous silica spheres are qualified for efficient recovery of DNA fragments from agarose gel.

Real-Time PCR for Detection and Quantification of the Protistan Parasite Perkinsus marinus in Environmental Waters

The protistan parasite Perkinsus marinus is a severe pathogen of the oyster Crassostrea virginica along the east coast of the United States. Very few data have been collected, however, on the abundance of the parasite in environmental waters, limiting our understanding of P. marinus transmission dynamics. Real-time PCR assays with SybrGreen I as a label for detection were developed in this study for quantification of P. marinus in environmental waters with P. marinus species-specific primers and of Perkinsus spp. with Perkinsus genus-specific primers. Detection of DNA concentrations as low as the equivalent of 3.3 x 10(-2) cell per 10-microl reaction mixture was obtained by targeting the multicopy internal transcribed spacer region of the genome. To obtain reliable target quantification from environmental water samples, removal of PCR inhibitors and efficient DNA recovery were two major concerns. A DNA extraction kit designed for tissues and another designed for stool samples were tested on environmental and artificial seawater (ASW) samples spiked with P. marinus cultured cells. The stool kit was significantly more efficient than the tissue kit at removing inhibitors from environmental water samples. With the stool kit, no significant difference in the quantified target concentrations was observed between the environmental and ASW samples. However, with the spiked ASW samples, the tissue kit demonstrated more efficient DNA recovery. Finally, by performing three elutions of DNA from the spin columns, which were combined prior to target quantification, variability of DNA recovery from different samples was minimized and more reliable real-time PCR quantification was accomplished.

Efficient recovery of DNA from peripheral blood for diagnostic analysis with a vacuum manifold

Efficient recovery of DNA from peripheral blood for diagnostic analysis with a vacuum manifold

Efficient recovery of DNA from peripheral blood for diagnostic analysis with a vacuum manifold

Efficient recovery of DNA from peripheral blood for diagnostic analysis with a vacuum manifold

Efficient Recovery of DNA From Peripheral Blood for Diagnostic Analysis With a Vacuum Manifold

Efficient Recovery of DNA From Peripheral Blood for Diagnostic Analysis With a Vacuum Manifold

Cationic lipid binding to DNA: characterization of complex formation.

We recently demonstrated that cationic lipids, added in monomer or micellar form, bind to DNA, resulting in the formation of a hydrophobic complex. This complex can serve as a well-defined intermediate in the preparation of DNA-lipid particles (DLPs) with many potential applications for delivery of polynucleotides in vitro and in vivo. To develop a better understanding of the factors governing complex formation, we have characterized the cationic lipid/DNA binding reaction. This was evaluated by measuring DNA and cationic lipid (DODAC) complex formation using the Bligh and Dyer extraction procedure. Efficient recovery of DNA (> 95%) in the organic phase was achieved when sufficient monocationic lipids interact with DNA phosphate groups. The rate of binding depends on the amount of DNA or cationic lipid present in the system. The time required to generate the hydrophobic complex was increased when < 10 micrograms of DNA or < 40 nmol of DODAC was present. Surprisingly, the rate of complex formation was contingent on the incubation period after partitioning the DNA/lipid mixture into organic and aqueous phases. These results suggest that the cationic lipid/DNA complex forms at the aqueous/organic interface and that DNA/lipid binding is dependent on multivalent interactions at this interface. A Scatchard analysis of DNA/DODAC binding demonstrated that the binding reaction exhibits a high degree of positive cooperativity. The apparent dissociation constant (Kn), using data obtained under conditions where DODAC binding to DNA approached saturation, indicated a high-affinity reaction (Kn > 10(-11) mol L-1). At this point, approximately 8400 mol of DODAC was bound per mole of DNA, which is equivalent to a charge ratio (+/-) of 0.585 for the 7.2 kb plasmid used and suggests that formation of the hydrophobic complex occurs at a stage prior to charge neutralization. The influence of other lipids on DNA/cationic lipid binding at the aqueous/organic interface was also studied. Cholesterol and DOPC had little effect on DNA/DODAC binding while the anionic lipids LPI, DOPS, and DMPG inhibited complex formation. The zwitterionic lipid DOPE, however, had a concentration-dependent effect on cationic lipid binding that was also dependent on the mixing order. We believe that this approach for evaluating lipid/DNA binding provides an effective procedure for assessing factors which control the dissociation of lipids from DNA and may be beneficial in the selection of lipids for effective use in gene transfection studies.

A new procedure for efficient recovery of DNA, RNA, and proteins from Listeria cells by rapid lysis with a recombinant bacteriophage endolysin

A method for the rapid lysis of Listeria cells, employing a recombinant Listeria bacteriophage A118 lytic enzyme (PLY118), is described. The procedure can be used with all listerial species. It enables fast, efficient, and gentle recovery of DNA, RNA, or native cellular proteins from small-scale (2- to 5-ml) cultures. Moreover, this approach should be very useful in analytical detection and differentiation of Listeria strains when the release of native nucleic acids or proteins is required.