High-quality Genomic DNA Research Articles

Testing the impact of Whole Genome Amplification on genome comparison using the polyploid flagellated Giardia duodenalis as a model

The availability of high quality genomic DNA in sufficient amounts to perform Next Generation Sequencing (NGS) experiments is challenging for pathogens that cannot be cultivated in vitro, as is the case for many parasites. Therefore, Whole Genome Amplification (WGA) of genomic DNA is used to overcome this limitation. In this study, we evaluated the effect of WGA using the intestinal flagellated protozoan Giardia duodenalis as a model, due to its genome compactness (12 Mb), the presence of two diploid nuclei with variable levels of allelic sequence heterogeneity (ASH), and the availability of reference genomes. We selected one isolate (ZX15) belonging to the same genetic group of the reference isolate WB, namely Assemblage A, sub-Assemblage AI. Genomic DNA from the ZX15 isolate (GEN dataset) and that obtained by WGA of 1 ng of the same genomic DNA (WGA dataset) were sequenced on a HiSeq Illumina platform. Trimmed reads from the GEN and WGA experiments were mapped against the WB reference genome, showing the presence of a very small number of mutations (846 and 752, respectively). The difference in the number of mutations is largely accounted by local variation in coverage and not by bias introduced by WGA. No significant difference were observed in the distribution of mutations in coding and non-coding regions, in the proportion of heterozygous mutations (ASH), or in the transition/transversion ratio of Single Nucleotide Variants within coding sequences. We conclude that the quantitative and qualitative impact of WGA on the identification of mutations is limited, and that this technique can be used to conduct comparative genomics studies.

Whole-Genome Bisulfite Sequencing and Epigenetic Variation in Cereal Methylomes.

Whole-genome bisulfite sequencing (WGBS) is a technique used for the analysis of genome-wide DNA methylation patterns (DNA methylomes) at a single-base resolution. Here, I describe a simple DNA extraction method from rice endosperm and the universal protocol of WGBS, MethylC-sequencing library preparation. The use of benzyl chloride allows for the extraction of high-quality genomic DNA from starchy endosperm, while sodium bisulfite converts unmethylated cytosine to uracil, whereas methylated cytosine is unchanged. The bisulfite conversion of whole genome sequencing libraries before the final amplification step allows for the discrimination of methylated from unmethylated cytosines in a genome-wide manner.

Comparison of DNA extraction methods from Geranium (Geraniaceae)

The genus Geranium (Geraniaceae); with about 320 species throughout the temperate regions, is chemically characterised by the presence of tannins, flavonoids, anthocyanins and essential oils which interfere with the extraction of pure genomic DNA. It is necessary to optimise the extraction protocols to reduce the effects of the presence of these compounds to the lowest level. The present study compares the plant genomic DNA extraction Kit (DNP™ Kit), CTAB DNA extraction method by Murray and Thompson and Sahu et al., from the extracting DNA point of view Geranium species. The results showed significant differences in DNA contents between the three methods. Quantity and quality of extracted genomic DNAs were compared by employing the spectrophotometer, Nano-Drop, agarose gel electrophoresis, and polymerase chain reaction (PCR) methods and molecular marker such as (ITS and trnL-F) and ISSR. The method of Sahu et al., provided the best results (200 ng/µL) in terms of quantity and quality of DNA, therefore, this method was taken and optimised for DNA extraction. Our results proposed that this method could be effective for plants with same polysaccharides, proteins and polyphenols components. The advantage of this method is that it omits the use of liquid nitrogen and toxic phenols which are expensive. The success of this method in obtaining high-quality genomic DNA has been demonstrated in the Geranium species group and the reliability of this method has been discussed.

Histopathological factors affecting the extraction of high quality genomic DNA from tissue sections for next-generation sequencing.

