DNA Extraction Protocol Research Articles

The PortaLyzer, a DIY tool that allows environmental DNA extraction in the field

The PortaLyzer is a portable homemade device that allows researchers to perform bead-beating steps commonly found in environmental DNA (eDNA) extraction protocols in the field without access to power. This allows researchers to preserve in situ organism abundance by beginning eDNA extraction quickly. The PortaLyzer is composed of a variable speed, battery-powered multi-tool and a vortexer adapter plate. We used the PortaLyzer, in conjunction with the Qiagen DNEasy PowerSoil Pro kit, to successfully field process samples taken from the forelands of the Sólheimajökull and Kvíárjökull glaciers in Iceland, as well as soil samples acquired from a prairie in Indiana. Additionally, we provide evidence that samples held in Buffer CD2 of the DNEasy PowerSoil Pro Kit may be transported to traditional lab spaces and processed up to one month after the initial protocol steps, and still provide an equivalent DNA quality and abundance yield as those processed the same day. These improvements to DNA extraction protocols give researchers more flexibility while sampling, shipping and processing eDNA samples.

Assessing the coral microbiome at the scale of tissue-specific habitats within the coral meta-organism

As sequencing techniques have advanced and become cheaper in recent years, there has been a rapid increase in the number of studies conducted into the role of the microbiome in coral health, physiology, and response to environmental change. However, there is substantial variation in the methodological approaches applied. For example, DNA extraction protocols and the types of tissues sampled from the coral meta-organism are known to influence the downstream analyses of the amplified microbial communities and subsequently the interpretation of the microbiome diversity, stability and role. Studies have generally focused on whole organisms, in which the coral sampling steps homogenize the meta-organism microhabitats, however other studies targeting specific microhabitats have identified sources of variation specific to distinct compartments of the coral’s microbial landscape. Here we present a comparative analysis of methodologies optimized for the generation of coral microbiome data from the coral tissues and whole coral fragments of two commonly studied branching coral genera with distinct tissue structure. We investigate the microbiome of the imperforate Pocillopora, where the coral tissue does not penetrate through the calcium carbonate matrix, and the perforate Acropora, where the coral tissues and skeleton are interwoven throughout the coral branch. Through comparing data generated from different DNA extraction protocols using fixed coral tissues isolated from the coral skeletal structure with fixed whole coral fragments, we identify sources of variation inherent to microbial data generated from different sample types, species, and extraction protocols.

P395 Clinical utility of semi-nested conventional PCR for diagnosis of mucormycosis in fresh clinical samples

