Commercial DNA Extraction Kits Research Articles

New metagenomic procedure for the investigation of the eukaryotes present in the digestive gland of Mytillus galloprovincialis

Mussels’ aquaculture is one of the most important cultures in Europe, with a high level of self-sufficiency. In Spain, the main cultivated species is M. galloprovincialis, whose production takes place primarily in Galicia. In that region, a Protected Designation of Origin certification exists to certify the high quality of the product and its sustainability. This species is also present in other regions making the control of its traceability complicated. In order to distinguish Galician mussels from the others, the eukaryotes present in its digestive gland could be a useful tool. However, to use them as biomarkers, having an adapted protocol to recover the diversity present in the digestive gland is essential. In this work, we have selected a new protocol to optimize the study of the diversity of eukaryotes associated with the digestive gland of M. galloprovincialis. This protocol is based on a combination of a pre-treatment and a commercial DNA extraction kit, the amplification of DNA with two newly designed primer sets to avoid the DNA from the mussel, and the use of next-generation sequencing. The results showed a significant reduction of mussel DNA amplification and enough DNA quality for the subsequent sequencing, increasing the number of sequences recovered from other eukaryotes. This new protocol offers then a good chance to determine the diversity of eukaryotes present in the digestive gland of M. galloprovincialis, and it can be used in future studies to examinate the seasonal variation of the present species and to evaluate its potential as geographical traceability tool.

Development of Loop-Mediated Isothermal Amplification for the Detection of Prototheca bovis Directly from Milk Samples of Dairy Cattle.

Prototheca bovis is an algal emerging pathogen in dairy farms causing refractory protothecal mastitis with increasing incidence worldwide and significant economic impact. P. bovis infects cows throughout the lactation cycle, including dry periods, and can persist in the udder and the environment for a long time. Since P. bovis does not respond to treatments with antibiotics, the suggested sanitary measure to restrict the spread is culling infected animals. A point-of-care test for early detection of the causative agent is critically needed to guide farm management and the appropriate treatment of mastitis. Loop-mediated isothermal amplification (LAMP) is a highly specific molecular method, time-saving, cost-effective and easy to perform in limited settings. This study aimed to develop a LAMP assay for P. bovis detection directly from milk samples; it was employed in conjunction with a commercial DNA extraction kit which was previously used to extract DNA from milk specimens containing microbes. The LAMP assay detected P. bovis DNA within 1h in milk samples spiked with P. bovis at a concentration of 50 cells/μL, enabling on-farm disease monitoring and decision-making based on a reliable diagnosis. The LAMP method will contribute to the accurate and rapid identification of P. bovis in asymptomatic or recurrent mastitis cases and consequently aid the implementation of targeted control measures and the reduction of losses in milk production.

Genetic differentiation of Hippa adactyla based on CO1 gene sequence in Sundaland

The Hippidae is a family of sand crabs that inhabit intertidal ecosystems, specifically in sandy areas scattered throughout the coast. Differences in habitat conditions cause variations in Hippidae members. Hippa adactyla is a member of the genus Hippa. This study aimed to map the genetic differentiation between Hippa adactyla populations in the Greater Sunda Islands. The sand crab (Hippa adactyla) samples were taken from Sumatra (Pariaman, Bengkulu, Lampung) and Java (Pangandaran, Pelabuhan Ratu, Banyuwangi) by handcatching then stored in alcohol 96%. Total genomic DNA was extracted from the muscle tissue of the samples using a commercial DNA Extraction KIT. The upper part of the COI gene (5' end) (±750 bp) was amplified using specific primers. Based on the K2P substitution model, the genetic distance of the Pangandaran population to the Bali Strait population is the same as that of the Sumatran population (0.062). Genetic diversity ranges from 0.000 to 0.123. Haplotype diversity was 0.928, consisting of 18 haplotypes.

An optimal genomic DNA extraction method for shoots of four Dendrocalamus species based on membership function analysis.

High-quality genomic DNA extraction is fundamental for the study of gene cloning and expression in plants. Therefore, this study evaluated several methods for extracting genomic DNA from shoots of four Dendrocalamus species to determine the optimal technique. Genomic DNA was extracted using three different methods: a commercial DNA extraction kit method, a modified cetyltrimethylammonium bromide method and a sodium dodecyl sulfate method. A membership function analysis was employed to compare these methods. The results demonstrated that the commercial DNA extraction kit method was the most effective and comprehensive approach for extracting genomic DNA from shoots of four Dendrocalamus species. Furthermore, this study provided valuable insights into optimizing techniques for extracting genomic DNA in other bamboo species.

