Genomic DNA Samples Research Articles

Clinical application and importance of one-step human CYP2C19 genotype detection.

BackgroundTo directly achieve cytochrome P450 2C19 gene (CYP2C19) classification using one-step real-time fluorescent PCR detection and to verify the capabilities of this method with nucleic acid extracted from whole blood samples.MethodsA human CYP2C19 genotyping kit based on one-step real-time fluorescent PCR detection was used to analyze whole blood or genomic DNA samples. This method was compared with pyrosequencing and another quantitative (q)PCR kit for its accuracy, repeatability, detection range analysis, sensitivity, specificity, and anti-interference analysis.ResultsThe one-step real-time PCR method achieved a 100% accuracy rate compared with pyrosequencing and the other qPCR kit. When detecting different concentrations of known genes, concentrations of each sample ranging from 0.2 to 125 ng/µL could be correctly detected. The genotypes of samples treated with anticoagulants, including EDTA and sodium citrate, and chyle blood samples could be correctly detected.ConclusionThe one-step detection method demonstrated high accuracy and a wide detection range. It also had high levels of repeatability, sensitivity, and specificity for the assessment of genomic DNA test samples.

Complete RHD next-generation sequencing: establishment of reference RHD alleles

AbstractThe Rh blood group system (ISBT004) is the second most important blood group after ABO and is the most polymorphic one, with 55 antigens encoded by 2 genes, RHD and RHCE. This research uses next-generation sequencing (NGS) to sequence the complete RHD gene by amplifying the whole gene using overlapping long-range polymerase chain reaction (LR-PCR) amplicons. The aim was to study different RHD alleles present in the population to establish reference RHD allele sequences by using the analysis of intronic single-nucleotide polymorphisms (SNPs) and their correlation to a specific Rh haplotype. Genomic DNA samples (n = 69) from blood donors of different serologically predicted genotypes including R1R1 (DCe/DCe), R2R2 (DcE/DcE), R1R2 (DCe/DcE), R2RZ (DcE/DCE), R1r (DCe/dce), R2r (DcE/dce), and R0r (Dce/dce) were sequenced and data were then mapped to the human genome reference sequence hg38. We focused on the analysis of hemizygous samples, as these by definition will only have a single copy of RHD. For the 69 samples sequenced, different exonic SNPs were detected that correlate with known variants. Multiple intronic SNPs were found in all samples: 21 intronic SNPs were present in all samples indicating their specificity to the RHD*DAU0 (RHD*10.00) haplotype which the hg38 reference sequence encodes. Twenty-three intronic SNPs were found to be R2 haplotype specific, and 15 were linked to R1, R0, and RZ haplotypes. In conclusion, intronic SNPs may represent a novel diagnostic approach to investigate known and novel variants of the RHD and RHCE genes, while being a useful approach to establish reference RHD allele sequences.

Determination of mir-155 and mir-146a expression rates and its association with expression level of TNF-α and CTLA4 genes in patients with Behcet’s disease

IntroductionMicroRNAs (miRNAs) are involved in the pathogenesis of inflammatory diseases. MiR-146 and miR-155 emerged as key regulators of the immune response. This study designed to analyze the miR-146a and miR-155 expression in patients with Behcet’s disease (BD) and investigated their association with the expression of tumor necrosis factor-alpha (TNF-α) and cytotoxic T lymphocyte associated antigen-4 (CTLA-4) genes. MethodologyIn a case-control study, 47 Iranian Azeri BD patients and 61 age- and sex matched healthy controls recruited to the study. Peripheral blood mononuclear cells (PBMCs) were isolated from EDTA blood tubes by Ficoll density-gradient centrifugation. Genomic DNA samples of BD and healthy controls were extracted using the rapid genomic DNA extraction method from the peripheral blood collected in tubes containing EDTA. Total RNA was extracted from the PBMCs according to the TRIzol protocol. MiR-146a, miR-155, TNF-α and CTLA-4 expression were studied using real-time PCR. ResultsMiR-155 and TNF-α expression was significantly increased, whilst CTLA-4 expression was significantly decreased in the PBMCs of BD patients. There was no significant difference in the miR-146a expression rate between BD patients and controls. A positive correlation between miR-155 and TNF-α expression and negative correlation between miR-155 and CTLA-4 expression were observed. No significant association was observed between the expression of miR-155, miR-146a, TNF-α and CTLA-4 genes with BD activity. MiR-155 and miR-146a expression rate were significantly higher in patients with uveitis and phlebitis, respectively. Discussion and conclusionThe expression of miR-155 increased in BD and associated with upregulation of TNF-α and downregulation of CTLA-4 genes.

