Antisense Primer Research Articles

Rapid Identification of Seven Common ABO Alleles by Two-Dimensional Polymerase Chain Reaction Technology

Introduction: The molecular biology detection technology of the human ABO blood group system makes up for the limitations in many aspects compared with conventional serological typing technology. This study aimed to establish a new method to identify seven common ABO alleles (ABO*A1.01, ABO*A1.02, ABO*A2.01, ABO*B.01, ABO*O.01.01, ABO*O.01.02, and ABO*O.02.01) by two-dimensional polymerase chain reaction (2D PCR). 2D PCR can identify multiple target genes in a closed test tube by labeling specific primers with tags homologous to the sequence of fluorescently labeled probes, and melting curve analysis is performed after the fluorescent probes are hybridized with tag complementary sequences in PCR-specific products. In this study, 2D PCR and PCR sequence-specific primer (PCR-SSP) were combined to discriminate different alleles in a single reaction, which has the characteristics of high throughput, and compared with other typing techniques; the typing results can be obtained without additional operations. Methods: The ABO*A1.01 allele genetic sequence was used as the reference sequence. The specific sense and antisense primers for seven common ABO alleles were designed on exons 6 and 7 according to the principle of 2D PCR and PCR-SSP. Single nucleotide polymorphism sites for identifying seven alleles were detected in FAM and HEX channels, respectively. Two hundred sixty DNA samples were enrolled for rapid ABO genotyping by 2D PCR, and 95 of them were selected for Sanger sequencing. The Kappa test was used to analyze the agreement of the methodologies. Results: These 7 alleles each had four characteristic melting valleys at different single nucleotide polymorphism loci. A total of 15 genotypes were detected, including ABO*A1.01/A1.02, ABO*A1.01/O.01.01, ABO*A1.01/O.01.02, ABO*A1.02/A1.02, ABO*A1.02/O.01.01, ABO*A1.02/O.01.02, ABO*B.01/B.01, ABO*B.01/O.01.01, ABO*B.01/O.01.02, ABO*O.01.01/O.01.01, ABO*O.01.01/O.01.02, ABO*O.01.02/O.01.02, ABO*A1.01/B.01, ABO*A1.02/B.01, and ABO*B.01/O.01. v (containing a rare ABO*O allele, based on the sequencing results). The Kappa test showed completely consistent results for 2D PCR and Sanger sequencing (Kappa = 1). Conclusion: The 2D PCR technique could be used for molecular typing of the ABO blood group, which was efficient, rapid, accurate, and economical.

PCR-Based Assembly of Gene Sequences by Thermodynamically Balanced Inside-Out (TBIO) Gene Synthesis.

The ability to enzymatically assemble DNA oligonucleotides into longer DNA duplexes in a process known as gene synthesis has wide-ranging applications in the fields of genetic engineering and synthetic biology. Thermodynamically balanced inside-out (TBIO) gene synthesis is one of several PCR-based primer extension genesynthesis protocols that have been developed. In TBIO gene synthesis, overlapping primers with equivalent melting temperatures (Tms) are designed so that the 5' half of the DNA is encoded by sense primers and the 3' half of the DNA molecule is encoded by antisense primers. Primer extension is initiated at the center of the DNA and continues bidirectionally to progressively elongate the DNA molecule. Here we provide the protocols necessary for performing TBIO gene synthesis to generate a DNA molecule of interest.

A novel genotyping method for rapid identification of the Le gene to select patients for diagnosis with CA19-9

BackgroundThe antigenic determinant of CA19-9 is synthesized by the α1,3/4fucosyltransferase encoded by the Le gene in the Lewis blood group system. Accordingly, a diagnosis with CA19-9 is not appropriate forLe-negative patients who possess the Le gene-mutated le alleles homozygously. MethodsA Le gene-specific PCR was undertaken to determine c59T>G by using a set of tag-sense and biotin-labeled anti-sense primers and a peptide nucleic acid-le-clamp which bound to G59 in the le alleles. Following mixing with streptavidin-coatedbluelatex beads, the PCR products were developed on a strip on which the complementary tag oligonucleotide to theLe gene-specific amplicon was immobilized. ResultsWhen the PCR products were developed on the strip, a clear line was rapidly observed in Le-positive but not in Le-negative individuals. In contrast, a significant number of cancer patients with Lewis-negative phenotype were found to possess CA19-9, while they were specifically genotyped asLe/-. No contradictory results were observed in cancer patients (n = 315) with respect to their Lewis genotypes and CA19-9 levels. Conclusionsc59T>G occurred commonly in the le alleles could be specifically and rapidly identified by the present method. This method appeared to be relevant forselecting cancer patientsto bediagnosed with CA19-9.

