Size Of DNA Fragments Research Articles

Fast high-throughput screening of glutathione S-transferase polymorphism by voltage programming-based multi-channel microchip electrophoresis

Glutathione S-transferase (GST) polymorphism (M1 = 215 bp and T1 = 480 bp) can cause liver damage and increase the risk of cancer. In this study, voltage programming (VP)-based microchip electrophoresis (ME) with a laser-induced fluorescence (LIF) detector was developed to detect specific sizes of DNA fragments. The optimum conditions for a single-channel microchip were as follows: 4 kV for 0–9.5 s, 1.5 kV for 9.5–15.5 s, and 4 kV for 15.5–30 s. Next, these conditions were applied to another microchip that was constructed with many channels making possible simultaneous parallel detection. Finally, GST genes extracted from human blood were amplified by polymerase chain reaction (PCR) and were introduced into the multi-channel microchip. Target DNA molecules amplified by only 10 PCR cycles could be detected by the VP-based multi-channel ME method, but not by slab gel electrophoresis (SGE). In addition, the migration time for ME was <15 s, which was 700 times faster than conventional SGE. The developed VP-based multi-channel ME method with LIF detection was demonstrated to be an effective, rapid analysis technique for highly sensitive and high-throughput screening of GST genes.

The Application of a Meiocyte-Specific CRISPR/Cas9 (MSC) System and a Suicide-MSC System in Generating Inheritable and Stable Mutations in Arabidopsis.

The CRISPR/Cas9 system has been widely used for generating targeted mutations in various species. In Arabidopsis, it largely relies on the edited cells where the Cas9 protein performs its activity to obtain heritable and stable mutated lines. Here, we designed an improved CRISPR/Cas9 system, named as the MSC (meiocyte-specific CRISPR/Cas9) system, in which the Cas9 expression is driven by an experimentally approved meiocyte-specific promoter (AtDMC1 promoter). Two endogenous genes, including vegetative gene AtDET2 and reproductive gene AtDMC1, were targeted. We obtained heterozygous T1 plants for targeted genes with high efficiency (64%). In the T2 generation, the homozygous plants were abundant with high efficiency (37%). Analysis of Sanger sequencing results of T2 generation revealed that heritable gene mutations were high (52%). Moreover, we showed that the MSC system could sufficiently delete a middle size DNA fragment (∼500 bp) between two cleavage sites with a high rate (64.15%) in the T1 plants, providing direct evidence for making complete knock-out or certain domain-depletion mutations. In addition, we further made a suicide-MSC system, which can edit the targeted endogenous gene and the exogenous Cas9 gene simultaneously, not only successfully avoiding the further destroy of alleles brought in by molecular complementary or genic allelic test, but also maintaining the stable mutated alleles for functional studies. In short, the two systems provide new approaches to generate mutations for gene functional studies.

Performance of commonly requested destructive museum samples for mammalian genomic studies

High-throughput sequencing methods have facilitated obtaining large amounts of data from degraded DNA, thus resulting in a dramatic increase in destructive sampling requests to museums. Because the tissues taken from museum specimens as sources of DNA are destroyed during analysis, consideration of the costs and benefits of loss of valuable specimen material relative to knowledge gained is required for any project utilizing destructive sampling. Variation exists in the preservation of DNA in historical specimens due to specimen age and type of museum preparation, among other factors. Thus, it is important to assess DNA yield and quality from different sources of museum specimens when considering the needs of a particular molecular project. We compared DNA derived from several common sources of museum specimens including bone, claw, skin, and soft tissue adherent to skeletal preparations. To account for differences in preparation type and therefore specimen preservation, we tested the performance of samples representing 3 taxonomic groups: mephitids, rodents, and marsupials. We also compared yields from 2 commonly used DNA extraction techniques. DNA quality was assessed by comparing average fragment size, concentration, and copy number of template DNA (for mitochondrial and nuclear markers) in genomic DNA extracts, as well as mitochondrial genome sequence coverage resulting from shotgun sequencing. We show that DNA quality derived from historic museum samples differs depending on specimen and sample type; however, all samples yielded high mitochondrial copy number except the skin and nail from the tanned specimen. Overall, claw samples produced the greatest number of high-quality sequencing reads with the least amount of bacterial contamination. We also found that high DNA concentrations did not necessarily result in high percentages of on-target reads; in fact, the samples that yielded the highest DNA quantities also had the highest amount of exogenous bacterial DNA. Our results indicate that most historical tissue types can be suitable for next-generation sequencing approaches, therefore providing multiple options for natural history collection staff and researchers when considering destructive sampling requests.