To enable the widespread application of genomic medicine, the extraction of genomic DNA from thin sections of archived formalin-fixed and paraffin-embedded (FFPE) tissue blocks for next-generation sequencing (NGS) is often necessary. However, there are currently no guidelines available on which specific regions of the microtome sections to use for macrodissection with respect to the histopathological factors observed under microscopic examination. The aim of this study was to clarify the relationship between histopathological factors and DNA quality, and to standardize the macrodissection method for more efficient implementation of NGS. FFPE tissue specimens of 218 patients from the Biomarker Research for Anti-EGFR Monoclonal Antibodies by Comprehensive Cancer Genomics study were used to investigate the relationship between 15 histopathological factors and the quantitative ratio of double-stranded DNA (dsDNA) to total nucleic acids, as well as the ∆ crossing point value of each tissue specimen. Multivariate logistic regression analysis revealed that specimen storage of ≥3 years was negatively associated with dsDNA quality (P=0.0007, OR: 4.30, 95% CI: 1.85-10.04). In contrast, the presence of a mucus pool was positively associated with dsDNA quality (P=0.0308, OR: 0.23, 95% CI: 0.06-0.87). Metastatic tumors and longer specimen storage periods were significantly associated with lower ∆Cp values (P=0.0007, OR: 4.43, 95% CI: 1.87-10.49; and P=0.0003, OR: 5.51, 95% CI: 2.18-13.95, respectively). Therefore, macrodissection should not be performed on specimens exhibiting histopathological factors associated with poor DNA quality. In particular, the use of tissue blocks with a storage period of <3 years allows the extraction of genomic DNA suitable for NGS.

Abstract 4665: Lineage-tracing technology to understand the molecular events in a mouse model of tongue squamous cell carcinoma (SCC) carcinogenesis

Abstract Oral cavity squamous cell carcinomas (OCSCCs) account for over 450,000 cases per year worldwide, present a high metastatic potential, and have a survival rate ≤ 50%. There is a high incidence of OCSCC, but the mechanisms of carcinogenesis remain unclear. Therefore, the aim of this research was to delineate the mutational landscape in developing tumors and OCSCCs by combining a lineage tracing approach and a mouse model of tongue carcinogenesis, previously established by our group. We used K14-CreERTAM; Rosa26LacZ mice, which allowed the long term basal stem cells of the epithelium to be permanently marked at the time of a two-day tamoxifen treatment. We then induced carcinogenesis by treating mice for 10 weeks with the carcinogen 4-nitroquinoline 1-oxide (4-NQO), which recapitulates key phenotypic and molecular features observed in human OCSCC. We performed Laser Capture Microdissection (LCM) to isolate and retrieve high-quality genomic DNA exclusively from each clone of permanently labeled, long-lived LacZ+ stem cells; each clone of cells arises from one LacZ+ stem cell. We then performed exome sequencing of 3 to 5 mice per group, sacrificed immediately after the 10-week treatment with 4-NQO (pre-neoplastic), and mice sacrificed &amp;gt;19 weeks after the end of 4-NQO treatment (SCC). As a reference genome for alignment and germline mutation exclusion, we included 3 mice of the same genetic background that did not receive 4-NQO. After 10 weeks of 4-NQO treatment the tongues were hyperplastic, with occasional necrosis and mild atypia, whereas all mice evaluated &amp;gt;19 weeks post 4-NQO treatment showed tongue SCCs. The total numbers of somatic mutations and LOH (loss of heterozygosity) identified using the mutation calling tool VarScan were 75% (4615±2170 vs 1148±76) higher in SCC compared to pre-neoplastic tongue samples. Among the mutations predicted as high and moderate impact, the levels of SNVs (single nucleotide variants) vs. indels were similar between SCCs and pre-neoplastic samples, 98.4% and 96.5%, respectively. By detailed analysis of the mutation types we showed that the pre-neoplastic tissues exhibited a much higher proportion of LOH events than the SCCs, 34.4% and 7.6%, respectively. Some of the genes affected by LOH in pre-neoplastic tissues were identified as tumor suppressors. Our findings suggest that a predominance of LOH in pre-neoplastic tongue tissue may result in loss of tumor suppressor genes, potentially defining the early steps of carcinogenesis in tongue SCCs. In conclusion, following the fate of long term tongue epithelial stem cells has allowed us to identify genomic perturbations occurring in the tongues of 4-NQO-treated mice and to find potential early biomarkers for OCSCC. Grant ID: R01CA205258; Funding Agency: NIH/NCI. Citation Format: Marta Melis, Tuo Zhang, Theresa Scognamiglio, Lorraine J. Gudas. Lineage-tracing technology to understand the molecular events in a mouse model of tongue squamous cell carcinoma (SCC) carcinogenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4665.