Study of angiotensin-converting enzyme insertion/deletion polymorphism in Egyptians with Chronic Kidney Diseases: A case-control study

Kidney disease is clinically characterized by increasing rates of urinary albumin excretion, starting from normo-albuminuria, which progresses to microalbuminuria, macro albuminuria nephropathy, and eventually to end stage renal disease. This study aimed to estimate the frequencies of different polymorphisms of ACE gene in chronic kidney disease, and Healthy control. A case-control study design was carried out on 207 patients they were classified into 104 nephrotic syndrome and 103 chronic kidney injury patients in addition to 102 healthy subjects. The I/D polymorphism of the ACE gene was determined sequences of the sense and antisense primers PCR was performed in a final volume of 50 μl. Results: Genotype frequencies 2nd PCR (II, ID and DD-genotypes) in control group were found to be 12, 74 and 58; respectively while genotype frequencies 2nd PCR (II, ID and DD-genotypes) in CKD group were 0, 45 and 58; respectively. In Chronic Kidney Disease (CKD) patients, there were significant increase in [Creatinine (mg/dL), Cholesterol (mg/dL), T.G (mg/dL), LDL (mg/dL), Uric Acid (mg/dL)], 7.70 ± 2.98, 236.45 ± 42.19, 256.41 ± 43.11, 149.78 ± 12.78, 6.68 ± 0.82; respectively compared to control group (p<0.01). But there was a significant decrease in albumin 3.02 ± 0.419, compared to control group (p<0.01). From this study we conclude that there is a correlation between polymorphism of angiotensin converting enzyme gene and chronic kidney disease, especially DD genotype, which increases the chances of development to the stage of renal failure.

A novel regulatory role of trappc9 and L-plastin in osteoarthritis

A novel regulatory role of trappc9 and L-plastin in osteoarthritis

Targeted Locus Amplification (TLA): A Novel Next Generation Sequencing (NGS) Technology to Detect New Molecular Markers and Monitoring Minimal Residual Disease (MRD) in Mantle Cell and Follicular Lymphoma

Background: Minimal residual disease (MRD) monitoring by PCR methods is a strong and standardized predictor of clinical outcome in mantle cell (MCL) and follicular (FL) lymphoma [Pott C., Blood 2010; Ladetto M, Blood 2013]. However, some technical limitations hamper its feasibility in daily clinical routine. First of all, current techniques allow the identification of a molecular marker only in 75-80% of MCL and 55-60% of FL patients. All the other “no marker” cases cannot be studied for MRD purposes. Next generation sequencing (NGS) tools might overcome these limitations. Among them, the recently developed targeted locus amplification (TLA) technology [de Vree P.J., Nat Biotechnol 2014] that selectively amplifies and sequences entire genes on the basis of the crosslinking of physically proximal DNA loci, was able to identify novel candidate oncogenes and gene fusions in acute lymphoblastic leukemia. [Kuiper R.P.; Vroegindeweij EM, 57th ASH Communication, Blood 2015].The aim of this study was to test the TLA approach in MCL and FL diagnostic samples, with at least one translocation confirmed by FISH and a failure in molecular marker identification, with the aim to increase the rate of success in marker screening for MRD purposes. Moreover, the performances of the newly TLA molecular markers were compared to the standardized quantitative PCR (qPCR) approach for MRD monitoring.Methods: Genomic DNA was extracted from bone marrow (BM) and peripheral blood (PB) samples of MCL and FL patients, enrolled in prospective clinical trials of the Fondazione Italiana Linfomi (FIL). All patients provided written informed consent for the research use of the biological samples. Libraries for TLA (Cergentis, Utrecht) were prepared using only one couple of antisense primers targeting the immunoglobulin heavy chain (IGH) enhancer locus, as previously described [Hottentot QP, Methods Mol Biol. 2017], and sequenced on MiSeq platform (Illumina, San Diego). MRD monitoring was carried out by qPCR designing allele specific oligonucleotides (ASO) and consensus probes based on the new breakpoint sequences obtained from TLA NGS. Finally, the efficiency of TLA novel markers to monitor MRD was compared to MRD data obtained from clonal IGH rearrangements detected and tracked by standardized ASO qPCR. [Van der Velden VHJ, Leukemia 2007].Results: TLA was firstly tested on 21 t(11;14)-positive, BCL-1/IGH major translocation cluster (MTC) negative MCL baseline samples (8 BM and 13 PB): in 86% of cases (18/21) a novel BCL-1/IGH breakpoint was identified by NGS. In these cases, TLA described new rearrangements between “no-MTC” BCL1 loci and JH1, JH4, JH5 or JH6 regions on chromosome 14. Therefore, additional 8 FL baseline BM samples, resulting t(14;18) FISH positive and BCL-2/IGH MBR/mcr/“minor rearrangements”-negative by PCR, were tested obtaining a 62% (5 out of 8 samples) success rate. Also in these cases, new and not yet described BCL2/IGH rearrangements were found. Additionally, to assess the specificity of the technology, the MBR-positive control sample (DOHH2 cell line) was tested and correctly sequenced, as well. Interestingly, among the new TLA positive FL cases, in one sample, the NGS technology highlighted the breakpoint on the DH region of chromosome 14, describing an unusual t(14;18) translocation. 5/29 cases (17%) in which TLA was not able to identify a new marker were characterized by a low tumor infiltration, mainly <5% by flow cytometry (median 4.4%, range 0.03-8%). Secondly, a validation of the NGS sequences was performed by ASO primers designed on the new TLA breakpoints and MRD was monitored by ASO qPCR. Overall, in 14 MCL cases where also a classic IGH-based marker was available, the MRD results were superimposable between the two markers (Fig1A), with good and overlapping performances in terms of overall correlation (r2=0,86; Fig 1B). In FL samples, ASO qPCR confirmed the high tumor burden of the analyzed tissues (Fig 1C). Finally, an extension of the analyzed samples series and MRD validation is currently ongoing among additional FL patients.Conclusion: The TLA NGS technology allowed to identify a translocation-based novel molecular marker in FISH positive t(11;14) and t(14;18) MCL and FL baseline samples, where the classical Sanger sequencing failed a marker identification. These new breakpoints can reliably be used for the design of ASO qPCR primers for MRD detection in previously not traceable patients [Display omitted] DisclosuresKlous:Cergentis BV: Employment. Yilmaz:Cergentis BV: Employment. Vitolo:Gilead: Honoraria; Mundipharma: Honoraria; Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Honoraria. Luminari:PFIZER: Speakers Bureau; GILEAD: Speakers Bureau; TEVA: Membership on an entity's Board of Directors or advisory committees; CELGENE: Honoraria, Membership on an entity's Board of Directors or advisory committees; ROCHE: Membership on an entity's Board of Directors or advisory committees; TAKEDA: Membership on an entity's Board of Directors or advisory committees. Federico:takeda: Honoraria, Research Funding. Splinter:Cergentis BV: Employment.