Abstract 5593: The fragment size and levels of cell-free DNA provide prognostic information in patients with advanced pancreatic cancer

Abstract Purpose: To investigate the prognostic value of cell-free DNA (cfDNA) fragment size and cfDNA levels in patients with advanced pancreatic cancer. Methods: Blood samples were acquired from patients with locally advanced or metastatic pancreatic cancer at baseline (n = 61) and one month (n = 48) after initiation of chemotherapy. All samples were processed by Lymphoprep™ (Axis Shield) density centrifugation followed by DNA isolation from the plasma fraction using the QIAamp Circulating Nucleic Acid kit (Qiagen). The cfDNA fragment size and cfDNA levels were then determined using the High Sensitivity DNA kit on an Agilent 2100 Bioanalyzer. A cohort of healthy age-matched volunteers (n = 28) constituted the control group. Results: Both the cfDNA fragment size (median 167 vs 176.5 bp; p &amp;lt; 0.001) and the cfDNA levels (median 4.48 vs 0.33 ng/mL plasma; p &amp;lt; 0.001) were significantly different between patient and healthy control samples. A pre-treatment cfDNA fragment size of ≤167 bp (median) was associated with both shorter progression-free survival (PFS) (4 vs. 7.7 months; log-rank p = 0.002) and overall survival (OS) (4.6 vs. 10.5 months; log-rank p = 0.001). Similarly, cfDNA levels above the median value were associated with both shorter PFS (3.3 vs. 7.7 months; log-rank p &amp;lt; 0.001) and OS (5.4 vs. 8.8 months; log-rank p = 0.001) for samples obtained before initiation of chemotherapy. Survival analysis on the combination of fragment size and cfDNA levels showed a significantly longer PFS (8.1 vs 4.6 vs 2.6 months; log-rank p &amp;lt; 0.001) and OS (10.6 vs 7.9 vs 3.8 months; log-rank p = 0.001) for patients negative for both parameters compared to patients with 1 or 2 positive parameters, respectively. Analyses of plasma samples obtained one month after initiation of chemotherapy indicated a trend towards an association with PFS (p = 0.066 and p = 0.025), but not OS (p = 0.504 and p = 0.213) for cfDNA fragment size and cfDNA levels, respectively. Univariate cox regression using both the continuous and the categorical variable for cfDNA fragment size and cfDNA levels, and the categorical variable for the combination test, confirmed the results from the Kaplan-Meier estimates. Conclusion: Determination of cfDNA fragment size and cfDNA levels is a non-invasive and simple method for predicting prognosis in patients with advanced pancreatic cancer. Further investigations in larger patient cohorts are needed to elaborate the consistency and clinical impact of this finding. Citation Format: Morten Lapin, Satu Oltedal, Kjersti Tjensvoll, Tove Buhl, Rune Smaaland, Nils Glenjen, Bjørnar Gilje, Oddmund Nordgård. The fragment size and levels of cell-free DNA provide prognostic information in patients with advanced pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5593.