Poster session 3, September 23, 2022, 12:30 PM - 1:30 PM ObjectivesWe aim to assess the clinical utility of the semi-nested conventional PCR in smear positive, culture-negative clinical samples for diagnosis of mucormycosis.MethodsThis prospective study was conducted for a period of 3 months (April-June 2021). A total of 218 clinical samples were included from patients in whom smear was positive for aseptate hyphae, but the culture failed to grow within 2 days of incubation or smear was negative but had high suspicion of mucormycosis. Molecular diagnosis was attempted using semi-nested PCR with Mucorales-specific primers targeting 18S region, described previously by Bialek et al.1. Phenol-chloroform based manual DNA extraction protocol was optimized in the mycology laboratory of the department of medical microbiology, PGIMER, Chandigarh. This method was applicable to different types of clinical samples to yield good quality DNA with minimal chances of extraneous contamination (Fig. 1). Amplified PCR products were further sequenced to identify the causative species (Fig. 2).ResultsAmong 218 patients with suspected mucormycosis included in this study, the majority were rhino-orbito-cerebral mucormycosis (ROCM), (77.7%, n = 169), followed by pulmonary mucormycosis (19.2%, n = 42), cutaneous (0.02%, n = 4), and gastro-intestinal (GI) mucormycosis (0.01%, n = 3). In 24 samples, the presence of both septate and aseptate hyphae was seen under microscopic examination raising the possibility of mixed infection. On microscopic examination, 90.3% samples (197/218) had aseptate hyphae while the remaining samples were smear-negative but had strong clinical suspicion of mucormycosis. The molecular technique was able to identify causative agent in 154 culture-negative samples (81.9%, 154/188) and 52.4% (11/21) in smear-negative cases. Among 218 patients, only 20 samples show delayed growth of Mucorales, and on comparison with molecular results 100% concordance was observed. However, 10 patients with strong clinical suspicion for mucormycosis were negative by both conventional and molecular methods. The low culture positivity necessities the molecular diagnosis based on in-house semi-nested PCR using above-mentioned primers followed by Sanger sequencing. In the case of 24 mixed infections with aseptate-septate hyphae, Mucorales-specific PCR correctly identified 23/24 (95.8%) Mucorales as a causative agent. The overall turn-around-time from the sample receiving to diagnosis was ˂48 h. Overall, R. arrhizus (85/143, 59.4%) was most commonly associated with ROCM, while R. microsporus (13/38, 34.2%), and R. homothallicus (5/38, 13.1%) were seen mainly with pulmonary mucormycosis. Apophysomyces and Saksenaea genus were associated with GI and cutaneous mucormycosis.ConclusionsThe molecular technique utilizing semi-nested PCR, followed by Sanger sequencing was able to identify Mucorales species 81.9% of culture-negative cases. Optimized manual DNA extraction protocol is suitable for different sample types, with minimum chances of extraneous contamination and offers low cost with a shorter turnaround time (TAT).

A Simple and Cost-Effective Protocol for DNA Isolation from Herbarium Specimens

We developed a new CTAB-based seven-step DNA extraction protocol to isolate DNA from herbarium specimens. DNA was extracted from five species belonging to five genera from four families of angiosperms. The spectrophotometric measurement of double-stranded DNA demonstrated DNA of sufficient quality and quantity for PCR analysis. DNA was tested with universal primers and amplification success was 95%. The protocol is applicable to different taxonomic groups of herbarium specimens, fresh and silica-dried samples, and requires only common laboratory consumables and equipment. It is possible to extract 30 samples in less than two hours and more than 120 samples in a working day. The cost of consumables per sample is less than in many traditional protocols and commercially available kits.

Effects of source sample amount on biodiversity surveys of bacteria, fungi, and nematodes in soil ecosystems

Bacteria, fungi, and nematodes are major components of soil ecosystems, playing pivotal roles in belowground material cycles and biological community processes. A number of studies have recently uncovered the diversity and community structure of those organisms in various types of soil ecosystems based on DNA metabarcoding (amplicon sequencing). However, because most previous studies examined only one or two of the three organismal groups, it remains an important challenge to reveal the entire picture of soil community structure. We examined how we could standardize DNA extraction protocols for simultaneous DNA metabarcoding of bacteria, fungi, and nematodes. Specifically, in an Illumina sequencing analysis of forest and farmland soil samples, we performed DNA extraction at five levels of soil-amount (0.5, 2, 5, 10, and 20 g). We then found that DNA extraction with the 0.5 g soil setting, which had been applied as default in many commercial DNA extraction kits, could lead to underestimation of α-diversity in nematode community. We also found that dissimilarity (β-diversity) estimates of community structure among replicate samples could be affected by soil sample amount. Based on the assays, we conclude that DNA extraction from at least 20 g of soil is a standard for comparing biodiversity patterns among bacteria, fungi and nematodes.