GH Receptor Exon 3 Genotype and Echocardiographic Abnormalities in Patients With Active Acromegaly

Background: The GH receptor (GHR) exon 3 polymorphism occurs at a genomic level. Approximately 50-60% of the population is homozygous for the exon-3 containing genotype (+3/+3), 30-40% are heterozygous (+3/-3) and 10-20% are homozygous for the exon-3 lacking genotype (-3/-3). Some studies suggest that children homo- and heterozygous for the GHR exon 3 lacking genotype (-more efficient 3/-3 and +3/-3, respectively) respond better to treatment with exogenous rhGH and there is also in vitro evidence showing a more efficient signal transduction through this exon 3 deleted isoform. Some studies have found that patients with acromegaly harboring the exon 3-deleted genotype may have a higher prevalence of diabetes and hypertension. Hypothesis and Objective: Patients with active acromegaly harboring the exon 3-lacking GHR genotype may have more echocardiographic abnormalities than those who are homozygous for the exon 3 containing genotype. Patients and Methods: This is a cross-sectional study of patients with active acromegaly, defined by an IGF-1 level > 1.3 times the upper limit of normal (x ULN), who underwent transthoracic echocardiography. Exon-3 GHR genotype was determined by PCR using previously described sense and antisense primers. Results: The cohort consisted of 28 patients, 54% female, with a mean age of 51 ± 12 years. Mean disease duration at the time of echocardiographic examination was 4.48 ± 4.7 years; median basal GH and IGF-1 were 12 ± 26 ng/mL and 2.4 ± 1.04 x ULN. The prevalence of hypertension and diabetes were 43% and 36%, respectively. Fifty three percent of the patients were homozygous for the exon 3-containaing genotype (+3/+3), 18% were homozygous for the exon 3-lacking genotype (-3/-3) and 29% were heterozygous (+3/-3). Clinical and biochemical features did not differ between patients with the different GHR genotypes, except for hypertension that was more prevalent in the +3/+3 genotype group (60% vs 23%, p= 0.04). The frequency of the different echocardiographic parameters was similar among groups (left ventricular hypertrophy 33% vs 15%, p= 0.27; diastolic dysfunction 47% vs 31%, p= 0.39; subclinical systolic dysfunction 42% vs 54%, p= 0.54; left ventricular ejection fraction 59±10% vs 60±16%, p= 0.83); aortic valve abnormalities 19% vs 15%, p=0.63; mitral valve abnormalities 46% vs 15%, p=0.07). Conclusions: Echocardiographic abnormalities in patients with active acromegaly do not differ among patients with the different GHR exon 3 genotypes. The clinical spectrum of acromegaly varies considerably. Although such variability is usually related to the severity of the hypersomatotropinemia, in many patients this is not the case.