Abstract LB-024: The effects of chromatin structure on variation in fragment size of cell free DNA

Abstract Liquid biopsy using cell-free DNA (cfDNA) has established its clinical utilities in multiple clinical cohorts. However, the exact mechanism by which cell-free DNA (cfDNA) is generated remains undetermined. The fragment size of cfDNA sheds important insights on elucidating the process. Previous studies qualitatively concluded that tumor-derived cfDNA is shorter than cfDNA from healthy cells, but the exact size distribution of tumor-derived cfDNA remains unclear. To better understand the mechanism of cfDNA generation and directly study cfDNA of tumor origins, we analyzed the deep targeted sequencing results of cfDNA from 605 patients with various cancer types, and developed a method that separates tumor cfDNA through identification of somatic variant alleles-containing fragments. We show that tumor-derived cfDNA has an increased proportion of fragments less than 150bp compared to healthy controls, while the most common size is still 166 bp. It has been speculated that differences in nucleosome wrapping may contribute to differing fragment lengths of cfDNA.To better understand the relationship between the magnitude of chromatin packaging and cfDNA length, we quantitatively analyzed the size distribution of cfDNA collected from 5 cancer patients in genomic regions with the varying degree of chromatin openness. We discovered that the fragment size of cfDNA was closely associated with chromatin inaccessibility and chromosome condensation as measured by transcriptional start sites, DNase I hypersensitivity, histone modifications, and Giemsa banding, or as implied by chromosomal sub-nuclear localization. The shortening occurs at both 5' and 3' ends of cfDNA fragments without bias. Taken together, these results collectively established a positive correlation between cfDNA fragment size and chromatin inaccessibility. Citation Format: Hui Xia, Xingya Li, Hua Bao, Xiangyuan Ma, Xue Wu, Yang W. Shao, Feixiang Wu, Shucai Zhang. The effects of chromatin structure on variation in fragment size of cell free DNA [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-024.

Abstract 3408: Characteristics of circulating tumor DNA in lung cancer patients

Abstract Liquid biopsy using circulating tumor DNA (ctDNA) has been spread world-wide. We established fully automatic sensitive mutation detection system, mutation-biased PCR and quenched probe system (MBP-QP) method, and accomplished multicenter prospective study to investigate the utility of ctDNA in lung cancer patients who acquired resistance to epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKI). The results of the clinical study showed that ctDNA was frequently detected in lung cancer patients with distant metastasis, and detection of ctDNA was associated with poor prognosis. In metastatic animal model using immunodeficient mice, NOJ mice, the amount or ctDNA was associated with tumor progression such as tumor burden and metastasis. Based on these evidences, we hypothesized that ctDNA has some biological effects on tumor progression. In order to examine that, we analyzed biological and clinical characteristics of ctDNA. ctDNA was extracted from 1000μl plasma by automated DNA extraction system using cellulose magnetic beads. Compared to circulating free DNA (cfDNA) isolated from healthy volunteers, size distribution of ctDNA or cfDNA using a capillary electrophoresis system was different, that is one peak around the size of 170 bp in healthy individuals, and two peaks, 170 bp and 5 kb in advanced lung cancer patients. We next examined 130 plasma samples from 92 lung cancer patients in addition to 18 benign pulmonary disease patients and 20 healthy individuals at Saga University Hospital. The DNA concentration quantified by Quantus®, the fluorescent measurement of dsDNA intercalated dye, was higher in lung cancer patients compared to those in benign pulmonary disease patients and healthy individuals. Among lung cancer patients, DNA concentration was increased in those with advanced stages, especially in presence of metastasis. In addition, 5 kb fragments were significantly increased in these cases compared to 170 bp fragments. To investigate which fragment contained tumor-derived DNA, 170 bp and 5 kb fragments were separately isolated, and EGFR mutation, L858R was examined. L858R was detected in both ctDNA fragments, 170 bp and 5 kb, indicating that both sized DNA fragments contain tumor-derived DNA. Although the 170 bp short fragments of ctDNA are well known as an apoptotic product, the origin of 5 kb long fragments has not been clarified. We have examined whole ctDNA sequence using next generation sequencing, and compared two fragments. In addition, origins of each fragment have been investigated, and biological effects on tumor progression will be analyzed. Citation Format: Tomonori Abe, Chiho Nakashima, Akemi Sato, Eisaburo Sueoka, Shinya Kimura, Naoko Aragane. Characteristics of circulating tumor DNA in lung cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3408.