Moving environmental DNA (eDNA) technologies from benchtop to the field using passive sampling and PDQeX extraction

AbstractEnvironmental DNA (eDNA) metabarcoding has shown great promise as an effective, non‐invasive monitoring method for marine biomes. However, long filtration times and the need for state‐of‐the‐art laboratories are restricting sample replication and in situ species detections. Methodological innovations, such as passive filtration and self‐contained DNA extraction protocols, have the potential to alleviate these issues. We explored the implementation of passive sampling and a self‐contained DNA extraction protocol by comparing fish diversity obtained from active filtration (1 L; 0.45 μm cellulose nitrate [CN] filters) to five passive substrates, including 0.45 μm CN filters, 5 μm nylon filters, 0.45 μm positively charged nylon filters, artificial sponges, and fishing net. Fish diversity was then compared between the PDQeX Nucleic Acid Extractor and the conventional Qiagen DNeasy Blood & Tissue protocol. Experiments were conducted in both a controlled mesocosm and in situ at the Portobello Marine Laboratory, New Zealand. No significant differences in fish diversity were observed among active filtration and more porous passive materials (artificial sponges and fishing net) for both the mesocosm and harbor waters. For the in situ comparison, all passive filter membranes detected a significantly lower number of fish species, resulting from partial sample drop‐out. While no significant differences in fish eDNA signal diversity were observed between either DNA extraction methods in the mesocosm, the PDQeX system was less effective at detecting fish for the in situ comparison. Our results demonstrate that a passive sampling approach using porous substrates can be effectively implemented to capture eDNA from seawater, eliminating vacuum filtration processing. The large variation in efficiency observed among the five substrate types, however, warrants further optimization of the passive sampling approach for routine eDNA applications. The PDQeX system can extract high‐abundance DNA in a mesocosm with further optimization to detect low‐abundance eDNA from the marine environment.

Real-time PCR, and Recombinase Polymerase Amplification combined with SYBR Green I for naked-eye detection, along with Propidium Monoazide (PMA) for the detection of viable patulin-producing fungi in apples and by-products

Nowadays, the application of rapid molecular-based methods such as PCR/qPCR and isothermal techniques like Recombinase Polymerase Amplification (RPA), has increased in order to overcome some of the drawbacks of traditional culture-based methods due to their high sensitivity and specificity. One of the main limitations of these techniques is their inability to differentiate between live and dead microorganisms. Patulin is a mycotoxin typically produced by fungi belonging to Penicillium spp. that exerts acute and chronic toxic effects to humans and animals. In the present study, one real-time PCR assay, and a RPA coupled with SYBR Green I for naked-eye detection, were developed for the detection of patulin-producing fungi. Primers and a probe were designed based on the idh gene of the patulin metabolic pathway. Furthermore, propidium monoazide was implemented in the DNA extraction protocol for the specific detection of viable fungi. The developed assays were able to detect down to 1.25 pg/μL (qPCR) and 23.8 pg/μL (RPA-SG) of pure P. expansum DNA. Finally, when artificially inoculated apples, and by-products, were analysed the LOD50 of the qPCR was found to be 8.1 × 103 spores/5 g of food sample. In the case of the RPA-SG, the determined LOD50 was 5.8 × 104 spores/5 g.

Evaluation of DNA extraction protocols and real-time PCR-based methods for efficient investigation of pig traces in foods

Industrially processed foods are composed of a complex mixture of molecules combined under specific chemical and physical conditions. Besides their native interactions, most of the ingredients included in processed foods are highly transformed through extreme heat variations, grinding, freezing, pH, and pressure fluctuations in order to reach the desired final product. Due to their complex structure and high level of degradation, processed foods are difficult to analyse. Undeclared components are often detected in processed foods, and accurate diagnostic testing is required to protect those with health, cultural, and religious restrictions. Molecular biology techniques involving PCR are most frequently used for determining the authenticity of foods containing derivatives of living organisms. In the present work, we investigated four different DNA extraction protocols of three commercial kits, two different quantitative PCR (qPCR) techniques, and six different primer pairs. We analysed 96 extracts (12 samples from each of the eight products) by SYBR Green-based qPCR using the two most specific and sensitive primer pairs, and compared these results to those obtained with standard commercial kits that use dual dye-labelled probes. Adopting high-efficiency DNA extraction protocols, our findings highlighted the importance of targeting several small regions of the mitochondrial genome to effectively detect small traces of porcine products, and reduce the risk of false-negative results. Adopting these will ensure that consumers can make accurate and informed choices.

