High-quality Genomic DNA Research Articles

Genetic diversity and comparative study of genomic DNA extraction protocols in Tamarix L. species

The genus Tamarix consists of about 54 species that mainly grow in saline areas of deserts and semi-deserts. This genus is chemically characterized by the presence of tannins, flavonoids, anthocyanins and essential oils which interfere with the extraction of pure genomic DNA. Thus it is necessary to optimize extraction protocols to minimize the influence of these compounds to the lowest level. The present study compares the efficiency of five different approaches to extract total genomic DNA in Tamarix species, showing significant differences in the extracted DNA contents and quality,by using Kit (DNP TM Kit), CTAB DNA extraction method by Murray and Thompson, Sahu et al., Nalini et al. and Bi et al., for the extraction of DNA from Tamarix species. Our results showed significant differences in DNA contents between these five methods. The quantity and quality of extracted genomic DNA were checked by the spectrophotometer, Nano-Drop and and agarose gel electrophoresis analysis. Finally, a PCR-based method was also applied to verify the amplification efficiency for two molecular markers (ITS and ISSR).. In the present study, the genetic diversity of 96 Tamarix individuals species and 8 populations were studied using 10 ISSR markerswhile for nrDNA ITS 8 species samples were used. The method of Nalini et al., provided best results (207 ng/μL) in terms of quantity and quality ofDNA. Our results proposed that this method could be effective for plants with the same polysaccharides, proteins and polyphenols components. The advantage of this method is simple and fast as it does not involve time consuming steps such as incubation at higher temperatures, and also do not requires expensive chemicals such as proteinase K, liquid nitrogen. ,. The success of this method in obtaining high-quality genomic DNA has been demonstrated in the Tamarix species group and the reliability of this method has been discussed.

Genomic DNA extraction from minimal amount of dried mushroom samples

Aims: To develop a simple and rapid genomic DNA extraction technique for dried (≈ 1 year old) mushroom fruiting bodies that yields high-quality DNA, suitable for use by post-graduate institutions. Method: Small amounts (0.04 g) of pulverized dried mushroom sample were incubated in a Tris /EDTA/SDS lysis buffer (100mM:10mM: 2%) at 65°C to lyse the chitinous fungal cell walls. Genomic DNA purification was performed using chloroform isoamyl alcohol (24:1), and DNA was precipitated using 100% ethanol. Results: Genomic DNA was successfully extracted under 70 minutes from 16 samples morphologically identified as Panaeolus, Copelandia, Gymnopilus, Pluteus and Favolus species. DNA concentrations were on average of 696.9ng/µL. PCR successfully amplified the ITS-5.8S region. The protocol has been successfully used by numerous post-graduate students in our research programme. Conclusion: The rapid and easy protocol produced high-quality genomic DNA void of any inhibitors that is suitable for downstream molecular implications across multiple mushroom genera. Noticeably, this method requires only minute quantities (0.04g) of starting material and is ideal for student training in higher academic institutions.

The emergency medical service has a crucial role to unravel the genetics of sudden cardiac arrest in young, out of hospital resuscitated patients: Interim data from the MAP-IT study

BackgroundGenetics of sudden cardiac deaths (SCD) remains frequently undetected. Genetic analysis is recommended in undefined selected cases in the 2021 ERC-guideline. The emergency medical service and physicians (EMS) may play a pivotal role for unraveling SCD by saving biomaterial for later molecular autopsy. Since for high-throughput DNA-sequencing (NGS) high quality genomic DNA is needed.We investigated in a prospective proof-of-concept study the role of the EMS for the identification of genetic forms of SCDs in the young. MethodsWe included patients aged 1–50 years with need for cardiopulmonary resuscitation attempts (CPR). Cases with non-natural deaths were excluded. In two German counties with 562,904 residents 39,506 services were analysed. Paired end panel-sequencing was performed, and variants were classified according to guidelines of the American College of Medical Genetics (ACMG). Results769 CPR-attempts were recorded (1.95% of all EMS-services; CPR-incidence 68/100,000). In 103 cases CPR were performed in patients < 50y. 58% died on scene, 26% were discharged from hospital. 24 subjects were included for genotyping. Of these 33% died on scene, 37.5% were discharged from hospital. 25% of the genotyped patients were carriers of (likely) pathogenic (ACMG-4/-5) variants. 67% carried variants with unknown significance (ACMG-3). 2 of them had familial history for arrhythmogenic cardiomyopathy or had to be re-classified as ACMG-4 carriers due to whole exome sequencing. ConclusionThe EMS contributes especially in fatal OHCA-cases to increase the yield of identified genetic conditions by collecting a blood sample on scene. Thus, the EMS can contribute significantly to primary and secondary prophylaxis in affected families.