Abstract 3646: Investigation of origin of circulating free DNA: Is exosomal DNA the carrier

Abstract The usefulness of circulating free DNA (cfDNA) for analysis of genetic alterations is largely accepted. We accomplished multicenter prospective study to investigate sequential change of cfDNA in lung cancer patients who acquired resistance to epidermal growth factor receptor tyrosine kinase inhibitors. The detection frequency was elevated as cancer progressed, and the prognosis of the patients in which cfDNA was detected was poorer than those not detected. These results suggest that appearance of cfDNA was associated with tumor progression. To verify that, animal experiment using immunodeficient mice, NOJ mice, was performed. After transplantation with human lung cancer cell line, H1975 carrying EGFR L858R, and T790M, into dorsal flanks of these mice, systemic metastasis occurred. cfDNA was sequentially analyzed, resulting that the amount of cfDNA was correlated with tumor burden and metastatic status. In spite of these results, origin, kinetics or possible function on tumor progression of cfDNA has not been elucidated. We have reported there were large sized DNA fragments, around 5 Kb, which is longer than 170 bp of cfDNA conventionally detected in peripheral blood of advanced cancer patients. Exosomes, extracellular vesicles detected in peripheral blood has been reported to be involved in tumor progression through vesicle-mediated communication. In general, exosomes deliver protein, lipid and RNA, and few DNA was contained. However, some researchers reported a large proportion of cfDNA was localized in exosomes. To investigate the origin and localization of cfDNA in peripheral blood, we analyzed relationship between cfDNA and exosomal DNA. We isolated both of cfDNA and exosomal DNA simultaneously, from plasma of healthy individuals and advanced non-small cell lung cancer patients, and compared the DNA yield, DNA size distribution and EGFR mutation detection rate. Localization of DNA in exosome is also investigated using fluorescent dye. We used Total Exosome Isolation Kit® from plasma with/without proteinase K for isolation of exosome from 200 uL plasma, and extracted DNA using Maxwell RSC® circulating cell free DNA cartridge. The proportion of exosomal DNA /cfDNA (e/c DNA ratio) varied individually, but e/c DNA ratio was lower in plasma from cancer patients than healthy individuals. However, large sized fragments of cfDNA were observed in exosomal DNA in lung cancer patients. These results suggest that exosomal DNA might have some interaction with large sized cfDNA fragments observed in plasma isolated from advanced cancer patients. Citation Format: Chiho Nakashima, Tomonori Abe, Akemi Sato, Tomomi Nakamura, Kazutoshi Komiya, Eisaburo Sueoka, Shinya Kimura, Naoko Sueoka-Aragane. Investigation of origin of circulating free DNA: Is exosomal DNA the carrier [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3646.

Genetic Variations of Intra- and between-razor Clam Solen corneus Population Identified by PCR Analysis.

The author undertook PCR-founded genetic platform to investigate the hierarchical dendrogram of Euclidean genetic distances of one razor clam population, particularly for Solen corneus, which was further associated with those of the other clam population, by engaging with the precisely designed oligonucleotide primer sets. Seven oligonucleotides primers were used producing a total of 639 counted bands in population A and 595 in population B, respectively, ranging in size of DNA fragments from larger than approximately 50 bp to less than 1,100 bp. Their primers generated 39 specific fragments (6.10%) in population A and 47 (7.90%) in population B, respectively Comparatively, individuals of one razor clam population were fairly related to that of the other clam population, as shown in the hierarchical dendrogram of Euclidean genetic distances. The analysis of genetic variation between razor clam populations could offer important statistics for fisheries and mariculture. Generally the results showed specific and/or conserved genetic loci between razor clam populations. Specific markers established by the author will be valuable for the genetic analysis, species protection and increase of razor clam individuals in coastal region of the Korean Peninsula.

An Improved Method of Preparing High Efficiency Transformation Escherichia coli with Both Plasmids and Larger DNA Fragments.