Development and testing of an 18S rRNA phylogenetic microarray for marine sediments

There is increasing interest in finding new, more efficient methods for routine monitoring of anthropogenic effects on benthic biodiversity and ecosystems. A range of molecular methods have been developed for assessing biodiversity the last decades. Particularly interesting are microarrays targeting phylogenetic marker genes, such as the small subunit of ribosomal RNA in eukaryotes (18S rRNA). This method can detect a large number of taxonomic groups in several samples simultaneously within a relatively short time and has the potential for incorporation in automated remote sensing pipelines. In this study we developed and tested a microarray for eukaryotes in marine sediments. The probes were designed to target 18S rRNA OTUs obtained through metabarcoding of marine sediments. The resulting microarray was tested using both a spiked sample consisting of 50 plasmid-clones and further, samples of genomic DNA extracted from marine sediments. We developed a filtration pipeline to eliminate noise and reduce the number of false positives, making it possible to detect and quantify most of the OTUs with ≥ 0.1% abundance in the spiked sample. Our data indicated that the microarray was specific at higher taxonomic levels. However, the specificity decreased with increasing sequence similarity suggesting cross-hybridization between closely related OTUs. When using genomic DNA isolated from marine sediment there was a positive correlation between hybridization intensity signals and abundance of sequencing reads, suggesting a quantitative behavior of the microarray. Overall, the data suggest a potential for microarrays as a tool for high throughput sediment monitoring.

Multiplex testing for Factor II and Factor V mutations in thrombophilia: technical verification and clinical validation of the cobas\xae Factor II and Factor V test

Laboratory testing for thrombophilia is complicated but essential for diagnosis. In 2017, the cobas® Factor II and Factor V Test (cobas F2F5 test) was launched for use with the cobas z 480 analyzer. This qualitative polymerase chain reaction test enables multiplex Factor II and Factor V testing with flexible reporting and workflow efficiency. Here, we report the results from studies investigating the performance of the cobas F2F5 test. Technical performance verification, clinical validation, external laboratory performance, and workflow comparison studies were performed. Fresh and frozen whole-blood and genomic DNA (gDNA) samples were tested, and several manual and automated DNA isolation methods were used. Bidirectional Sanger sequencing was used to verify genotypes identified by the cobas F2F5 test. One hundred percent agreement between the cobas F2F5 test and Sanger sequencing was observed for all genotypes. An external laboratory using remnant clinical samples also yielded 100% agreement between cobas F2F5 test results and their routine testing method. The cobas F2F5 test reduced the total sample processing time compared with the LightCycler® 1.2 platform (98.6 vs 420.2 min; 96 samples). Hemoglobin, extraction buffer, and ethanol contamination of the gDNA sample can lead to invalid results. The cobas F2F5 test has a high degree of accuracy for identification of Factor II and Factor V genotypes. This multiplex testing with short sample processing time can reduce handling errors and increase efficiency. Both manual and automated DNA isolation methods can be used with the cobas F2F5 test.