A Cost-effective DNA Isolation Strategy from Crustaceans Enables the First Molecular Phylogenetic Identification of Moina macrocopa from India

We report the first barcode identification of water flea species Moina macrocopa (strain JSK1) from the Indian subcontinent. The strain isolated from a freshwater pond near Mumbai by collective efforts of CUBE (Collaborative Undergraduate Biology Education), was morphologically characterized as Moina species. Keeping up with the growing demand of affordable science in colleges and to avoid hazardous technologies, we established a home grown porcelain-abrasion DNA extraction strategy for crustaceans, and compared it against routinely used protocols employing liquid nitrogen or insect-DNA isolation kits. Our method successfully yielded high quality genomic DNA from Moina, comparable to published protocols as further analysed by restriction digestion, ligation and PCR efficiency. We also sequenced two barcoding loci namely, COI and ITS sequences, duly submitted and curated by Genbank (accession numbers MH734122.1 and MH745035.1). Molecular phylogenetic analyses identified the Indian species as Moina macrocopa and related it closely to the Russian clade. Protein prediction analyses of the variations in COI revealed that the missense mutations lie in the aliphatic membrane helices and should not affect the catalytic capacity of cytochrome oxidase. The strain JSK1 is currently disseminated and maintained collectively by CUBE students across India, towards developing it as a model system.

Retrieving high-quality genomic DNA from formalin-fixed paraffin-embedded tissues for multiple molecular analyses

Formalin-fixed paraffin-embedded (FFPE) tissues represent the biggest source of archival materials for molecular biology research and pathology investigations. Nevertheless, fixation by formalin may cause denaturation and modification of macromolecules constraining DNA quality and its downstream applications. In this study, we developed a fast, simple, and cost-effective phenol/chloroform-based protocol for the extraction of high-quality DNA from 101 FFPE colorectal cancer tissue blocks that can be used in multiple molecular studies. DNA samples extracted using this phenol/chloroform protocol and the QIAamp DNA FFPE Tissue kit were evaluated for the quantity, quality, and amplificability. Spectrophotometer analyses revealed significantly higher quality and quantity of DNA samples obtained with the phenol/chloroform protocol as compared to those of the QIAamp DNA FFPE Tissue kit. In addition, the amplificability of these samples as assessed by conventional and multiplex polymerase chain reaction (PCR), followed by sequencing and fragment analyses presented an absolute success rate. Additionally, it is able to amplify a DNA fragment of 725 base-pairs at an adequate amount for downstream applications. This fast, simple, and cost-effective method may facilitate the molecular analyses of a large number of FFPE specimens that best suits the needs of the overall study design in terms of the quality and quantity of the extracted DNA.

DNA isolation and amplification of raru leaf (Cotylelobium melanoxylon Pierre and Cotylelobium lanceolatum Craib)

Cotylelobium lanceolatum Craib and Cotylelobium melanoxylon Pierre or locally known as raru are trees used as ingredients for traditional Batak drinks called “tuak”. Destructive and uncontrolled harvesting of raru leads to a decline in its population and genetic diversity. Morphological distinctions for C. melanoxylon and C. lanceolatum have been conducted even though there are some weaknesses of this identification, thus molecular based marker are tools which is preferred for solved the problem. However, an appropriate DNA isolation method is basic and important step for molecular studies to obtain high-quality genomic DNA so that further analysis can proceed. Therefore, our study focused to obtain the successful DNA isolation method using the modified CTAB (Cetyl Trimethyl Ammonium Bromide) method and its amplification on three candidate of DNA barcodes i.e. trnL-trnF, trnH-psbA, ITS1-ITS4. In total six genomic DNA were isolated from the silica-gel dried leaf of the two raru species and amplified with trnL-trnF, trnH-psbA, and ITS1-ITS4 primer-pairs. The results showed that the modified CTAB method proved to be successful and applicable for the extraction of DNA of C. melanoxylon Pierre and C. lanceolatum Craib. Furthermore, the trnL-trnF primer-pair was successfully amplified compared with trnH-psbA and ITS1-ITS4 indicating that the primer-pair can be used as a DNA barcode candidate for further DNA sequencing and analysis.