The high-throughput, cost-efficient transformation systems determine the success of gene cloning and functional analysis. Among various factors that affect this transformation systems, the competence ability of target cells is one of the most important factors. We found antimicrobial peptides LFcin-B can increase the permeability of the cell membrane, and their lethal antibacterial properties can be inhibited by moderately high concentrations of Ca2+ and Mn2+. In this study, we established a convenient and rapid method (CRM) by adding small concentrations of (0.35mg/L) and moderately high concentrations of MnCl2 (50mM) and CaCl2 (30mM) in transformation buffer. The transformation efficiency of E.coli cells (DH5α, JM109 and TOP10) prepared by CRM were comparable with electroporation for plasmid transformation (3.1 ± 0.3 × 109 cfu/µg). Unlike competent cells prepared using other chemical methods, those obtained using CRM method are extremely competent for receiving larger size DNA fragments (> 5000bp) into plasmid vectors. The competent E.coli cells prepared by CRM method are particularly useful for most high-efficiency transformation experiments under normal laboratory conditions.

Automated DNA extraction using cellulose magnetic beads can improve EGFR point mutation detection with liquid biopsy by efficiently recovering short and long DNA fragments.

The clinical utility of plasma DNA for detecting cancer-specific mutations has rapidly achieved recognition, but reliability has not been established because of relatively low mutation-detection rates compared with those from tissue re-biopsy. To address this shortcoming we examined efficiency, in terms of mutation detection, of an automated DNA extraction system that uses cellulose magnetic beads. A fully automated, highly sensitive point-mutation-detection method, mutation-biased PCR and quenching probe (MBP-QP) system, was used for this study. Plasma DNA was extracted from 61 plasma samples collected from patients with advanced non-small cell lung cancer. Extraction was performed manually with 200 μl plasma (200-M) by using a silica membrane spin column system or an automated system using 200 μl (200-A) or 1000 μl (1000-A) plasma. Median DNA yield quantified by real-time PCR was 4.4, 4.5, and 17.3 ng with the three methods, respectively. Sensitivity for detecting epidermal growth factor receptor (EGFR) L858R point mutation was 36.6%, 58.5%, and 77.5%, and specificity was 93.3%, 100%, and 96.7%, respectively. Concordance rates were 60.6%, 76.1%, and 85.7%. The size distribution of plasma DNA with automated extraction was bimodal with modes at about 170 bp and 5 Kb, and plasma DNA of both sizes included tumor-derived DNA. In this report, we demonstrate that automated DNA extraction using cellulose magnetic beads can improve mutation-detection rates with plasma DNA in association with two overall sizes of DNA fragments recovered by this DNA isolation system. Examining the biological characteristics of these fragments will be the subject of further investigation.

Reduced Neuronal Transcription of Escargot, the Drosophila Gene Encoding a Snail-Type Transcription Factor, Promotes Longevity.

In recent years, several genes involved in complex neuron specification networks have been shown to control life span. However, information on these genes is scattered, and studies to discover new neuronal genes and gene cascades contributing to life span control are needed, especially because of the recognized role of the nervous system in governing homeostasis, aging, and longevity. Previously, we demonstrated that several genes that encode RNA polymerase II transcription factors and that are involved in the development of the nervous system affect life span in Drosophila melanogaster. Among other genes, escargot (esg) was demonstrated to be causally associated with an increase in the life span of male flies. Here, we present new data on the role of esg in life span control. We show that esg affects the life spans of both mated and unmated males and females to varying degrees. By analyzing the survival and locomotion of the esg mutants, we demonstrate that esg is involved in the control of aging. We show that increased longevity is caused by decreased esg transcription. In particular, we demonstrate that esg knockdown in the nervous system increased life span, directly establishing the involvement of the neuronal esg function in life span control. Our data invite attention to the mechanisms regulating the esg transcription rate, which is changed by insertions of DNA fragments of different sizes downstream of the structural part of the gene, indicating the direction of further research. Our data agree with the previously made suggestion that alterations in gene expression during development might affect adult lifespan, due to epigenetic patterns inherited in cell lineages or predetermined during the development of the structural and functional properties of the nervous system.

Association and Expression of Virulence from Plasmids of the Group B Strain in Pseudomonas syringae pv. eriobotryae.