Biochemical defence responses of resistant and susceptible rice genotypes against blast pathogen Magnaporthe oryzae

Rice blast is the leading fungal disease which is caused by Magnaporthe oryzae that contributes for the significant decline in the rice yield throughout the globe. There is a need for the understanding of biochemical changes in rice plant during blast infection for the development of novel disease control strategies. In the present study, we isolated M. oryzae from the local paddy fields and the fungal isolates (VCF and PON) were identified by ITS-PCR using genomic DNA samples. Further, we inoculated resistant (BR2655 and TUNGA) and susceptible (INTAN and HR12) rice cultivars with PON and VCF isolates. PON isolate showed relatively high virulence compared to VCF and standard MTCC fungal strains. Therefore, we evaluated the effect of PON on the total protein content and plant defence-related key enzymes (peroxidase, polyphenol oxidase, phenylalanine ammonia lyase, β-glucosidase, chitinase and lipoxygenase) activities between 24- and 120-hour post-inoculation (hpi). The results demonstrated the decrease in total protein content in all the inoculated cultivars. In addition, we observed the variation in the activity of peroxidase, polyphenol oxidase, β-glucosidase, chitinase and lipoxygenase at different time points in all the tested rice plants compared to respective controls. However, no significant difference was observed in the phenylalanine ammonia lyase activity relative to its control. Taken together, this study emphasizes on the variation in the activities of plant defence enzymes in different plant cultivars against the tested fungal pathogen and also implementation of defence enzymes as biochemical markers for resistant breeding.

Bisulfite-Free, Nanoscale Analysis of 5-Hydroxymethylcytosine at Single Base Resolution.

High-resolution detection of genome-wide 5-hydroxymethylcytosine (5hmC) sites of small-scale samples remains challenging. Here, we present hmC-CATCH, a bisulfite-free, base-resolution method for the genome-wide detection of 5hmC. hmC-CATCH is based on selective 5hmC oxidation, chemical labeling and subsequent C-to-T transition during PCR. Requiring only nanoscale input genomic DNA samples, hmC-CATCH enabled us to detect genome-wide hydroxymethylome of human embryonic stem cells in a cost-effective manner. Further application of hmC-CATCH to cell-free DNA (cfDNA) of healthy donors and cancer patients revealed base-resolution hydroxymethylome in the human cfDNA for the first time. We anticipate that our chemical biology approach will find broad applications in hydroxymethylome analysis of limited biological and clinical samples.

Novel rare variations in IRF6 in subjects with non-syndromic cleft lip and palate and dental agenesis.

Subjects with cleft lip and palate (CLP) present high prevalence of dental agenesis. Among candidate genes for these phenotypes is IRF6. However, genetic studies do not analyze dental agenesis as a phenotype associated with cleft. Therefore, we investigated the frequency of rare and novel variations in IRF6 in subjects with non-syndromic unilateral cleft lip and palate (NSUCLP), with and without dental agenesis. Genomic DNA samples of 100 subjects with NSUCLP with and without dental agenesis and 50 controls were sequenced. IRF6 mutational screening was conducted by direct sequencing. Ten new and rare missense variations were identified, two in the group cleft with agenesis and eight in the group cleft without agenesis, and none were found in control group. In silico analysis revealed four variations as potentially deleterious, being two in the group with cleft and agenesis and two in the group with cleft without agenesis. The study identified novel IFR6 variations in subjects with NSUCLP with or without associated dental agenesis. The hypothesis of a higher frequency of deleterious variations in the subjects with cleft associated with dental agenesis, when compared to the group of cleft without agenesis and control without cleft, was not supported.

Association of long interspersed nucleotide element-1 and interferon regulatory factor 6 methylation changes with nonsyndromic cleft lip with or without cleft palate.

To examine the possible associations between methylation changes in the promoter regions of long interspersed nucleotide element-1 (LINE-1) and interferon regulatory factor 6 gene (IRF6) and nonsyndromic cleft lip with or without cleft palate (NSCL/P). A case-control investigation was performed to compare 37 infants affected by NSCL/Ps with 60 babies without cleft malformations. Their genomic DNA samples were obtained, and the LINE-1 and IRF6 methylation levels were measured by using Sequenom MassArray. Unconditional logistic regression was adopted to estimate the odds ratio. Infants with NSCL/Ps had a higher methylation level at LINE-1 and IRF6 promoter regions than controls. High levels of LINE-1 (≥64.07%) and IRF6 (≥6.46%) methylation were associated with an increased risk of NSCL/P (LINE-1, OR=2.63, 95% CI: 1.07-6.57; IRF6, OR=4.73, 95% CI: 2.10-13.07), and the associations remained to be significant after adjusting for potential confounders. Similar associations were also found for cleft lip only, cleft lip, and palate. Our study suggested that aberrant methylation of LINE-1 and IRF6 might contribute to the development of NSCL/Ps. Further studies are needed to replicate the findings.