Arabidopsis thaliana Mature Endosperm Dissection and Isolation of Genomic DNA from MatureSeed Tissues.

We describe methods to separate endosperms and embryos from Arabidopsis thaliana mature seeds in large amountsand to isolate high-quality genomic DNA from those tissues. The resulting materials are suitable for analysis of DNA methylation by bisulfite sequencing or histone modifications by chromatin immunoprecipitation (ChIP).

Whole genome sequencing of Theileria parva using target capture

Protozoan parasite isolation and purification are laborious and time-consuming processes required for high quality genomic DNA used in whole genome sequencing. The objective of this study was to capture whole Theileria parva genomes directly from cell cultures and blood samples using RNA baits. Cell culture material was bait captured or sequenced directly, while blood samples were all captured. Baits had variable success in capturing T. parva genomes from blood samples but were successful in cell cultures. Genome mapping uncovered extensive host contamination in blood samples compared to cell cultures. Captured cell cultures had over 81 fold coverage for the reference genome compared to 0–33 fold for blood samples. Results indicate that baits are specific to T. parva, are a good alternative to conventional methods and thus ideal for genomic studies. This study also reports the first whole genome sequencing of South African T. parva.

A low-cost pipeline for soil microbiome profiling.

Common bottlenecks in environmental and crop microbiome studies are the consumable and personnel costs necessary for genomic DNA extraction and sequencing library construction. This is harder for challenging environmental samples such as soil, which is rich in Polymerase Chain Reaction (PCR) inhibitors. To address this, we have established a low‐cost genomic DNA extraction method for soil samples. We also present an Illumina‐compatible 16S and ITS rRNA gene amplicon library preparation workflow that uses common laboratory equipment. We evaluated the performance of our genomic DNA extraction method against two leading commercial soil genomic DNA kits (MoBio PowerSoil® and MP Biomedicals™ FastDNA™ SPIN) and a recently published non‐commercial extraction method by Zou et al. (PLoS Biology, 15, e2003916, 2017). Our benchmarking experiment used four different soil types (coniferous, broad‐leafed, and mixed forest plus a standardized cereal crop compost mix) assessing the quality and quantity of the extracted genomic DNA by analyzing sequence variants of 16S V4 and ITS rRNA amplicons. We found that our genomic DNA extraction method compares well to both commercially available genomic DNA extraction kits in DNA quality and quantity. The MoBio PowerSoil® kit, which relies on silica column‐based DNA extraction with extensive washing, delivered the cleanest genomic DNA, for example, best A260:A280 and A260:A230 absorbance ratios. The MP Biomedicals™ FastDNA™ SPIN kit, which uses a large amount of binding material, yielded the most genomic DNA. Our method fits between the two commercial kits, producing both good yields and clean genomic DNA with fragment sizes of approximately 10 kb. Comparative analysis of detected amplicon sequence variants shows that our method correlates well with the two commercial kits. Here, we present a low‐cost genomic DNA extraction method for soil samples that can be coupled to an Illumina‐compatible simple two‐step amplicon library construction workflow for 16S V4 and ITS marker genes. Our method delivers high‐quality genomic DNA at a fraction of the cost of commercial kits and enables cost‐effective, large‐scale amplicon sequencing projects. Notably, our extracted gDNA molecules are long enough to be suitable for downstream techniques such as full gene sequencing or even metagenomics shotgun approaches using long reads (PacBio or Nanopore), 10x Genomics linked reads, and Dovetail genomics.

Optimization of high molecular weight DNA extraction methods in shrimp for a long-read sequencing platform.