Pseudomonas syringae pv. eriobotryae causes serious stem canker in loquat (Eriobotrya japonica) trees. This study was conducted to determine whether plasmids are involved with its virulence. The strain NAE89, which belonged to the B group, harbored two plasmids at approximately 6.2 and 50 Mdal that caused stem canker and halo leaf spots on loquat plants. Following digestion with BamHI and ligation into the BamHI cloning site of the broad range host cosmid pLAFR3, four DNA fragments at 3.8, 6.6, 12.3, and 22.8 kb were generated. Although the plasmid-encoded virulence gene psvA was undigested with the BamHI, the halo leaf spot gene may be adjacent to the psvA gene was digested. A pLAFR3 cosmid clone was introduced into the non-pathogenic PE0 and NAE89-1 strains by triparental matings and the pathogenicity was recovered. As a result, the pLAFR3 cosmid clone was introduced into the largest size DNA fragment of 22.8 kb and determined to be the causal agent of canker on the stem of the loquat. This study revealed that the psvA gene, previously found in the 50 Mdal plasmid, was also observed in the 22.8 kb DNA fragment.

Analysis of human blood plasma cell-free DNA fragment size distribution using EvaGreen chemistry based droplet digital PCR assays

BackgroundPlasma cell-free DNA (cfDNA) fragment size distribution provides important information required for diagnostic assay development. We have developed and optimized droplet digital PCR (ddPCR) assays that quantify short and long DNA fragments. These assays were used to analyze plasma cfDNA fragment size distribution in human blood. MethodsAssays were designed to amplify 76,135, 490 and 905 base pair fragments of human β-actin gene. These assays were used for fragment size analysis of plasma cell-free, exosome and apoptotic body DNA obtained from normal and pregnant donors. ResultsThe relative percentages for 76, 135, 490 and 905 bp fragments from non-pregnant plasma and exosome DNA were 100%, 39%, 18%, 5.6% and 100%, 40%, 18%,3.3%, respectively. The relative percentages for pregnant plasma and exosome DNA were 100%, 34%, 14%, 23%, and 100%, 30%, 12%, 18%, respectively. The relative percentages for non-pregnant plasma pellet (obtained after 2nd centrifugation step) were 100%, 100%, 87% and 83%, respectively. ConclusionNon-pregnant Plasma cell-free and exosome DNA share a unique fragment distribution pattern which is different from pregnant donor plasma and exosome DNA fragment distribution indicating the effect of physiological status on cfDNA fragment size distribution. Fragment distribution pattern for plasma pellet that includes apoptotic bodies and nuclear DNA was greatly different from plasma cell-free and exosome DNA.

Interactions of tetracationic porphyrins with DNA and their effects on DNA cleavage

The interaction of tetracationic porphyrins with DNA was studied using UV–Vis absorption, fluorescence spectroscopy and viscometry, and the particle sizes were determined. Аs cationic porphyrins, two isomer porphyrins, 3,3′,3″,3‴-(5,10,15,20-Porphyrintetrayl)tetrakis(1-methylpyridinium) (TMPyP3) and 4,4′,4″,4‴-(5,10,15,20-Porphyrintetrayl)tetrakis(1-methylpyridinium) (TMPyP4), were studied. They differ in the position of NCH3+ group in phenyl ring of the porphyrins and hence, in degree of freedom of rotation of the phenyl rings about the central macrocycle. It was found that intercalated complexes are formed at DNA/porphyrin molar ratios (R) of 2.2 and 3.9 for TMPyP3 и TMPyP4, respectively. Decreasing R up to 0.4 and 0.8 for TMPyP3 и TMPyP4, respectively, leads mainly to formation of outside complexes due to π-π stacking between the porphyrin chromophores interacting electrostatically with phosphate framework of DNA. Each type of the obtained complexes was characterized using Scatchard approach. It was ascertained that the affinity of TMPyP4 to DNA is stronger than TMPyP3, meanwhile the wedge effect of the latter is higher. The differences between the porphyrin isomers become more evident at irradiation of their complexes with DNA. It was established that irradiation of the intercalated complexes results in DNA fragmentation. In the case of TMPyP4, DNA fragments of different size are formed. The irradiation of the outside DNA/porphyrin complexes leads to cleavage of DNA (TMPyP3 and TMPyP4) and partial destruction of the complex due to photolysis of the porphyrin (TMPyP3).