Immobilization of DNA with nitrogen mustard–biotin conjugate for global epigenetic analysis

We report the quantitative analysis of 5-methylcytosine, a representative epigenetic modification in genomic DNA, with an enzyme-linked immunosorbent assay (ELISA). We synthesized a novel hetero-bifunctional linker molecule consisting of nitrogen mustard and biotin to capture DNA on the surface of biosensing devices. The molecule can successfully immobilize genomic DNA on a streptavidin coated 96-well microplate, which was then employed for immunochemical epigenetic assessment. We achieved the sensitive and quantitative detection of 5-mC in genomic DNA samples. The CpG methylation ratios obtained from our system for mouse brain and mouse small intestine genomes were 79% and 82%, respectively. These numbers are in good agreement with the previously reported methylation ratio of 75–85%, which was identified by whole genome bisulfite sequencing. Accordingly, the present technology using our novel bifunctional linker molecule provides a fast, easy, and inexpensive method for epigenetic assessment, without the need for any conventional bisulfite treatment, polymerase chain reaction (PCR), or sequencing.

MS 마커를 활용한 지역별 오계 유전자원의 다양성 및 유연관계 분석

The aim of this study was to evaluate the genetic diversity and relationships of Ogye populations in Korea. A total of 243 genomic DNA samples from 6 Ogye population (Yeonsan Ogye; YSO, Animal Genetic Resources Research Center Ogye; ARO, Chungbuk Ogye; CBO, Chungnam Ogye; CNO, Gyeongbuk Ogye; GBO, Seoul National University Ogye; SUO) and 3 introduced chicken breeds (Rhode Island Red; RIR, White Leghorn; LG, Cornish; CN) were used. Sizes of 25 microsatellite markers were decided using GeneMapper Software(v 5.0) after analyzing ABI 3130XL. A total of 153 alleles were observed and the range was 2 to 10 per each locus. The mean of expected and observed heterozygosity and PIC (Polymorphism Information Content) value was 0.53, 0.50, 0.46 respectively. The lowest genetic distance (0.073) was observed between YSO and SUO, and the highest distance (0.937) between the RIR and CBO. The results of clustering analysis suggested 3 clusters (<italic>ΔK</italic>=7.96). Excluding GBO population, 5 Ogye populations (YSO, ARO, CBO, CNO, SUO) were grouped in same cluster with high genetic uniformity (0.990, 0.979, 0.989, 0.994, 0.985 respectively). But GBO population was grouped in cluster 1 with low genetic uniformity (0.340). The results of this study can be use to basic data for the genetic evaluation and management of Ogye populations in Korea.

P060 Characterization of 108 genomic DNA reference materials for 11 HLA LOCI: A GeT-RM collaborative project