Plasmid DNA extraction by double magnetic beads method and its application

Plasmids are the most commonly used gene carriers in the field of gene synthesis and sequencing. However, the main problems faced by traditional plasmid DNA extraction technology are low extraction throughput and high production cost, so they cannot meet the growing demand. In this study, a double-magnetic-bead method (DMBM) for plasmid extraction was developed based on the principle of plasmid extraction. The effects of the input of magnetic beads, the size of plasmid DNA fragments, and the volume of bacterial on plasmid DNA extraction were explored. In addition, the quality, throughput, and cost of plasmid DNA extraction were also compared between this technique and the commercial plasmid DNA extraction kits. The results showed that the DMBM can meet the needs of extracting plasmid DNA with different cell densities and fragment lengths. Moreover, the sensitivity and quality of plasmid extraction by the DMBM method were both superior to those of the centrifugal adsorption column method. In addition, this technique could be applied on a 96-channel automated nucleic acid extractor, resulting in higher purity of the extracted plasmid DNA, 80% reduction in extraction time, and 57.1% reduction in cost. It also reduces manual operations, achieving high-throughput and low-cost plasmid DNA extraction, thus may facilitate gene synthesis and sequencing.

Comparison of DNA Extraction Methods for the Detection of Canned Tuna Species with DNA Mini-Barcoding

Tuna is susceptible to species mislabeling due to its high demand, quick rate of production, and wide range of price points. DNA barcoding, a sequencing-based technique, allows for the detection of species mislabeling by targeting a standardized region of DNA. A mitochondrial control region (CR) DNA barcode has been found to be capable of species discrimination for tuna, but it is challenging to recover the entire DNA fragment from canned tuna. While a short fragment of CR, referred to as a “mini-barcode,” has shown some success with canned tuna species identification, more research is needed to improve identification rates. The objective of this study was to determine the optimal DNA extraction method for species identification of canned tuna using CR mini-barcoding. Four commercial DNA extraction kits were compared using a sample set of 24 different cans of tuna labeled as albacore, light tuna, skipjack, or yellowfin. All samples were tested in duplicate. The greatest success was found using the Qiagen DNeasy Blood and Tissue Kit and the Qiagen DNeasy Mericon Food Kit, which resulted in species identification for 42% of the samples. In comparison, the MP Biomedicals FastPrep-24 + Machery-Nagel NucleoSpin Tissue Kit resulted in species identification for 30% of the samples and the Qiagen DNeasy Blood and Tissue Kit + PowerClean Pro Cleanup Kit resulted in species identification for 21% of the samples. Overall, the top-performing DNA extraction methods for use with CR mini-barcoding of canned tuna products were determined to be the DNeasy Blood and Tissue Kit and the DNeasy Mericon Food Kit.

Rapid Diagnostic of Streptococcus suis in Necropsy Samples of Pigs by thrA-Based Loop-Mediated Isothermal Amplification Assay.

Streptococcus (S.) suis presents a serious threat to the pig industry as well as food safety and public health. Although several LAMP assays have been developed for the identification of S. suis, no universal assay is so far available for the field-suitable examination of clinical pig specimens. Based on the thrA housekeeping gene, a new loop-mediated isothermal amplification (LAMP) assay was developed and validated for the detection of S. suis in the brain and joints of pigs. For this LAMP assay, two different methods for the extraction of DNA from brain and joint swabs were compared. Using the LPTV boiling method, the detection limit of LAMP was 1.08 CFU/reaction, while the detection limit was 53.8 CFU/reaction using a commercial DNA extraction kit. The detection limits of thrA-LAMP in combination with the LPTV boiling method were 104-105 CFU/swab in the presence of brain tissue and 103-104 CFU/swab in the presence of joint tissue. The diagnostic quality criteria of LAMP were determined by the examination of 49 brain swabs and 34 joint swabs obtained during routine diagnostic necropsies. Applying the LPTV boiling method to brain swabs, the sensitivity, specificity, and positive and negative predictive values of thrA-LAMP were 88.0, 95.8, 95.7, and 88.5% using cultural investigation as a reference method, and 76.7, 100, 100, and 73.1% using real-time PCR as a reference method. Based on these results, the thrA-LAMP assay combined with the LPTV boiling method is suitable for rapid detection of S. suis from brain swabs.