Multiplexed Reduced Representation Bisulfite Sequencing with Magnetic Bead Fragment Size Selection.

Reduced representation bisulfite sequencing (RRBS) is a technique for assessing genome-wide DNA methylation in an organism whose genome has been fully sequenced. It allows researchers to target gene regions with particular CpG densities, thereby selecting the desired genomic contexts. Here, we describe an approach that uses magnetic beads to accomplish this selection. In addition, the use of indexed, methylated adapters enables up to 12 samples to be pooled, and subjected to multiplexed RRBS in a single-sequencing lane. First, genomic DNA is fragmented via restriction endonuclease digestion that ensures at least two CpG loci per fragment. The fragmented DNA is then end-repaired and A-tailed. Indexed, methylated adapters are ligated to the A-tailed DNA fragments to create a DNA library. A combination of negative and positive selections, using magnetic beads that preferentially bind to larger DNA fragments, ensures that only the desired sizes of adapter-ligated DNA fragments are included in a library. This allows researchers to dictate what types of genomic regions will be sequenced, since fragment size depends on the proximity of restriction sites. The DNA libraries are then quantified, and up to 12 libraries are pooled in order to be sequenced on a single lane of an Illumina HiSeq2500. The pools are next treated with sodium bisulfite, and then PCR amplified. A final bead cleanup removes any residual contaminants prior to sequencing, which is followed by base calling and alignment to a sequenced genome.

Higher DNA insert fragment sizes improve mitogenomic assemblies from metagenomic pyrosequencing datasets: an example using Limenitidinae butterflies (Lepidoptera, Nymphalidae)

A large number of diverse mitogenomic sequences can be obtained more easily and affordably via mitochondrial metagenomics, which generates high-throughput sequences directly from sheared DNA extractions and assembles mitogenomic sequences using a few bioinformatic processing steps. However, following de novo assembly analysis, the optimal DNA fragment insert size is unclear. In this study, four extracted Limenitidinae butterfly DNA samples were sonically fragmented, and two fragment size ranges (200–400 and 400–600 bp) of each sample were tagged with different barcodes, producing pyrosequencing datasets. The results show that the datasets generated from longer DNA insert fragments result in better coverage and more complete mitogenomic sequences, and the phylogenetic analysis shows high support at nodes, revealing that Athyma butterflies do not represent a monophyletic group. Therefore, we recommend using longer insert DNA fragment sizes to generate high-throughput datasets for obtaining complete mitogenomic sequences which can improve phylogenetic studies.

Genetic Diversity among Bread Wheat Cultivars for Drought Tolerance

The study was carried out for genetic diversity evaluation among fifteen bread wheat cultivars for drought tolerance locally cultivated in Iraq-Kurdistan Region using a valuable diagnostic tool Random Amplified Polymorphic DNA (RAPD) technique. The genotypes revealed different levels of polymorphism for the loci detected using fifteen RAPD primers; only ten primers related to drought due to band exhibition. They generate a total of 257 bands, amplified band sizes ranged between (137 – 1875) bp with 95.72% polymorphism. The lowest bands produced by OPF-6 (5 bands), while OPD-8 generates 64 bands as a high score. The genetic distance was calculated using un-weighted pair group of arithmetic means (UPGMA). The cluster analysis was performed and the resulted dendrogram classified the 15 wheat genotypes into two main clusters with Jaccard’s similarity coefficient ranging between (0.29 - 0.85). The first group includes Rizgary, Rabeaa, Azady, Adena, Abu-xreb and Sham-6, while the second group consists of nine wheat genotypes; Aras, Tammuz-2, Floakwa, Abehade, Sabeer beg, Ebaa-95, Panda, Ebba-99 and Ezz. The highest similarity 0.85 was found between Tmmuz-2 and Aras and the lowest 0.29 was recorded between Ebba-99 and Sham-6. Unique DNA fragments of different sizes were detected in a particular cultivar but not in the others. Such fragments could be used as markers for their respective cultivars. RAPD-PCR technique showed considerable potential for estimating genetic diversity among studied wheat cultivars. This study is important for the development of drought tolerant wheat cultivars with wider and diverse genetic background to achieve improved wheat productivity

Acid-free glyoxal as a substitute of formalin for structural and molecular preservation in tissue samples.