Improve availability of publicly available, renewable, and well-characterized genomic DNA reference materials to support molecular HLA typing assay development, validation and verification, quality control, and proficiency testing. The Centers for Disease Control and Prevention’s (CDC) Genetic Testing Reference Materials Coordination Program (GeT-RM) together with three clinical laboratories and the Coriell Cell Repositories characterized genomic DNA from 108 cell lines for HLA-A, B, C, DRB1, DRB3, DRB4, DRB5, DQA1, DQB1, DPA1, and DPB1. DNA was isolated by the Coriell Cell Repositories and distributed to the 3 laboratories. Methodologies used were: PCR-SSO-LABType® SSO (One Lambda, Canoga Park, CA), SSP – Olerup SSP® (Stockholm, Sweden), SBT-AlleleSEQR® HLA-SBT (Abbott Molecular, Des Plaines, IL) and NGS - HoloType HLA™ kits (Omixon Inc., Budapest, Hungary). Each of the 11 genes was tested by 2 or 3 methods. Consensus genotype was the NGS result, which provided the highest level of resolution, and was consistent with the results obtained by the parallel testing using other methods. HLA typing results using SBT, SSP, SSO and NGS were 99.95 % concordant. Of the 1905 alleles typed, only one discrepancy was observed. The discrepant result was found to be due to an error in SBT/SSO typing. Five unique novel alleles with differences in exonic regions were identified and three were submitted to the WHO Nomenclature Committee. Ambiguities persisted in 50/1905 allele calls, most due to alternative cis/trans combinations of exons 2 and 3 of DPB1 (n = 21 pairs: 42 alleles) and 8 caused by polymorphisms in a non-covered region (exon1) of the DRB1and DPB1 genes. Thirty HLA-A, 54-B, 30-C, 36-DRB1, 4-DRB3, 4-DRB4, 3-DRB5, 19-DQA1, 17-DQB1, 10-DPA1 and 29-DPB1 alleles are represented in the panel. These alleles cover a high percentage of HLA specificities present in five ancestry groups in the USA population. These genomic DNA samples are publicly available from the National Institutes of General Medical Science (NIGMS) Repository at the Coriell Cell Repositories. M. Bettinotti: 3. Speaker’s Bureau; Company/Organization; One Lambda Thermo Fisher. D. Ferriola: 7. Other (Identify); Company/Organization; Licensing proceeds of the HLA-NGS protocol by CHOP to Omixon. J.L. Duke: 7. Other (Identify); Company/Organization; receiving proceeds from the licensing of the HLA-NGS protocol by CHOP to Omixon. D. Monos: 4. Scientific/Medical Advisor; Company/Organization; Omixon. 7. Other (Identify); Company/Organization; receiving proceeds from the licensing of the HLA-NGS protocol by CHOP to Omixon.

P100 Complete single mica gene sequencing-based typing using next generation sequecers

Aim The MICA gene located in the HLA gene cluster region close to HLA-B is highly polymorphic. It is associated with solid organ transplant rejection and bone marrow transplant GVHD. To test a novel Next Generation Sequencing (NGS) based MICA high resolution typing method, we used both Sanger Based Typing (SBT) and NGS in a parallel double blind study. Methods Genomic DNA samples from 54 healthy Han people in southern China were genotyped by both SBT and NGS methods. Well established MICA SBT was performed using an amplicon covering the MICA gene from exon 2 to exon 5 (2.2kb) and MICA-Seq specific software. NGS was performed with a PCR product that includes the entire length of the MICA gene from mid 5’UTR to the end of the 3’UTR (12.6 Kb). Library preparation was performed with the standard library preparation method and reagents from Omixon’s V2 Holotype HLA kits and sequenced on an Illumina MiSeq using paired-end 2x250 bp V2 chemistry. MICA typing was assigned using Omixon Twin software and GenDx’s NGSEngine. Results 2.04 million reads with an average quality score ⩾Q30 were generated. 93.3% of reads produced were high quality and used for MICA consensus assembly with an average coverage depth of 300 across the exonic regions. 26 MICA genotypes consisting of 17 unique alleles among 54 samples were identified, and were 100% concordant between NGS and SBT. MICA NGS was able to resolve all ambiguities obtained by MICA SBT. Alleles initially typed as ambiguous MICA*008:01/04 by SBT were determined to be MICA*008:01:01, *008:01:02 and *008:04. MICA*008:04 was the most frequent at 19.4% (21/108 alleles) by NGS. In addition, 4 samples typed ambiguously as MICA*002:01/MICA*008 or MICA*023/MICA*035, and 3 samples typed as MICA*002:01/MICA*010 or MICA*022/MICA*025 by SBT were resolved by NGS. Furthermore a new *008:01 allele was found with 5 differences in intron 1 and 1 difference in intron 5. This new allele was represented three times in the population of 108 allele calls. Additionally we were able the complete the sequencing of exon 1 for *009:01 and *033 and exon 6 for *007:01 and *033 alleles. Conclusions A NGS based MICA typing method has been established and is capable of resolving ambiguities observed with SBT, identifies new alleles and allows the completion of IMGT reported incomplete allele sequences.