Modified CTAB method for high-quality genomic DNA extraction from ship barnacle (Balanus sp.)

Presence of high amount of polysaccharide and polyphenolic compounds in barnacles restricts the isolation of high molecular weight good quality DNA. CTAB, which is a detergent used to isolate DNA from high polysaccharide containing plants, is used in this study to extract quality DNA from ship barnacle. This modified CTAB method is simple, efficient and quick enough to be carried out within 2 h. Taking DNAzol ® isolated DNA as a reference, restriction digestion and PCR was performed to prove the absence of any enzyme inhibitory substances in the isolate. Traditional phenol-chloroform and salting out method failed to even isolate the DNA from barnacle sample. The molecular weight of the modified CTAB extracted DNA was higher than that of DNAzol ® extracted DNA which makes it better candidate for analyses like whole genome sequencing.

Optimized Nuclear Pellet Method for Extracting Next-Generation Sequencing Quality Genomic DNA from Fresh Leaf Tissue.

Next-generation sequencing (NGS) is a revolutionary advancement allowing large-scale discovery of functional molecular markers that has many applications, including plant breeding. High-quality genomic DNA (gDNA) is a prerequisite for successful NGS library preparation and sequencing; however, few reliable protocols to obtain such plant gDNA exist. A previously reported nuclear pellet (NP) method enables extraction of high-yielding gDNA from fresh leaf tissue of maize (Zea mays L.), but the quality does not meet the stringent requirements of NGS. In this study, we optimized the NP method for whole-genome sequencing of rice (Oryza sativa L.) through the integration of simple purification steps. The optimized NP method relied on initial nucleus enrichment, cell lysis, extraction, and subsequent gDNA purification buffers. The purification steps used proteinase K, RNase A, phenol/chloroform/isoamyl alcohol (25:24:1), and chloroform/isoamyl alcohol (24:1) treatments for protein digestion and RNA, protein, and phenol removal, respectively. Our data suggest that this optimized NP method allowed extraction of consistently high-yielding and high-quality undegraded gDNA without contamination by protein and RNA. Moreover, the extracted gDNA fulfilled the quality metrics of NGS library preparation for the Illumina HiSeq X Ten platform by the TruSeq DNA PCR-Free Library Prep Kit (Illumina). We provide a reliable step-by-step guide to the extraction of high-quality gDNA from fresh leaf tissues of rice for molecular biologists with limited resources.

All in One High Quality Genomic DNA and Total RNA Extraction From Nematode Induced Galls for High Throughput Sequencing Purposes.

Meloidogyne spp. are plant-parasitic nematodes that form a very complex pseudo-organ, called gall, which contains the giant cells (GCs) to nourish them. During the last decade, several groups have been studying the molecular processes accompanying the formation of these structures, combining both transcriptomics and cellular biology. Among others, it was confirmed that a generalized gene repression is a hallmark of early developing GCs formed by Meloidogyne javanica in Arabidopsis and tomato. One of the main mechanisms behind this gene repression involve small RNAs (sRNAs) directed gene silencing. This is supported not only by the described action of several microRNAs differentially expressed in galls, but by the differential abundance of 24-nucleotide sRNAs in early developing Arabidopsis galls, particularly those rasiRNAs which are mostly involved in RNA-directed DNA methylation. Their accumulation strongly correlates to the repression of several retrotransposons at pericentromeric regions of Arabidopsis chromosomes in early galls. However, the contribution of this global gene repression to GCs/galls formation and maintenance is still not fully understood. Further detailed studies, as the correlation between gene expression profiles and the methylation state of the chromatin in galls are essential to raise testable working hypotheses. A high quality of isolated DNA and RNA is a requirement to obtain non-biased and comprehensive results. Frequently, the isolation of DNA and RNA is performed from different samples of the same type of biological material. However, subtle differences on epigenetic processes are frequent even among independent biological replicates of the same tissue and may not correlate to those changes on the mRNA population obtained from different biological replicates. Herein, we describe a method that allows the simultaneous extraction and purification of genomic DNA and total RNA from the same biological sample adapted to our biological system. The quality of both nucleic acids from Arabidopsis galls formed by M. javanica was high and adequate to construct RNA and DNA libraries for high throughput sequencing used for transcriptomic and epigenetic studies, such as the analysis of the methylation state of the genomic DNA in galls (MethylC-seq) and RNA sequencing (RNAseq). The protocol presents guidance on the described procedure, key notes and troubleshooting.