An Efficient Extraction Method Allowing the Genetic Evaluation of Host DNA from Samples Collected for Virus Infection Diagnosis in Viral Transport Medium.

Introduction: During the COVID-19 pandemic, an extraordinary number of nasopharyngeal secretion samples inoculated in viral transport medium (VTM) were collected and analyzed to detect SARS-CoV-2 infection. In addition to viral detection, those samples can also be a source of host genomic material, providing excellent opportunities for biobanking and research. Objective: To describe a simple, in-house-developed DNA extraction method to obtain high yield and quality genomic DNA from VTM samples for host genetic analysis and assess its relative efficiency by comparing its yield and suitability to downstream applications to two different commercial DNA extraction kits. Methods: In this study, 13 VTM samples were processed by two commercial silica-based kits and compared with an in-House-developed protocol for host DNA extraction. An additional 452 samples were processed by the in-House method. The quantity and quality of the differentially extracted DNA samples were assessed by Qubit and spectrophotometric measurements. The suitability of extracted samples for downstream applications was tested by polymerase chain reaction (PCR) amplification followed by amplicon sequencing and allelic discrimination in real-time PCR. Results: The in-House method provided greater median DNA yield (0.81 μg), being significantly different from the PureLink® method (0.14 μg, p < 0.001), but not from the QIAamp® method (0.47 μg, p = 0.980). Overall satisfactory results in DNA concentrations and purity, in addition to cost, were observed using the in-House method, whose samples were able to produce clear amplification in PCR and sequencing reads, as well as effective allelic discrimination in real-time PCR TaqMan® assay. Conclusion: The described in-House method proved to be suitable and economically viable for genomic DNA extraction from VTM samples for biobanking purposes. These results are extremely valuable for the study of the COVID-19 pandemic and other emergent infectious diseases, allowing host genetic studies to be performed in samples initially collected for diagnosis.

Evaluation of A Simple DNA Extraction Method and Its Combination with Loop-Mediated Isothermal Amplification Assays for Rapid Plasmodium knowlesi Diagnosis.

The initial and vital stage in the diagnosis of malaria involves extracting DNA. The efficiency of malaria testing is restricted by the multiple steps involved in commercial DNA extraction kits. We attempted to improve an existing loop-mediated isothermal amplification (LAMP) for the detection of Plasmodium knowlesi by using a simple DNA extraction approach, making it a feasible option for mass screening. We utilized a simple nucleic acid extraction method directly from whole blood for the detection of P. knowlesi, taking only 5 min to complete. The extracted DNA was evaluated by two fluorescent-based LAMP and one colorimetric-based LAMP assay. The detection limit for both SYTO-LAMP and SYBR green-LAMP was 0.00001% and 0.0001% parasitemia, respectively. Meanwhile, neutral red-LAMP had a detection limit of 0.01% parasitemia. Combining this simple and inexpensive DNA extraction method, SYTO-LAMP could serve as an alternative molecular diagnosis for the detection of P. knowlesi and other human Plasmodium spp.

Abridged solid-phase extraction with alkaline Poly(ethylene) glycol lysis (ASAP) for direct DNA amplification

Complexity of sample preparation decelerate the development of sample-in-answer-out devices for point-of-need nucleic acid amplification testing. Here, we present the consolidation of alkaline poly(ethylene) glycol-based lysis and solid-phase extraction for rapid and simple sample preparation compatible with direct on-bead amplification. Simultaneous cell lysis and binding of DNA were achieved using an optimised reagent comprising 15% PEG8000, 0.5 M NaCl, and 3.5 mM KOH. This was combined with direct, on-bead amplification using 1.5 μg beads per 20 μL PCR reaction mix. The novel single reagent, 5-min method improved the detection limit by 10 and 100-fold compared with commercial DNA extraction kits and the original alkaline PEG lysis method, respectively. The sensitivity can be further enhanced by one amplification cycle with an ethanol wash or by extending the incubation to 10 min before collecting the magnetic particles. Both methods successfully detected a single copy of Escherichia coli DNA. In biological fluids (saliva, sweat, and urine), the 5-min method was delayed by about one cycle compared to the 15-min method. The proposed methods are attractive for incorporation in the workflow for point-of-need testing of biological samples by providing a practical and chemical method for simple alternative DNA sample preparation.