P077Imputing allelic variants for five human leukocyte antigen genes using a high-throughput genotyping array

Aim To assess the feasibility of imputing human leukocyte antigen (HLA) alleles in African cohorts based on high-throughput genotyping of single-nucleotide polymorphisms (SNPs) in the human major histocompatibility complex (MHC). Methods For two African cohorts representing eastern and southern Africa, genomic DNA samples extracted from peripheral blood were first used for genotyping HLA-A , HLA-B , HLA-C , HLA-DRB1 and HLA-DQB1 alleles, all with 4-digit resolution through a combination of PCR-based genotyping techniques, including direct sequencing of locus-specific amplicons. SNP genotypes were further defined by a widely-used commercial array (the Illumina ImmunoChip). Paired HLA and SNP data from the first 400 individuals in each cohort served as a reference (the training set) for validation in the rest of samples. All imputation procedures were done using the HIBAG algorithm. Results A total of 7539 SNPs spanning the extended human MHC passed various quality control procedures and then merged with fully-resolved HLA alleles in 490 Rwandans and 1100 Zambians. Overall, SNP-based imputation of 2-digit allele groups at the five target loci showed success rates between 93.9% and 98.3%, while success rates in imputing 4-digit alleles ranged from 85.4% to 97.2%. Two major HLA-DRB1 allele groups (DRB1*04 and DRB1*10) in Zambians were the only ones with suboptimal imputation probabilities ( Conclusions In the era of high-throughput genomics, SNP-based imputation of HLA variants may offer a quick and affordable alternative for initial assessment of 2-digit allele groups. However, reliable imputation of 4-digit allele remains difficult even for some common alleles of broad interest.

Clonal immunoglobulin λ light-chain gene rearrangements detected by next generation sequencing in POEMS syndrome.

Polyneuropathy, organomegaly, endocrinopathy, M-protein, and skin changes (POEMS) syndrome is a rare plasma cell dyscrasia characterized by polyneuropathy, organomegaly, endocrinopathy, extravascular fluid overload, M protein, and a myriad of skin changes. The pathogenesis is poorly understood, but monoclonal plasma cells are λ-restricted and these immunoglobulin λ light chain variable (IGLV) region genes are derived from only two germlines, either IGLV1-44 or 1-40. Here we analyzed the clonal IGLV gene rearrangements of genomic DNA samples of bone marrow mononuclear cells using next-generation sequencing (NGS) to understand the clonal composition of IGLV genes in patients with POEMS syndrome (n = 30). The dominant IGLV gene rearrangement of POEMS syndrome-specific germline sequences were significantly increased in 11 POEMS patients (36.7%; IGLV1-44: n = 9, IGLV1-40: n = 2). In some cases, IGLV gene rearrangement clone was not detected as significant increase but was detected using cDNA samples by heteroduplex (HD) analysis and Sanger sequencing, suggesting that the quite small number of monoclonal plasma cells may produce large quantity of mRNA of monoclonal proteins. However, significant increase of dominant clone sizes was not directly linked to the initial disease status. On the other hand, in cases with significantly increased dominant clones, they decreased and increased accompanying with disease remission and relapse. These data demonstrate that monoclonal plasma cells are related to the pathogenesis of POEMS syndrome.

Naked eye detection of the Mycobacterium tuberculosis complex by recombinase polymerase amplification-SYBR green I assays.

Rapid diagnosis of Mycobacterium tuberculosis (Mtb) is key to controlling the spread of tuberculosis, which is a global health concern. In this study, isothermal recombinase polymerase amplification (RPA) was developed to detect specific targets of Mtb, IS6110 and IS1081. Additionally, SYBR Green I was used for endpoint detection of the RPA products by the naked eye. A total of 146 genomic Mtb DNA samples and 24 genomic nontuberculous mycobacteria (NTM) DNA samples were amplified at IS6110 and IS1081 by RPA. After a complete amplification, the RPA amplicons were examined by agarose gel electrophoresis (RPA-AGE) and SYBR Green I (RPA-S) assays. The performance of the RPA assays was evaluated by comparing them to a conventional PCR. The RPA assay demonstrated to have a good capability to differentiate Mtb from NTM with a very short turnaround time at a constant temperature. Compared to conventional PCR, the sensitivities and specificities of RPA-AGE for IS6110 and IS1081 were 100%. The specificity of RPA-S was 100% for both targets; however, its sensitivities for IS6110 and IS1081 were 97.95% and 99.32%, respectively. The limits of detection of IS6110 RPA-AGE and RPA-S were 0.05 and 0.5ng, respectively, while the LODs of IS1081 RPA-AGE and RPA-S were 0.00005 and 0.05ng, respectively. Both RPA assays showed a satisfying diagnostic specificity, with no cross-reaction with other bacteria. A rapid, sensitive, naked eye RPA assay can be integrated into point-of-care diagnosis for Mtb detection, especially in remote areas where laboratory instrument resources are limited.