A method for improving the efficiency of DNA extraction from clotted blood samples.

BackgroundThe efficient and rapid extraction of high‐quality genomic DNA from clotted blood samples, which normally have a low yield and poor quality, is an important factor in genomic research. The objective of this study was to develop a simple and safe technique for dispersing the blood clots by the ball bearing metal shots. Normally, such clot samples may not have an acceptable yield by conventional DNA extraction methods. Also, in the present study, we have further investigated to improve salting‐out DNA extraction methods.MethodsInitially, 500 µL phosphate‐buffered saline (PBS) (1×) and two ball bearing metal shots were added to each tube of the clotted blood sample and then were gently rotated in an electric laboratory rotator for 1 hour at room temperature (18‐25°C). Genomic DNA was then extracted from samples using a modified salting‐out method and a modified QIAamp® DNA Blood Midi Kit and was compared with QIAamp® DNA Blood Midi Kit as a control. An assessment of the concentration and quality of the extracted DNA was performed using the UV‐visible spectrophotometer. The isolated DNA proved amenable to PCR amplification and gel electrophoresis.ResultsThe yield and purity of DNA obtained by these three methods were significantly different (P < 0.001), with a higher yield in the modified salting‐out method.ConclusionsOur proposed modified salting‐out method is simple and efficient for the isolation of DNA from old blood clot samples. It is both easy to use and is of low cost in routine laboratory tasks.

Standardization of DNA extraction protocol and screening of begomovirus in chilli and tomato plants

Extraction of high-quality genomic DNA is an important requisite for a number of molecular- and genome-based studies. Isolation of good quantity and quality of genomic DNA from plant materials is a major concern for molecular biologists as many of them contain large amount of polysaccharides, phenolics and secondary metabolites. The presence of polyphenols and polysaccharides in plants results in a brown–dark brown-coloured, contaminated DNA which is sticky and viscous in nature. In our present study, we performed DNA isolation from two plants (tomato and chilli) using Dellaporta, modified CTAB, phenanthroline-based, CaCl2-based DNA extraction protocols (with and without high-salt Tris-EDTA buffer treatment). For chilli plants, Dellaporta method with high-salt TE buffer treatment was found to be better than other methods, and for tomato plants modified CTAB and phenanthroline-based methods without high-salt TE treatment were better. To further confirm the applicability, total DNA was isolated from 99 chilli samples using Dellaporta method with high-salt TE treatment. The plants were then screened for geminivirus and 49 were found to be positive. Furthermore, one of the PCR products of Deng primer was cloned and analysis of the clones showed the presence of insert, thus confirming the applicability of the DNA extraction method for detection of virus, PCR and cloning.

Extracting the invisible: obtaining high quality DNA is a challenging task in small arthropods.