Review of the Efficiency of Ten Different Commercial Kits for Extracting DNA from Soil Mixed Biological Samples

Soil-mixed bodily fluids are the most common kind of evidence at outdoor crime scenes. This biological evidence contains DNA, which is a key component of forensic science’s ability to prove an accused person’s guilt because it connects the victim and suspect to the crime scene and aids in identifying the offender and victim. The yield of DNA is significantly influenced by factors including temperature, humidity, storage environment, time since deposition, etc. DNA degradation is caused by a variety of microbes, bacteria, humic acid, and other substances present in soil. Nowadays for DNA extraction, a variety of commercial DNA extraction kits was used now. This paper’s objective is to compare the efficiency of ten different commercial kits used to extract mixed DNA samples. It has been observed that samples stored at a low temperature (-20 °C) are the best for soil blood mixture samples. Compared to samples paired with other types of soil (silt, clay, and marshland), sand soil had the largest production of DNA using the QIAmp investigator kit (Qiagen). Blood Miniprep kit extractions were mostly inhibited, the control that amplified confirms that this kit was the worst in terms of DNA extraction potency. The samples with fewer dirt particles had a much greater yield of DNA.

High‐throughput genotyping in estimating genetic resources and detecting pathogens in aquaculture

High‐throughput genotyping in estimating genetic resources and detecting pathogens in aquaculture

82 Responses to a combined exercise and transport stressor following dietary supplementation of anti-endotoxin IgY in mature horses

Immunoglobulin Y elicits positive effects in domestic livestock with gastrointestinal or inflammatory diseases; however, there are limited studies in horses. To test the hypothesis that dietary anti-endotoxin IgY (IgY) has a positive effect on gastrointestinal health and mitigates systemic inflammation in horses undergoing exercise, 30 stock-type horses (578 ± 59 kg BW; 14 ± 3 yr) were used in a completely randomized design. Horses were stratified by BW, age, and sex and then randomly assigned to treatments: 0 g/d IgY (CON; n = 10), 1 g/d IgY (TRT1; n = 10), or 2 g/d IgY (TRT2; n = 10). Horses were fed every 12 h, with dietary treatments (Camas Inc., Le Center, MN) top-dressed on the morning concentrate for 33 d. Horses were maintained in dry lots with ad libitum access to Coastal bermudagrass hay. Horses were exercised 5 d/wk for 1 h. On d 0 and 30, BW and BCS were recorded, and blood was collected for complete blood cell count (CBC) and serum blood chemistry. On d 32, horses were transported 270 km and underwent a 3.2 km exercise stressor on concrete at a walk with maximum 15 min of trot. Whole blood, serum, and fecal samples collected via rectal palpation were obtained pre-, post-, and 24 h post-trailering. Fecal pH was determined by a portable meter, and serum cytokine (TNFα, IL-4, IL-10, IL-1β, IL-17, IFN-α, IFN-γ) and chemokine (CCL2, 3, 5 and 11) concentrations weremeasured using a multiplex platform. Whole blood DNA was extracted using a commercial DNA extraction kit. For gut permeability analysis, 16s rDNA was analyzed via qPCR and calculated as fold change from d 0. Data were analyzed using PROC MIXED of SAS v9.4. Dietary IgY did not affect BW, BCS, blood chemistry, or CBC (P ≥ 0.17). Fecal pH decreased over time (P ≤ 0.01). There was no treatment × time interaction for 16S rDNA (P = 0.45). Dietary treatment affected circulating 16S rDNA (P < 0.01) where TRT2 (1.89-fold) was greater than CON (0.70-fold), and TRT1 (1.24-fold) was intermediate. A significant time effect (P = 0.001) was observed for 16S rDNA where fold change decreased after the stressor from 1.90-fold for pre-, to 1.02 for post-, and 0.92 for 24 h post- trailering. There tended to be a treatment × time interaction (P = 0.08) for CCL3, as CON was greater at 24 h and TRT2 declined post-trailering, returning to baseline by 24 h. Levels of CCL5 decreased (P < 0.01) and TNFα tended to decline (P = 0.06) through 24 h post trailering in all treatment groups. Other cytokines and chemokines were non-detectible. Despite greater circulating 16s rDNA with higher treatment dosages, there was no effect on systemic inflammatory markers.