Optimised germination protocol for jackfruit seeds and evaluation of methods for extraction of DNA suitable for genetic analysis

Jackfruit (Artocarpus heterophyllus) is an underutilised plant that is promising in curbing food and nutritional security in sub-Saharan Africa. However, high level of secondary metabolites in its tissues significantly hampers its genetic characterisation for breeding purposes. Primarily, the compounds react with DNA during the extraction process, thus reducing its yield and quality. The utilisation of leaves from jackfruit seedlings is a potentially effective approach of addressing the challenge, however, limited information is available on efficient jackfruit seed germination procedures. Elucidating effective methods of jackfruit seed germination, and optimising protocols for DNA extraction is crucial in promoting its genetic characterisation studies for identification of superior varieties for propagation. The objective of this study was to evaluate methods of jackfruit seed germination, and DNA extraction procedures using jackfruit leaves. Pre-treatment of seeds with 3% hydrogen peroxide was effective in enhancing seed germination within a short time, compared to distilled water and 3% hydrochloric acid. We optimised a DNA extraction technique by combining CTAB-SDS based approach with an enhanced solvent extraction method. The technique yielded high quantity and quality of DNA from jackfruit leaves, which was appropriate for downstream polymerase chain reaction analysis. The sequence-related amplified polymorphism (SRAP) and simple sequence repeat (SSR) amplifications confirmed the effectiveness of the optimised CTAB-SDS based protocol for extraction of high quality DNA.

Improved method of DNA extraction from leaf and rhizome samples of black turmeric (Curcuma caesia) for molecular analysis

Curcuma caesia belongs to the genus Curcuma and the family of Zingiberaceae, which is a very important but unexplored medicinal plant. It is locally known as black turmeric or kali halide and is mainly used by the local tribal community as traditional medicine for the health sector. The rhizome of black turmeric has wide applications in the economic pharma sector due to essential active ingredients. This research aims to standardize a rapid, simple and efficient protocol for DNA extraction in Curcuma caesia which can be used for another genus of Zingiberaceae to obtain DNA from leaf samples. Changes in the concentration of components of DNA extraction buffer have improved the quantity of DNA from leaf samples compared to rhizomes. Extracted DNA samples also proved more efficient in PCR amplification of DNA barcode primers. The protocol developed in the present study is more efficient for leaf samples of Curcuma caesia compared to rhizome samples.

Optimization of a DNA extraction method using Subepidermis from Austrocylindropuntia and Opuntia-Opuntioideae

The taxonomic identification of species through analysis of the variation of orthologous DNA sequences, complement the information obtained with morphological characters. Cytogenetic studies indicate that polyploid taxa occur in the subfamily Opuntioideae, Opuntia ficus-indica, contributing to morphological variability in the individuals of a population, and influencing the correct identification of species. However, the lengths of the sequences in Opuntioideae are affected by the extraction of pure DNA. Different extraction methods were evaluated and modified, and a procedure was established to obtain good quality DNA, free of inhibitors for gene amplification by polymerase chain reaction. The ratio A260/A280 and A260/A230 ranged from 1.6 to 2.1, revealing absence of contamination with the modified protocol for DNA extraction from cotton leaves. This method is inexpensive compared to those of commercial manufacturers and, therefore, can be applied in studies with limited resources.