BackgroundThe application of an appropriate extraction method is a relevant factor for the success of all molecular studies.MethodsSeven different DNA extraction methods suitable for high-throughput DNA sequencing with very small arthropods were compared by applying nine different protocols: three silica gel based spin methods, two cetyltrimethyl ammonium bromide (CTAB) based ones (one with an additional silica membrane), a protein precipitation method and a method based on a chelating resin (applying different protocols). The quantity (concentration) and quality (degradation, contamination, polymerase chain reaction (PCR) and sequencing success) of the extracted DNA as well as the costs, preparation times, user friendliness, and required supplies were compared across these methods. To assess the DNA quantity, two different DNA concentration measurements were applied. Additionally, the effect of varying amounts of starting material (different body sizes), variable lysis temperatures and mixing during DNA extraction was evaluated.ResultsAlthough low DNA concentrations were measured for all methods, the results showed that—with the exception of two methods—the PCR success was 100%. However, other parameters show vast differences. The time taken to perform DNA extraction varied from 20 min to 2.5 h (Chelex vs. CTAB) and the costs from 0.02 to 3.46 € (Chelex vs. QIAamp kit) per sample. High quality genomic DNA was only gained from four methods. Results of DNA quantity measurements further indicated that some devices cannot deal with small amounts of DNA and show variant results.DiscussionIn conclusion, using Chelex (chelating resin) turned out as a rapid, low-cost method which can provide high quality DNA for different kinds of molecular investigations.

An efficient method for DNA extraction from cyanobacteria isolated from hypersaline and marine environments.

Cyanobacteria are ancient organisms surviving on the earth due to their simple nutritional requirements and ability to produce distinct secondary metabolites that can combat detrimental environmental impacts. In order to understand these abilities of cyanobacteria at the molecular level, it is necessary to extract high-quality genomic DNA. However, the presence ofsecondary metabolites and exopolysaccharides hinders the DNA extraction from these organisms, especially from hypersaline environments. Here we have developed and compared a new method with two known methods of DNA extraction from environmental isolates. The results clearly indicate that the new optimized method yielded large amount of DNA with high purity. Additionally, the extracted DNA showed reduced degradation and excellent overall quality, which can be used directlyfor downstream purposes such as PCR and sequencing.

Extraction of high-quality genomic DNA from different plant orders applying a modified CTAB-based method

BackgroundReliable measurement of DNA concentration and purity is important for almost all molecular genetics studies. Different plant species have varying levels of polysaccharides, polyphenols, and other secondary metabolites which combine with nucleic acids during DNA isolation and further affect the quality of the extracted DNA. The current extraction protocol is based upon the conventional cetyl trimethylammonium bromide (CTAB) method with further modifications for the extraction of DNA from variable plant seeds and crops belonging to seven different orders. The principle modifications currently employed for DNA extraction involved the use of higher CTAB concentration and higher levels of 2-β-mercaptoethanol. Additionally, higher concentrations of sodium chloride and potassium acetate were added simultaneously with absolute ice cold isopropanol for the precipitation of DNA free from polysaccharides.Results and conclusionThe prescribed modifications in the present method establish a quick and efficient standardized protocol for DNA extraction from different plant orders. The current extraction protocol, therefore, can be of great value for molecular analysis involving large numbers of different plant samples from different orders. These modifications consistently produced pure and high-quality DNA suitable for further molecular analysis. Successful PCR amplification with random amplified polymorphic DNA primer, NPTII gene, and the complete digestion of the isolated DNA with the HindIII restriction enzyme validated the quality of the isolated DNA. Moreover, it reflects the efficiency of the protocol and proves its suitability for further applications for the assessment of food safety, detection of genetically modified (GM) crops, and conservation of biodiversity.

DNA Extraction and Optimization of ISSR-PCR Reaction System for Pyracantha

Three different DNA extraction methods (CTAB, improved CTAB, and nuclear DNA method) were compared in order to isolate high-quality genomic DNA from fresh leaves of Pryacantha. The concentration of Mg2+, Taq polymerase, dNTPs, primer, and template DNA, greatly influencing ISSR-PCR of Pyracantha, were optimized by orthogonal design in this study, and the annealing temperature was also improved. The results showed that the high-quality genomic DNA was obtained by the improved CTAB method and was suitable for ISSR study. The optimal PCR system for ISSR analysis was as follows: total volume 25 µL, 2.5 µL 10×buffer, 1.0 mmol·L−1 Mg2+, 0.15 mmol·L−1 dNTPs, 0.1 umol·L−1 primer, 1.2 U Taq DNA polymerase, and 80 ng template DNA. The reaction procedure was as follows: pre-degeneration at 94 °C for 5 min, degeneration at 94 °C for 1 min, annealing at 51.0 °C∼59.2 °C for 1 min (annealing temperature depend on different primer), extension at 72 °C for 1 min, 40 cycles, final extension at 72 °C for 5 min and preservation at 4 °C.