A simple PCR-SSP method for detection of HLA-B*15:02, *15:13, and *15:21

Abstract Objectives This study aims to develop a simple polymerase chain reaction (PCR) sequence-specific primer method, which will be used to genotype HLA-B*15:02, HLA-B*15:13, and HLA-B*15:21. Methods DNA was extracted from whole blood using a commercial DNA extraction kit. New specific primers were designed. The PCR was optimized for reproducibility and specificity. Parameters investigated included concentrations of MgCl2, primers concentration, and annealing temperature, to produce specific bands of interest. Known DNA samples were selected at random and tested using the PCR method. Results Simultaneously six duplex PCRs were successfully optimized using 1.0 mM MgCl2 and an annealing temperature of 62 °C. The method was also reproducible and specific. The results from the method showed 100% concordance with known DNA samples. Conclusions This study has successfully developed a simple and specific method to simultaneously genotype HLA-B*15:02, HLA-B*15:13, and HLA-B*15:21 using in-house developed PCR-SSP.

Evaluation of five commercial DNA extraction kits using Salmonella as a model for implementation of rapid Nanopore sequencing in routine diagnostic laboratories.

Oxford Nanopore long-read sequencing offers advantages over Illumina short reads for the identification and characterization of bacterial pathogens for outbreak detection and surveillance activities within a diagnostic public health laboratory context. Compared to Illumina, Nanopore is more cost-effective for small batches, has a lower capital cost and has a faster turnaround time, in addition to the ability to assemble complete bacterial genomes. The quantity and quality of DNA required for Nanopore sequencing are greater than for Illumina, and the DNA extraction methods recommended for obtaining high-molecular-weight DNA are different from those typically used in diagnostic laboratories. Using a Salmonella isolate with a previously closed PacBio genome as a model Enterobacteriaceae organism, we evaluated the quantity, quality and fragmentation of five commercial DNA extraction kits. Nanopore sequencing performance was evaluated for the top three methods: Qiagen EZ1 DNA Tissue, Qiagen DNeasy Blood and Tissue, and a modified, in-house version of the MasterPure Complete DNA and RNA purification. To evaluate the effect of post-extraction DNA purification methods, we subjected extracted DNA from the three selected extraction methods to purification by AMPure beads or ethanol precipitation and compared these outputs with untreated DNA as a control. All methods are suitable for routine whole-genome sequencing (WGS), since all 60 replicates had very high genome recovery rates, with ≥98 % of the reference genome covered by mapped Nanopore reads. For 85 % of the replicates, assembly was able to produce a complete, circular chromosome using either Flye or Canu. In most cases, it is recommended to move directly from extraction to sequencing, as untreated DNA had the highest rates of genome closure regardless of extraction method. Using our evaluation criteria, the Qiagen DNeasy Blood and Tissue kit was found to be the best overall method due to its low cost, ability to scale from single tubes to 96-well plates, and high consistency in yield and sequencing performance.

Comparing earthworm biodiversity estimated by DNA metabarcoding and morphology-based approaches

Conventional identification based on morphology is today's tool of choice for monitoring earthworm diversity. However, it requires skills to distinguish specific features, which can become the bottleneck in soil ecological studies. DNA-based metabarcoding is increasingly used to monitor diversity of soil organisms. Few studies have compared this method with conventional methods. The aim of the study was to compare metabarcoding based on the mitochondrial 16S rRNA region with conventional morphology-based identification, where metabarcoding captured more species (eight) than identified by conventional method (five). Additionally, we tested the effects of two commercial DNA extraction kits: PowerSoil based on 0.25 g of soil and PowerMax based on 10 g of soil (both from Qiagen), where the PowerMax covered higher richness than PowerSoil.

Genomic DNA extraction and phenolic content of salty and tannic plant material of two mangrove tree species from the mexican pacific coast

Abstract Mangroves, unique tree and shrub species inhabiting coastal saline environments, exhibit distinctive ecological and morpho-physiological traits, including forming pure intertidal stands and possessing specialized mechanisms for salt excretion and aerial root respiration. These species produce valuable antioxidants, such as phenols and tannins, with significant pharmacological, ecological, and toxicological implications. This study aimed to develop a highly efficient DNA extraction protocol for mangrove leaves rich in salt and tannins. The commonly used CTAB extraction protocol and a commercial DNA extraction kit were modified to enhance DNA purity and yield. The antioxidant capacity of the samples was assessed using various protocols. The results demonstrated that the modified commercial kit outperformed other methods in extracting DNA from mangrove leaves, effectively overcoming challenges associated with high salt and phenolic compounds that could impede next-generation sequencing (NGS) analysis. Furthermore, the findings revealed an inverse relationship between tannin concentration and DNA extraction yield. This study offers a valuable resource for DNA extraction protocols from tannin-rich plant materials. In conclusion, the modified commercial kit represents the most efficient and effective approach for extracting DNA from tannin-rich mangrove leaves.