First Report of Lasiodiplodia gilanensis Causing Twig and Leaf Blight on Machilus thunbergii in Zhejiang Province of China.

Machilus thunbergii Sieb. et Zucc. (Lauraceae) is a broad-leaved evergreen tree and an endangered species in warm-temperate and subtropical areas of China. It also has high ornamental, timber-use, and soil-conservation functions. In recent years, brown to dark-brown lesions were found on leaves or on twigs of M. thunbergii, which caused decay of the cortex. We first found this disease on about 30% of M. thunbergii trees in April 2017 in Zhejiang Tongling Mountain National Forest Park at Wencheng, Wenzhou, Zhejiang province. Symptomatic material from leaves and stems was surface sterilized in 75% ethanol for 10 s and then in 3% NaClO for 3 min, rinsed three times in sterilized distilled water, dried, transferred onto potato dextrose agar (PDA), and incubated at 25°C. Fourteen pure isolates from symptomatic tissues were obtained by single-spore isolations. These isolates formed colonies with abundant villous aerial hyphae. The colonies initially were gray and then turned to black. The strains had black, scattered, or clustered pycnidia and cylindrical conidiophores that occasionally branched. The conidia of the strains were fusiform, thick walled, 31.2 ± 2.5 μm long, and 16.7 ± 1.5 μm wide (length-to-width ratio = 1.9 ± 0.2), and with obtuse apices tapering to a truncate base. Initially, conidia were single celled and transparent, and then they became double celled and the melanin gradually increased. The morphology was similar to the description of Lasiodiplodia gilanensis Abdollahzadeh, Javadi & A.J.L. Phillips (Abdollahzadeh et al. 2010). Genomic DNA samples of the 14 isolates were extracted using the cetyl trimethylammonium bromide method. The β-tubulin (TB) gene was amplified by polymerase chain reaction assay using primers bt2a/bt2b, and a portion of the translation elongation factor 1-alpha (EF1-α) gene was amplified using primers EF-1-986/EF-728. The amplified DNA fragments were then sequenced, and the sequences of all 14 isolates were identical. The sequences of TB (GenBank accession no. MG372749) and EF1-α (GenBank accession no. MG372751) in the representative strain HNDNYB-1 were 100 and 100% identical to the TB sequence of L. gilanensis strain 3K59 (GenBank accession no. KF955888) and the EF1-α sequence of L. gilanensis strain IRAN1501C (GenBank accession no. GU945341), respectively (Abdollahzadeh et al. 2010; Chen et al. 2014). Morphological and molecular results confirmed the isolate as L. gilanensis. Five isolates were selected for pathogenicity tests. Healthy twigs and leaves of M. thunbergii were collected in the field and wounded with a sterilized needle. A 5-mm-diameter mycelial plug of each isolate was excised from the margin of a 4-day-old colony from a PDA plate and placed to the wounded twigs or leaves, and the twigs and leaves were treated similarly with uncolonized plugs as a control. All plants were maintained in an artificial climate chamber at 25°C and 90% relative humidity with a 12-h photoperiod per day. After 5 days, typical lesions developed on inoculated twigs and leaves, whereas control plants remained asymptomatic. The L. gilanensis strains were only reisolated from inoculated plants, and then they were confirmed via morphological observations and genomic DNA testing as identical to the original inoculated strains. L. gilanensis has previously been isolated on California pistachio and pomegranate (Chen et al. 2014; Urbez-Torres et al. 2016), but to the best of our knowledge, this is the first report of this pathogen on M. thunbergii worldwide.