A Modified Method for Rapid Genomic DNA Extraction from Tuberose

The isolation of high yield and quality genomic DNA is crucial for studying the molecular genetics of plants. However, high contents of secondary metabolites, especially in medicinal and aromatic plants, interfere with the extraction of clean DNA, thereby rendering it useless for downstream analyses such as DNA amplification, restriction, sequencing and cloning. The chemotypic heterogeneity among plant species may not permit high quality DNA isolation with a single protocol, thus a species-specific extraction method is required for quality extractions. Here, we present a modified cetyltrimethylammonium bromide (CTAB) protocol for good quality DNA extraction from tuberose, which is an important plant for perfume and pharmaceutical industry due to its pleasant fragrance and essential oil content. In contrast to other CTAB methods, the modified procedure is rapid, omits the use of liquid nitrogen and phenol, uses inexpensive and less hazardous reagents, and requires only ordinary laboratory equipment. The procedure employed the high concentration of NaCl and use ofPVP-10 to get rid of problems associated with polysaccharides and polyphenols, respectively. The yield and quality of extracted DNA were fairly good and amenable for downstream analyses. Moreover, to our knowledge, the described method is the first report of a modified DNA extraction protocol for tuberose.

A High-throughput qPCR-based Method to Genotype the SOD1G93A Mouse Model for Relative Copy Number.

The most commonly used mouse model in ALS preclinical research expresses multiple copies of the human SOD1 (G93A) transgene. During the course of breeding, successive generations of mice can lose copies of the transgene. Because shorter lifespan of these mice is dependent on transgene copy number, it is essential to ensure that no low-copy, and therefore longer-lived, mice are included in preclinical studies. Existing techniques for SOD1G93A mouse genotyping are broadly based on creating a standard curve using a reference gene and deducing the relative amount of SOD1 by comparison with the standard curve. This type of technique is used in Alexander et al. (2004) , Vieira et al. (2017) and Maier et al. (2018) . However, it is not described in detail (see Note 1). This paper provides a detailed protocol for determining the relative copy number of the human SOD1 transgene. Briefly, the protocol involves first the extraction of high-quality genomic DNA from mouse ear tissue, creation of a genomic DNA concentration-based standard curve, and qPCR analysis of up to 88 samples at once alongside the standard curve with Gapdh as a reference gene. Analysis involves the normalization of each unknown sample using the standard curve followed by determination of the copy number of the sample relative to the cohort median. This protocol has been optimized to produce high-quality genomic DNA and consistent results, and the relative copy number cutoffs have been optimized and validated empirically by comparison of relative copy number and mouse lifespan.

DNA extraction from FFPE tissue samples - a comparison of three procedures.

Aim of the studyOne of the critical steps in molecular oncology diagnostics is obtaining high quality genomic DNA. Therefore, it is important to evaluate and compare the techniques used to extract DNA from tissue samples. Since formalin-fixed, paraffin-embedded (FFPE) tissues are routinely used for both retrospective and prospective studies, we compared three commercially available methods of nucleic acid extraction in terms of quantity and quality of isolated DNA.Material and methodsSlides prepared from 42 FFPE blocks were macro-dissected. Resulting material was divided and processed simultaneously using three extraction kits: QIAamp DNA FFPE Tissue Kit (QIAGEN), Cobas DNA Sample Preparation Kit (Roche Molecular Systems) and Maxwell 16 FFPE Plus LEV DNA Purification Kit (Promega). Subsequently, quantity and quality of obtained DNA samples were analysed spectrophotometrically (NanoDrop 2000, Thermo Scientific). Results of quantitative analysis were confirmed by a fluorometric procedure (Qubit 3.0 Fluorometer, Life Technologies).ResultsThe results demonstrated that the yields of total DNA extracted using either Maxwell or Cobas methods were significantly higher compared to the QIAamp method (p < 0.001). The Maxwell Extraction Kit delivered DNA samples of the highest quality (p < 0.01). However, the highest total yield of extracted DNA was achieved with the Cobas technique, which may be due to a higher volume of eluate compared to the Maxwell method.ConclusionsTo our knowledge, this is the first paper which directly compares three extraction methods: Cobas, Maxwell and QIAamp. The data herein provide information required for the selection of a protocol that best suits the needs of the overall study design in terms of the quantity and quality of the extracted DNA.