Tissue fixation in phosphate buffered formalin (PBF) remains the standard procedure in histopathology, since it results in an optimal structural, antigenic and molecular preservation that justifies the pivotal role presently played by diagnoses on PBF-fixed tissues in precision medicine. However, toxicity of formaldehyde causes an environmental concern and may demand substitution of this reagent. Having observed that the reported drawbacks of commercially available glyoxal substitutes of PBF (Prefer, Glyo-fix, Histo-Fix, Histo-CHOICE, and Safe-Fix II) are likely related to their acidity, we have devised a neutral fixative, obtained by removing acids from the dialdehyde glyoxal with an ion-exchange resin. The resulting glyoxal acid-free (GAF) fixative has been tested in a cohort of 30 specimens including colon (N = 25) and stomach (N = 5) cancers. Our results show that GAF fixation produces a tissue and cellular preservation similar to that produced by PBF. Comparable immuno-histochemical and molecular (DNA and RNA) analytical data were obtained. We observed a significant enrichment of longer DNA fragment size in GAF-fixed compared to PBF-fixed samples. Adoption of GAF as a non-toxic histological fixative of choice would require a process of validation, but the present data suggest that it represents a reliable candidate.

Simple Expression Domains Are Regulated by Discrete CRMs During Drosophila Oogenesis.

Eggshell patterning has been extensively studied in Drosophila melanogaster. However, the cis-regulatory modules (CRMs), which control spatiotemporal expression of these patterns, are vastly unexplored. The FlyLight collection contains >7000 intergenic and intronic DNA fragments that, if containing CRMs, can drive the transcription factor GAL4. We cross-listed the 84 genes known to be expressed during D. melanogaster oogenesis with the ∼1200 listed genes of the FlyLight collection, and found 22 common genes that are represented by 281 FlyLight fly lines. Of these lines, 54 show expression patterns during oogenesis when crossed to an UAS-GFP reporter. Of the 54 lines, 16 recapitulate the full or partial pattern of the associated gene pattern. Interestingly, while the average DNA fragment size is ∼3 kb in length, the vast majority of fragments show one type of spatiotemporal pattern in oogenesis. Mapping the distribution of all 54 lines, we found a significant enrichment of CRMs in the first intron of the associated genes’ model. In addition, we demonstrate the use of different anteriorly active FlyLight lines as tools to disrupt eggshell patterning in a targeted manner. Our screen provides further evidence that complex gene patterns are assembled combinatorially by different CRMs controlling the expression of genes in simple domains.

A multiplex PCR for detection of six viruses in ducks

In this study, six pairs of specific primers that can amplify DNA fragments of different sizes were designed and synthesized according to viral protein gene sequences published in GenBank. Then, a multiplex PCR method was established for rapid detection of duck hepatitis virus 1, duck plague virus, duck Tembusu virus, muscovy duck parvovirus, muscovy duck reovirus, and duck H9N2 avian influenza virus, and achieve simple and rapid detection of viral diseases in ducks. Single PCR was used to confirm primer specificity, and PCR conditions were optimized to construct a multiplex PCR system. Specificity and sensitivity assays were also developed. The multiplex PCR was used to detect duck embryos infected with mixed viruses and those with clinically suspected diseases to verify the feasibility of the multiplex PCR. Results show that the primers can specifically amplify target fragments, without any cross-amplification with other viruses. The multiplex PCR system can amplify six DNA fragments from the pooled viral genomes and specifically detect nucleic acids of the six duck susceptible viruses when the template amount is 102 copies/μl. In addition, the system can be used to detect viral nucleic acids in duck embryos infected with the six common viruses. The detection results for clinical samples are consistent with those detected by single PCR. Therefore, the established multiplex PCR method can perform specific, sensitive, and high-throughput detection of six duck-infecting viruses and can be applied to clinical identification and diagnosis of viral infection in ducks.