Dead migrants in the Mediterranean: genetic analysis of bone samples exposed to seawater

In April 2015, a fishing boat that departed from Libya with about 1,000 migrants on board sank in the Mediterranean Sea. Most of the migrants were packed in the hull of the boat and drowned in the shipwreck. After fifteen months, the ship was recovered from the seabed and brought to a Sicilian naval area for forensic investigations. Skeletal remains belonging to more than 700 people were retrieved. A selected sample composed of 80 victims was considered in order to evaluate the possibility of achieving genetic profiles useful for a positive identification from these challenging specimens. The molecular features of the DNA recovered from a significant number of real casework samples exposed to seawater for long periods of time were described for the first time. Three different DNA extraction protocols and three different commercial kits were employed in order to generate genetic profiles based on the characterization of 21 autosomal STR loci. The combination of multiple DNA extractions and the cross-checking of multiple PCR amplifications with different kits allowed to obtain reliable genetic profiles characterized by at least 16 STR markers in more than 70% of the samples. The factors that could have affected the different quality of the genetic profiles were investigated and the bone preservation was examined through microscopic and macroscopic analyses. The approach presented in this study could be useful in the management of the genetic analysis of bone samples collected in other similar DVI scenarios. The genetic profiles recovered from the bone samples will be compared in kinship analysis to putative relatives of the victims collected in Africa in order to obtain positive identifications.

Developing whole cell standards for the microbiome field

BackgroundEffective standardisation of the microbiome field is essential to facilitate global translational research and increase the reproducibility of microbiome studies. In this study, we describe the development and validation of a whole cell reference reagent specific to the gut microbiome by the UK National Institute for Biological Standards and Control. We also provide and test a two-step reporting framework to allow microbiome researchers to quickly and accurately validate choices of DNA extraction, sequencing, and bioinformatic pipelines.ResultsUsing 20 strains that are commonly found in the gut, we developed a whole cell reference reagent (WC-Gut RR) for the evaluation of the DNA extraction protocols commonly used in microbiome pipelines. DNA was first analysed using the physicochemical measures of yield, integrity, and purity, which demonstrated kits widely differed in the quality of the DNA they produced. Importantly, the combination of the WC-Gut RR and the three physicochemical measures allowed us to differentiate clearly between kit performance. We next assessed the ability of WC-Gut RR to evaluate kit performance in the reconstitution of accurate taxonomic profiles. We applied a four-measure framework consisting of Sensitivity, false-positive relative abundance (FPRA), Diversity, and Similarity as previously described for DNA reagents. Using the WC-Gut RR and these four measures, we could reliably identify the DNA extraction kits’ biases when using with both 16S rRNA sequencing and shotgun sequencing. Moreover, when combining this with complementary DNA standards, we could estimate the relative bias contributions of DNA extraction kits vs bioinformatic analysis. Finally, we assessed WC-Gut RR alongside other commercially available reagents. The analysis here clearly demonstrates that reagents of lower complexity, not composed of anaerobic and hard-to-lyse strains from the gut, can artificially inflate the performance of microbiome DNA extraction kits and bioinformatic pipelines.ConclusionsWe produced a complex whole cell reagent that is specific for the gut microbiome and can be used to evaluate and benchmark DNA extractions in microbiome studies. Used alongside a DNA standard, the NIBSC DNA-Gut-Mix RR helps estimating where biases occur in microbiome pipelines. In the future, we aim to establish minimum thresholds for data quality through an interlaboratory collaborative study.BEU9jL7xnwncT84Rof_ZoXVideo