Optimized protocol to isolate high quality genomic DNA from different tissues of a palm species

ABSTRACT The application of molecular techniques to tackle ecological and evolutionary questions requires genomic DNA in good quality and quantity. The quality of the isolated DNA, however, can be influenced by the tissue type and the way the sample was conserved and manipulated. Therefore, customizing protocols to improve the DNA isolation and locus amplification is crucial. We optimized a cheap and manual protocol of DNA extraction and microsatellites amplification using five different tissues of a palm species of the brazilian Atlantic Forest. We successfully extracted DNA from all five tissue types. Leaf, stem, and endocarp of non-dispersed seeds presented the highest rates of successful DNA extraction and microsatellite amplification; whereas root, endocarp of dispersed seeds, and embryo showed the lowest quality and quantity. Based on these results, we discussed the implications of using different tissues for studies about seed dispersal, pollination, and population genetics.

Increasing DNA Isolation Performance from Blood Cells Using a Modified Commercial Low-Cost Reagent.

Genomic studies facilitate comprehension of the pathophysiology, diagnosis, and treatment of chronic diseases. Such studies require sufficient and good quality DNA isolated from a large number of blood samples. This study attempts to obtain a high-quality genomic DNA isolated from a large number of blood samples using a simple and cheap method. The EasyPure® Genomic DNA Kit (Transgen Biotech) was modified to increase the amount of DNA recovery: a few steps and two additional column elutions were added to the original manufacturer´s procedure. The amount of DNA isolated from frozen blood samples increased by an average of 56%. Its 260/280 ratio and electrophoretic mobility properties make it suitable for genomic studies. A relatively low-cost commercial column and a simple modification of the manufacturer´s protocol, provided a simple and cheap procedure to isolate high-quality DNA from a large number of blood samples suitable for genomic studies.

Method for DNA Isolation From Sweetpotato Weevil (Coleoptera: Curculionidae) Collected in Pheromone-Baited Traps.

This study provides a protocol for the isolation of high-quality DNA from sweetpotato weevils (Cylas formicarius elegantulus (Summers)) collected from pheromone-baited aerial funnel traps. This study was based on our discovery that a 2-wk collection interval of sweetpotato weevils from pheromone traps did not permit isolation of intact high-quality genomic DNA. To test the effect of collection methods, i.e., sample collection interval and preservation method, on quality of isolated DNA, we placed freshly killed male sweetpotato weevils into aerial funnel traps in the field and removed subsamples at several times thereafter. DNA yield from freshly isolated (day = 0) samples was significantly greater than samples preserved in 70% ethanol or at -20°C, whereas there was no difference between 70% ethanol and -20°C storage. Likewise, DNA yield from freshly isolated (day = 0) samples was significantly greater than for later sampling times. Quality assessment of genomic DNA through gel electrophoresis and polymerase chain reaction (PCR) indicated isolation of high molecular weight DNA for all samples collected at t ≤ 7 d, but that DNA quality was degraded by 14 d. Our goal was to develop a reliable method for isolation of genomic DNA from field-collected sweetpotato weevil suitable for direct use in PCR. We discovered that it is critical to collect specimens from traps at an interval of 1 wk or less. Our findings allow for scheduling of sampling at reasonable intervals without the need for special materials. This has the added benefit of allowing individuals without special training to collect and prepare sweetpotato weevil specimens for genetic studies.