Detection of the Genotoxicity of Gentiana L. Extracts by Using RAPD-PCR and ISSR-PCR Techniques

Abstract: Backround: The RAPD- and ISSR-PCR techniques are offering an insight into the detecting the genotoxicity of Gentiana extracts. Objective: It is aimed with the present study, to detect the genotoxicity of methanol extracts of ten Turkish Gentiana L. taxa on germinated Allium cepa L. root tips. Methods: RAPD- and ISSR-PCR techniques were used for detection of genotoxicity of Gentiana extracts. Results: Four RAPD and three ISSR primers produced the reproducible polymorphic and monomorphic banding patterns among 10 RAPD and 10 ISSR primers for all DNA samples. It is not any serious alteration along with band intensity change, the disappearance of the bands, and appearance of the new bands in the band profiles amplified from the Gentiana extracts-treated genomic DNA sample of A. cepa. The most efficient results were obtained by RAPD-P9 and ISSR-1 primer among the seven productive primers. Discussion: It is not observed any variation in the RAPD- and ISSR-PCR band profiles in time and concentration-dependent manner. Conclusion: It is determined that the three different concentrations of Gentiana extracts did not interact with the A. cepa DNA. Key words: Gentiana L., Genotoxicity, RAPD-PCR, ISSR-PCR.

Cooperative Toehold: A Mechanism To Activate DNA Strand Displacement and Construct Biosensors.

Toehold-mediated DNA strand displacement has proven powerful in the construction of various DNA circuits, DNA machines, and biosensors. So far, many new toehold activation mechanisms have been developed to achieve programmed DNA strand displacement behaviors. However, almost all those toeholds are inflexible via either a covalently attached manner or a complementary hybridization strategy, which limit the versatility of DNA devices. To solve this problem, we developed a new toehold, named "cooperative toehold", to activate DNA strand displacement. On the basis of a base stacking mechanism, the cooperative toehold is comprised of two moieties with completely independent DNA sequences between each other. The cooperative toehold enabled one to continuously tune the rate of DNA strand displacement, as well as more sophisticated strand displacement reactions. The cooperative toehold has also been employed as a universal signal translator for biosensors to qualitatively determine RNA and ATP. Moreover, as a novel specific PCR monitoring system, cooperative toehold-mediated DNA strand displacement can detect the pUC18 plasmid in genomic DNA samples with an aM detection limit.

Genetic variants in the CNTNAP2 gene are associated with gender differences among dyslexic children in China

BackgroundIt is well known that males have a higher prevalence of developmental dyslexia (DD) than females. Although the mechanism underlying this gender difference remains unknown, the contactin-associated protein-like 2 (CNTNAP2) gene, which shows sex-specific patterns in some neurodevelopmental disorders, has attracted extensive attention. This study aimed to explore whether CNTNAP2 shows a sex-specific association with DD in a Chinese population. MethodsUsing genomic DNA samples of 726 students [372 cases (282 male, 90 female), 354 controls (267 male, 87 female)], we genotyped five SNPs of CNTNAP2. Gender-stratified logistic regression models were used to determine the relationships between the CNTNAP2 variants and DD. FindingsAfter adjustment for the false discovery rate (FDR), two SNPs (rs3779031, rs987456) of CNTNAP2 were associated with DD risk in females but not in males. Female participants carrying the rs3779031 G allele had a lower risk of DD than those with the A genotype [GG vs AA: OR (95%CI) = 0.281 (0.097–0.814)]. The rs987456 CC genotype was associated with a decreased risk of DD in females [CC vs AA+CA: OR (95%CI) = 0.222 (0.078–0.628)]. Furthermore, the interaction between CNTNAP2 (rs987456) and environmental factors (scheduled reading time) played a protective role in females [OR (95%CI) = 0.431 (0.188–0.987)]. InterpretationWe performed a genetic association study on CNTNAP2 variants and DD. The sex specificity of CNTNAP2 in DD, along with the gene-environment interaction may help us to understand gender differences in DD.