COMPARISON, VALIDATION, AND OPTIMIZATION OF INTERNAL GENOMIC DNA EXTRACTION PROTOCOL FOR CAMPYLOBACTER SPECIES

Campylobacter remains the leading cause responsible of human gastroenteritis worldwide. The current study aimed to compare the internal DNA extraction protocol with two commercially available kits, using C. jejuni (ATCC® 29428TM) and C. coli (ATCC®43478TM) and to validate and optimize the internal protocol through artificial contamination and confirmation of Campylobacter spp. from broiler chickens, turkeys, and beef meats samples. The extraction processes were carried out following the internal protocols steps and the manufacturer's instructions of PureLinkTM Genomic DNA Mini Kit and Wizard® Genomic DNA Purification Kit, respectively. The agarose gel electrophoresis system was used to control the DNA quality. After that, forty Campylobacter spp. isolates were confirmed, and finally, an artificial contamination of the aforementioned reference strains at three different concentrations was performed with sterilized minced meats. This is the first work comparing the PureLinkTM Genomic DNA Mini Kit and Wizard® Genomic DNA Purification Kit with internal genomic DNA extraction protocol for Campylobacter spp. The results indicated that the internal protocol provided similar efficiency to the two kits. All confirmed isolates were successfully amplified, in which28 isolates were C. coli and 12 were C. jejuni, as revealed by biochemical tests. A positive amplification was also observed in the three contaminated food matrices, after enrichment, at all examined doses. Except some reactions that were negative at 1 CFU/mL of C. jejuni and C. coli. This was explained by the detection limits of both internal protocol and qPCR. Based on our findings, three crucial steps in determining the extraction of DNA quality of this protocol were amended. Hence, the study highlighted the importance of validating simpler, cheaper and faster DNA extraction protocol, for each laboratory, as part of future risk assessment, control and monitoring programs of Campylobacter frequency required in molecular studies.

Developing and deploying an efficient genotyping workflow for accelerating maize improvement in developing countries.

Background: Molecular breeding is an essential tool for accelerating genetic gain in crop improvement towards meeting the need to feed an ever-growing world population. Establishing low-cost, flexible genotyping platforms in small, public and regional laboratories can stimulate the application of molecular breeding in developing countries. These laboratories can serve plant breeding projects requiring low- to medium-density markers for marker-assisted selection (MAS) and quality control (QC) activities. Methods: We performed two QC and MAS experiments consisting of 637 maize lines, using an optimised genotyping workflow involving an in-house competitive allele-specific PCR (KASP) genotyping system with an optimised sample collection, preparation, and DNA extraction and quantitation process. A smaller volume of leaf-disc size plant samples was collected directly in 96-well plates for DNA extraction, using a slightly modified CTAB-based DArT DNA extraction protocol. DNA quality and quantity analyses were performed using a microplate reader, and the KASP genotyping and data analysis was performed in our laboratory. Results: Applying the optimized genotyping workflow expedited the QC and MAS experiments from over five weeks (when outsourcing) to two weeks and eliminated the shipping cost. Using a set of 28 KASP single nucleotide polymorphisms (SNPs) validated for maize, the QC experiment revealed the genetic identity of four maize varieties taken from five seed sources. Another set of 10 KASP SNPs was sufficient in verifying the parentage of 390 F 1 lines. The KASP-based MAS was successfully applied to a maize pro-vitamin A (PVA) breeding program and for introgressing the aflatoxin resistance gene into elite tropical maize lines. Conclusion: This improved workflow has helped accelerate maize improvement activities of IITA's Maize Improvement Program and facilitated DNA fingerprinting for tracking improved crop varieties. National Agricultural Research Systems (NARS) in developing countries can adopt this workflow to fast-track molecular marker-based genotyping for crop improvement.

Efficient, fast and low-cost strategies for DNA extraction from different nucleated sheep cells

DNA extraction is usually the first step to perform molecular studies. This process can be nonviable due to genomic DNA (gDNA) extraction commercial kits prices. Furthermore, available DNA extraction protocols generally have high specificity, limiting their use to specific sources of biological material. In order to reduce costs, optimize time and laboratory logistics, besides to demonstrate a versatile protocol, the present study worked on an efficient DNA extraction protocol from somatic and non-somatic cells, using biological material from sheep as a model. For that, gDNA was extracted from whole blood, spermatozoa, and hair bulb cells, collected from three adult sheep, transported at 5ºC and stored at -20ºC until lab procedures. After extraction, gDNA concentration and purity were evaluated in a nano spectrophotometer. gDNA concentration from whole blood was greater (p < 0.05) than extracted from hair bulb cells, which in turn was superior (p < 0.05) than in spermatozoa. Also, gDNA from whole blood and, followed by, sperm showed greater (p < 0.05) purity when compared to gDNA of hair bulb cells. Adapting a gDNA extraction protocol, originally developed for bovine whole blood, enabled to obtain and isolate gDNA in different nucleated sheep cells