Association of IFITM1 Promoter Methylation with Severity of SARS-CoV-2 Infection.

During viral infections such as SARS-CoV-2, epigenetic changes within the promoter region of the immune system genes would possibly occur and have an effect on the immune system response as well as disease outcome. We aimed to evaluate and compare the methylation level of the IFITM1 gene promoter in different stages of COVID-19 disease with a healthy control group. In this cross-sectional study, 75 COVID-19 patients (25 mild, 25 severe, and 25 critical in addition to 25 age- and gender-matched healthy volunteers) have been included. DNA was extracted from the peripheral white blood cells using a commercial DNA extraction kit. PCR was performed using two types of primers designed for the methylated and unmethylated forms of the IFITM1 gene promoter. The mean age of the patient and healthy volunteer groups was 52.733 ± 13.780 and 49.120 ± 12.490, respectively. Out of a hundred participants, 52 were male. The results demonstrated that severe (p = 0.03, OR 6.729) and critical (p = 0.001, OR 11.156) patients were much more likely to show methylation of the IFITM1 gene in contrast with mild patients. Moreover, IFITM1 methylation was significantly higher in COVID-19 patients in comparison with the healthy volunteer group (p = 0.004, OR 3.17). Furthermore, IFITM1 methylation in male patients with critical status, (p = 0.01) was significantly higher than in male patients with mild status. In addition, IFITM1 methylation of male (p = 0.03) and female (p = 0.01) critical patients was considerably higher compared to males and females of volunteer group. Increased methylation of the IFITM1 gene in the severe and critical stage of COVID-19 diseases may indicate the role of SARS-CoV-2 infection in increasing methylation of this antiviral gene. This might be involved in suppressing the immune system, promoting SARS-CoV-2 replication and disease outcome.

Plasmid Profile and Enterobacterial Repetitive Intergenic Consensus-PCR Characterization of Salmonella Infantis Isolates Recovered From Poultry Sources

Background: Salmonella is known as one of the most important bacterial agents infecting both humans and animals. Salmonella Infantis has been reported as one of the 15 most prevalent serovars worldwide. Despite its clinical importance, there is little information on the molecular characteristics of S. Infantis in Iran. Objectives: This study was conducted to characterize S. Infantis isolates collected from poultry sources in the last decade. The isolates were typified by plasmid profile and enterobacterial repetitive intergenic consensus (ERIC-PCR). Methods: Forty S. Infantis isolates from poultry sources were subjected to plasmid profile and ERIC-PCR characterization. We used a commercial plasmid extraction kit to extract and purify plasmid DNA which then was separated by gel electrophoresis and viewed under a UV transilluminator. For ERIC-PCR, a commercial bacterial chromosomal DNA extraction kit was used. In this study, we chose ERIC2 primer for the ERIC-PCR test. Results: The plasmid profile revealed that 35% of isolates did not contain any plasmids, but the rest (65%) carried a variable number of plasmids with different molecular weights. Six plasmid profiles were found among 40 S. Infantis isolates. Using ERIC2 primer, 7 profiles were found among 40 S. Infantis isolates in ERIC-PCR. Bands with molecular weights ranging from 400 to 3000 bp were observed. Conclusion: This study provided some genetic data on S. Infantis isolates recovered from poultry sources, and these data can be used for a broader epidemiological study nationwide. These findings showed that although plasmid and ERIC profiles are valuable in epidemiological studies, they have some limitations, too.

A simple and rapid technique of template preparation for PCR.

Many techniques have been developed for extracting DNA, but most are often complex, time-consuming, and/or expensive. In this study, we describe a simple, rapid and cost-effective technique for preparing DNA template for PCR. This technique involves 0.1 M potassium hydroxide treatment at 100°C for 10 min followed by centrifugation. The suspended centrifuged sediments were shown as excellent templates for PCR. Templates prepared using this technique worked for diverse microorganisms belonging to bacteria, fungi and oomycetes and their amplification efficiencies were comparable to/better than those prepared using common but relatively more complex, time-consuming, and/or expensive methods, including commercial DNA extraction kits. Furthermore, this technology is suitable for high-throughput batch processing and for amplifications of long DNA fragments. Flow cytometry and scanning electronic microscopy analyzes showed that this technique generated primarily damaged cells and cell-bound DNA, not free naked DNA. This technique provides a great convenience for simple PCR template preparation.