Marine organisms are important to global food security as they are the largest source of animal proteins feeding mankind. Genomics-assisted aquaculture can increase yield while preserving the environment to ensure sufficient and sustainable production for global food security. However, only few high-quality genome sequences of marine organisms, especially shellfish, are available to the public partly because of the difficulty in the sequence assembly due to the complex nature of their genomes. A key step for a successful genome sequencing is the preparation of high-quality high molecular weight (HMW) genomic DNA. This study evaluated the effectiveness of five DNA extraction protocols (CTAB, Genomic-tip, Mollusc DNA, TIANamp Marine Animals DNA, and Sbeadex livestock kits) in obtaining shrimp HMW DNA for a long-read sequencing platform. DNA samples were assessed for quality and quantity using a Qubit fluorometer, NanoDrop spectrophotometer and pulsed-field gel electrophoresis. Among the five extraction methods examined without further optimization, the Genomic-tip kit yielded genomic DNA with the highest quality. However, further modifications of these established protocols might yield even better DNA quality and quantity. To further investigate whether the obtained genomic DNA could be used in a long-read sequencing application, DNA samples from the top three extraction methods (CTAB method, Genomic-tip and Mollusc DNA kits) were used for Pacific Biosciences (PacBio) library construction and sequencing. Genomic DNA obtained from Genomic-tip and Mollusc DNA kits allowed successful library construction, while the DNA obtained from the CTAB method did not. Genomic DNA isolated using the Genomic-tip kit yielded a higher number of long reads (N50 of 14.57 Kb) than those obtained from Mollusc DNA kits (N50 of 9.74 Kb). Thus, this study identified an effective extraction method for high-quality HMW genomic DNA of shrimp that can be applied to other marine organisms for a long-read sequencing platform.

Development of an SSR-based DNA fingerprinting method for black wattle (Acacia mearnsii De Wild)

Background: The most commonly used method for extracting DNA from plant leaf tissue involves cetyl trimethylammonium bromide but some species, such as Acacia mearnsii, contain high levels of secondary metabolites and polysaccharides that interfere with this process. Various modifications have been proposed for effective removal of these biomolecules but these methods can be time consuming. Therefore, this study was initiated to optimise the cetyl-trimethylammonium bromide protocol for the extraction of high-quality genomic DNA and to develop a fingerprinting tool using cross species transferable simple sequence repeat markers for genetic diversity studies in A. mearnsii.&#x0D; Methods: Five CTAB-based modification were examined and 49 cross-species microsatellite markers, developed for several Acacia species, were tested in four multiplex panels of A. mearnsii populations.&#x0D; Results: The modified protocol yields high quantity and quality DNA from A. mearnsii leaves using high concentration of NaCl to remove polysaccharides and polyvinylpolypyrrolidone (PVPP) to eliminate polyphenols during DNA purification. In addition, omitting the selective precipitation and NaCl gradient steps in the extraction protocol, enabled us to extract DNA 10–20 min faster than the normal protocol. Of the tested microsatellite loci, 11 were successful in amplifying sharp and high-intensity bands in all the four multiplex panels and were polymorphic. The level of polymorphism ranged from 0.115 to 0.794, with a mean 0.50 and mean number of alleles varied from 2 to 10, with overall mean of 6 alleles per locus. The mean observed and expected heterozygosity ranged from 0.058 to 0.970 and 0.102 to 0.796, respectively. The 11 microsatellite loci that were effectively amplified from A. mearnsii DNA were adequate in detecting genetic variation among the tested populations.&#x0D; Conclusions: These PCR-based, multi-allelic, co-dominant microsatellite markers provide a powerful tool for genetic, breeding and conservation studies in A. mearnsii.