Genetic protocols for DNA extraction from white-tailed deer cast antlers to confirm individuality

White-tailed deer (Odocoileus virginianus) are the most sought-after deer species in America. The antlers of mammals, such as deer, are one of the fastest regenerative tissues in the world and are grown and naturally cast every year. Research on cast antlers have been used for a variety of purposes including population comparisons and impacts of deer health due to climatic stressors. When investigating cast antlers, it is important to confirm individuality of match sets in addition to antlers of the same individual between years. Therefore, individuality must be confirmed genetically, and protocols must be developed and established to do so. Our objectives were to 1) establish a genetic protocol to harvest DNA from cast antlers using connective tissue, and 2) determine individuality from subsequent years and match sets. When fresh antlers are cast, they leave behind a viable connective tissue from which DNA can be extracted. The DNA was successfully extracted from the skin rings harvested from naturally cast antlers. This study developed viable methods to confirm individuality, which aid researchers and wildlife biologists in a better understanding of the white-tailed deer herd as they set management goals and harvest regulations.

Molecular Identification of Cystoisospora Belli in Patients Infected with the Human Immunodeficiency Virus

Infection with Cystoisospora belli causes severe diarrhea, bile duct involvement, and Extra-intestinal spread in patients with acquired immunodeficiency syndrome (AIDS). This study included eight adult patients with AIDS and chronic diarrhea diagnosed stool cystosisporiasis (oocysts) and / or duodenal biopsies (asexual and sexual stages in epithelium). Identification was carried out at the molecular level using a nested-polymerase chain reaction (PCR) technique that amplifies a fragment of the ribosomal ribonucleic acid (RNA) small subunit gene, using DNA extracted from feces and biopsies. This work allowed us to make the diagnosis of cystosisporiasis through analysis coproparasitological, optimize the DNA extraction protocol from samples fecal and implement the nested -PCR technique for the diagnosis of C. belli in biological samples from infected patients.

Protocol Optimization of DNA Extraction from Banana Fruits

Background: DNA extraction is the process by which the DNA is extracted and purified from the living or dead cells or tissues. There are many different kits commercially available for DNA extraction, with varying protocols. This study aimed at determining the optimal conditions for the extraction of high yield DNA from banana fruits.
 Methodology: DNA was extracted from banana fruits by precipitation method. The DNA yield was determined using the Nanodrop DNA quantification technique.
 Results: The optimal sodium chloride salt concentration for DNA extraction from banana fruits was 4mM and the amount of DNA extracted increased as the temperature decreased. The highest yield was obtained between 4 0C to 0 0C.
 Conclusion: We report for the first time a protocol with an optimal salt concentration (NaCl, 4mM) for quality DNA yield from banana fruits. A critical consideration is required to establish an optimal range of moderate temperatures and DNA concentrations for better quality and desired yields. There may not be a universal protocol for DNA extraction from all plant materials.

A rapid and inexpensive genotyping method using dried blood spots for mutational analysis in a mutant mouse model: an update.

Dried blood spot (DBS) testing is a well-known method of bio-sampling by which blood samples are blotted and dried on filter paper. The dried samples can then be analyzed by several techniques such as DNA amplification and HPLC. We have developed a non-invasive sampling followed by an alternative protocol for genomic DNA extraction from a drop of blood adsorbed on paper support. This protocol consists of two separate steps: (1) organic DNA extraction from the DBS, followed by (2) DNA amplification by polymerase chain reaction (PCR). The PCR-restriction fragment length polymorphism (PCR-RFLP) is an advantageous and simple approach to detect single nucleotide polymorphisms (SNPs). We have evaluated the efficiency of our method for the extraction of genomic DNA from DBS by testing its performance in genotyping mouse models of obesity and herein discuss the specificity and feasibility of this novel procedure. Our protocol is easy to perform, fast and inexpensive and allows the isolation of pure DNA from a tiny amount of sample.