SILEX: a fast and inexpensive high-quality DNA extraction method suitable for multiple sequencing platforms and recalcitrant plant species

BackgroundThe use of sequencing and genotyping platforms has undergone dramatic improvements, enabling the generation of a wealth of genomic information. Despite this progress, the availability of high-quality genomic DNA (gDNA) in sufficient concentrations is often a main limitation, especially for third-generation sequencing platforms. A variety of DNA extraction methods and commercial kits are available. However, many of these are costly and frequently give either low yield or low-quality DNA, inappropriate for next generation sequencing (NGS) platforms. Here, we describe a fast and inexpensive DNA extraction method (SILEX) applicable to a wide range of plant species and tissues.ResultsSILEX is a high-throughput DNA extraction protocol, based on the standard CTAB method with a DNA silica matrix recovery, which allows obtaining NGS-quality high molecular weight genomic plant DNA free of inhibitory compounds. SILEX was compared with a standard CTAB extraction protocol and a common commercial extraction kit in a variety of species, including recalcitrant ones, from different families. In comparison with the other methods, SILEX yielded DNA in higher concentrations and of higher quality. Manual extraction of 48 samples can be done in 96 min by one person at a cost of 0.12 €/sample of reagents and consumables. Hundreds of tomato gDNA samples obtained with either SILEX or the commercial kit were successfully genotyped with Single Primer Enrichment Technology (SPET) with the Illumina HiSeq 2500 platform. Furthermore, DNA extracted from Solanum elaeagnifolium using this protocol was assessed by Pulsed-field gel electrophoresis (PFGE), obtaining a suitable size ranges for most sequencing platforms that required high-molecular-weight DNA such as Nanopore or PacBio.ConclusionsA high-throughput, fast and inexpensive DNA extraction protocol was developed and validated for a wide variety of plants and tissues. SILEX offers an easy, scalable, efficient and inexpensive way to extract DNA for various next-generation sequencing applications including SPET and Nanopore among others.

Natal Origin Identification of Green Turtles in the North Pacific by Genome-Wide Population Analysis With Limited DNA Samples

Although studies using genome-wide single nucleotide polymorphisms (SNPs) are growing in non-model species, it is still difficult to prepare sufficient high-quality genomic DNA (gDNA) for population genomic analyses in many wild species. In this study, we first analyzed the population structure of green turtles in the North Pacific; four regions in Japan (the Yaeyama, Okinawa, Amami, and Ogasawara Islands), the Southeast Asia, Taiwan, the Federated States of Micronesia, the Republic Marshall Islands, and the Central or Eastern Pacific (C/E Pacific) using ≃1 ng of gDNA from green turtle field samples—including ones from dead carcasses—and multiplexed inter-simple sequence repeat (ISSR) genotyping by sequencing (MIG-seq). In addition, we explored whether the genome-wide SNP data narrowed down the natal origins of foraging turtles that were difficult to identify by mitochondrial DNA (mtDNA) haplotype alone. The overall nesting population structure observed from genome-wide SNPs was consistent with results from mtDNA, and the population isolation of the C/E Pacific and Ogasawara Islands from the other North Pacific populations was highlighted. However, the population boundaries among the Northwestern Pacific, other than those of the Ogasawaras, were not clear. The uniqueness of the Ogasawara population in genome-wide SNP data enabled the identification of turtles that were more likely to have been born on the Ogasawara Islands. Our results show that genome-wide SNP data are more practical in identifying the natal origins of turtles that were difficult determine by the conventional mtDNA-basis mixed stock analysis. This study also showed that MIG-seq can be expected to meet the potential demand to utilize many preserved or fragmented gDNA samples for population genomics in many marine megafaunas for which sample collections are difficult.

Extraction of high-quality genomic DNA and identification of different DNA barcoding markers for chickpea (Cicer arietinum L.)

The genetic studies of individual plants, especially self-pollinated species like chickpea need to be evaluated at the DNA level with the help of molecular markers for identifying genetic variations among the plants. High-quality DNA extraction is a prerequisite for genetic studies. Extraction of intact genomic DNA with high – molecular mass is essential for the study of many molecular biology applications like Polymerase Chain Reaction, endonuclease restriction digestion, southern blot analysis, and also for the construction of a genomic library. Several plant DNA extraction methods are available, even though the DNA isolation methods that give good yield employing both quantity and quality is quite difficult especially for self-pollinated crops like a chickpea. This work was focused on developing a standard protocol for the extraction of genomic DNA and identifying different barcoding markers. The result revealed that the CTAB extraction method with slight modification in protocol had been optimized for DNA isolation. The purified DNA, which was isolated through the CTAB method, had excellent spectral qualities and is efficiently digested by a restriction endonuclease, and is found to be more suitable for long-fragment PCR amplification. DNA barcoding is considered as a promising tool because it provides a practical and standard identification of plants. The isolated DNA sample was processed with a classical DNA barcoding approach by amplifying and sequencing with a universal primer. According to the result, among the different barcoding markers studied, the RbcL and Mat K were found to given the best result for molecular species identification in chickpea.

The Influence of DNA Extraction and Lipid Removal on Human Milk Bacterial Profiles.

Culture-independent molecular techniques have advanced the characterization of environmental and human samples including the human milk (HM) bacteriome. However, extraction of high-quality genomic DNA that is representative of the bacterial population in samples is crucial. Lipids removal from HM prior to DNA extraction is common practice, but this may influence the bacterial population detected. The objective of this study was to compare four commercial DNA extraction kits and lipid removal in relation to HM bacterial profiles. Four commercial DNA extraction kits, QIAamp® DNA Microbiome Kit, ZR Fungal/Bacterial DNA MiniPrep™, QIAsymphony DSP DNA Kit and ZymoBIOMICS™ DNA Miniprep Kit, were assessed using milk collected from ten healthy lactating women. The kits were evaluated based on their ability to extract high quantities of pure DNA from HM and how well they extracted DNA from bacterial communities present in a commercial mock microbial community standard spiked into HM. Finally, the kits were evaluated by assessing their extraction repeatability. Bacterial profiles were assessed using Illumina MiSeq sequencing targeting the V4 region of the 16S rRNA gene. The ZR Fungal/Bacterial DNA MiniPrep™ and ZymoBIOMICS™ DNA Miniprep (Zymo Research Corp., Irvine, CA, USA) kits extracted the highest DNA yields with the best purity. DNA extracted using ZR Fungal/Bacterial DNA MiniPrep™ best represented the bacteria in the mock community spiked into HM. In un-spiked HM samples, DNA extracted using the QIAsymphony DSP DNA kit showed statistically significant differences in taxa prevalence from DNA extracted using ZR Fungal/Bacterial DNA MiniPrep™ and ZymoBIOMICS™ DNA Miniprep kits. The only difference between skim and whole milk is observed in bacterial profiles with differing relative abundances of Enhydrobacter and Acinetobacter. DNA extraction, but not lipids removal, substantially influences bacterial profiles detected in HM samples, emphasizing the need for careful selection of a DNA extraction kit to improve DNA recovery from a range of bacterial taxa.

Data on the Draft Genome Sequence of Bacillus sp. Strain AN2 Isolated from Agricultural Soil in Brazil

Aims: This research aims to report the genome sequence of the Bacillus sp. strain AN2, isolated from agricultural soil from Rio de Janeiro, Brazil.&#x0D; Place and Duration of Study: Laboratory of Environmental Biotechnology, Western Rio Janeiro State University in Brazil, between January 2019 and December 2019.&#x0D; Methodology: High-quality genomic DNA was extracted using a GenElute Bacterial Genomic DNA kit. The Nextera XT DNA Library Prep Kit was used for genomic library construction. Paired-end sequence reads were generated by an Illumina MiSeq instrument with the 600 cycles MiSeq Reagent Kit v3. Sequence data were assembled with A5-MiSeq pipeline software and the contigs were annotated by Rapid Annotation using Subsystems Technology (RAST).&#x0D; Results: The obtained genome sequence of Bacillus AN2 included 21 contigs with a calculated size of 3,681,081 bp in length. The G + C content for the draft genome is 41.4%. A total of 3824 coding sequences (CDS) were predicted and encoded at least 88 tRNAs.

A scalable, efficient, and safe method to prepare high quality DNA from mycobacteria and other challenging cells

The rapid development in sequencing technology is creating an increase in demand for largely intact DNA as starting material as very long strands of DNA are sequenced directly to generate reads that are thousands of bases long. Organisms with thick cell walls are difficult to lyse, often impacting both DNA recovery and quality. Consequently, most mycobacterial DNA extraction methods require bead-beating steps or toxic chemicals. Here we present an updated method that yields abundant, high quality genomic DNA from M. tuberculosis and diverse nontuberculous mycobacterial (NTM) species, in addition to complex biological communities from a variety of sources. This method eliminates the time-consuming phenol and chloroform extraction and ethanol precipitation steps, and high quality DNA from up to 96 samples can be extracted in about 2–3 h of hands-on time. This DNA is suitable for long and short read sequencing technologies as well as PCR and qPCR amplification.

Optimization of Chelex 100 resin-based extraction of genomic DNA from dried blood spots.

Dried blood spots (DBS) are widely utilized as part of universal newborn screening and as a means of transporting samples from field sites. We use DBS from African field sites to assess for rare maternal-fetal cell exchange during pregnancy known as microchimerism. We aimed to develop a protocol to maximize the quantity of high-quality genomic DNA (gDNA) extracted from DBS. The total gDNA yield obtained from control DBS utilizing a Qiagen-based protocol and a Chelex® 100 resin-based protocol was first compared. Variations of the Chelex® protocol were subsequently tested to develop an optimized protocol. The gDNA was quantified by qPCR targeting the human beta-globin gene. DNA yield for a given experimental condition was normalized to a Chelex® control performed on the same day, and the total yields were compared using a Student’s t-test. The control Chelex® protocol yielded 590% more DNA than the QIAamp® DNA Blood Mini Kit . The absolute efficiency of the control Chelex® protocol was 54%, compared to an absolute efficiency of 9% for the QIAamp® DNA Blood Mini Kit. Modification of the Chelex® protocol to include a second heat precipitation from the same DBS increased the gDNA yield by 29% (P < 0.001). Our optimized protocol including this modification increased the absolute efficiency of extraction to 68%. The gDNA extracted using the Chelex® protocol was stable through repeated freeze–thaw cycles. In a mock microchimerism experiment, rare donor alleles at a frequency of 10 in 100 000 could be identified in gDNA from DBS extracted using the optimized Chelex® protocol. Our findings may be of significance for a diverse range of applications that utilize DBS and require high-quality DNA, including newborn screening programs, pathogen and drug resistance screening from remote field sites, forensics, and rare allele detection.

Optimized Extraction of Insect Genomic DNA for Long-Read Sequencing.

Long-read sequencing technologies continue to increase the length of reads, and at present can average read lengths of >20 kb up to 60–80 kb. Now the challenge is to extract genomic DNA of sufficient fragment size and quality to support longer read lengths. We developed a successful method to consistently obtain high-quality long genomic DNA from insects. The optimal developmental stage of insects for genomic DNA extraction was determined to be the pupal stage, eliminating DNA from ingested food and reducing contamination by chitinous material that can interfere with extraction. Improved results were obtained by a modified procedure of a commercial genomic DNA extraction kit. Initially, soft pupal tissue of the red flour beetle, Tribolium castaneum, was disrupted in the kit lysis buffer using Teflon micropestles. Modifications to the kit protocol also included gentle mixing by inversion of the tube, instead of harsh vortexing steps, and using wide-bore pipette tips in transferring fractions containing genomic DNA. Data from one sample were provided as an example of successful downstream library production and sequencing. While the technique has been optimized for insects, extractions from tissues of other organisms using these modified procedures also may improve long-read sequencing results.

A simplified quick microbial genomic DNA extraction via freeze-thawing cycles.

Effective isolation of high-quality genomic DNA is one of the essential steps in molecular biology, biochemistry, and genetic studies. Here we describe a simplified procedure based on repeated freeze-thawing cycles to isolate genomic DNA from different organisms of microbes (Burkholderia pyrrocinia JK-SH007, Bacillus pumilus HRl0, Botrytis cinerea) and nematodes (Bursaphelenchus xylophilus). The DNA extraction buffer includes 10% of CTAB; 4% of NaCl (W/V); 20mM of ethylenediamine tetraacetic acid; 100mM of Tris-HCl, pH 8.0 and 1% of polyvinylpyrrolidone. The released DNA was purified from the mixture using a phenol/chloroform mixture and precipitated in 70% ethanol to remove proteins, carbohydrates, phenols, RNA, etc. Our method is a reproducible, simple, and rapid technique for routine DNA extractions from various microorganisms and nematodes. Furthermore, the low cost of this method could be an economic benefit to large-scale studies.