Gene Pyramiding for Brown Planthopper Resistance-related Traits, Early Maturity and Aroma of Rice Assisted by Molecular and Phenotypic Markers

The brown planthopper (BPH)-resistant rice, early maturity, and aromatic are expected by both farmers and consumers. The traits have been combined through gene pyramiding and some promising rice genotypes obtained. However the genetic control of BPH resistance and maturity is quantitatively inherited, it is therefore both molecular and phenotypic assessments would be tremendously helpful in selecting promising genotypes. The study aimed to obtain genotypes with such valuable traits. Rice lines were analyzed using molecular markers i.e., RM586, RM589, RM8213 (BPH resistant gene markers); RM7610 and RM19414 (early maturity markers), and IFAP (Internal Fragrant Antisense Primer) for detecting aromatic, and INSP (Internal Non-fragrant Sense Primer) for non-aromatic rice. Phenotypic assessment was performed for brown planthopper resistant-related traits, such as chlorophyll content, stomatal conductance, and trichome density. Other evaluations were heading date and aroma (using 1.7% KOH solution). Results showed that molecular markers for evaluating BPH resistance genes (Bph3, Bph4, Qbph4, and Bph17), aroma (fgr gene), and heading date (Hd2 and Hd3 genes) could differentiate genotypes, and they serve as perfect markers, except for heading date markers. Seven genotypes i.e., #2, #3, #4, #5, #6, #10, and #11 were related to all traits expected based on molecular marker analysis. Meanwhile, genotypes #1, #2, #4, #6, and #11 were similar to their parents based on phenotypic analysis. Pyramiding program based on molecular and phenotypic markers enables us to combine three valuable traits into one rice genotype as presented in this study

Sensory Test and Molecular Marker Based-Selection for Aromatic Rice in the F3 Progenies

Aromatic rice is a special type of rice that highly preferred by people in Asia due to the presence of aroma. Aroma in rice is determined by 2-acetyl-1-pyrroline (2AP) compound which is controlled by a recessive fgr gene. A hybridization between cv. Sintanur (aromatic rice) and PTB33 (non-aromatic, resistant to brown planthopper/BPH) has been done in order to develop aromatic rice lines that resistant to BPH. In the F2 progeny, molecular marker-based selection and bioassay for the brown planthopper resistant lines have been carried out; however selection for the aromatic trait has not been performed yet. The objective of this study was to obtain the F3 progeny’s individual with aromatic trait. Sensory test was conducted by KOH 1.7% solution, meanwhile molecular markers applied were ESP (External Antisense Primer), IFAP (Internal Fragrant Antisense Primer), INSP (Internal Non fragrant Sense Primer) and EAP (External Antisense Primer). Eighty-eight plants from two selected (SP#31 and SP#224) F3 lines progenies derived from cv. Sintanur and PTB33 have been evaluated in this study. Detection by molecular markers found seventy-five genotypes (85.23%) were homozygous recessive (aromatic rice) and one was heterozygous (non-aromatic). Eighty-five (96.59%) genotypes were aromatic as detected by sensory test alone. Seventy-two (81.82%) genotypes were categorized as aromatic rice based on sensory test and molecular markers. Due to inconsistency results from each method alone, it is advised both methods to be applied to ensure the reliability and the accuracy since aroma in rice is affected by genetic composition and environment conditions. Selected genotypes will be continued for breeding program in developing aromatic rice with improved agronomic traits.

Efficient Method for Molecular Characterization of the 5' and 3' Ends of the Dengue Virus Genome.

Dengue is a mosquito-borne disease that is of major importance in public health. Although it has been extensively studied at the molecular level, sequencing of the 5′ and 3′ ends of the untranslated regions (UTR) commonly requires specific approaches for completion and corroboration. The present study aimed to characterize the 5′ and 3′ ends of dengue virus types 1 to 4. The 5′ and 3′ ends of twenty-nine dengue virus isolates from acute infections were amplified through a modified protocol of the rapid amplification cDNA ends approach. For the 5′ end cDNA synthesis, specific anti-sense primers for each serotype were used, followed by polyadenylation of the cDNA using a terminal transferase and subsequent PCR amplification with oligo(dT) and internal specific reverse primer. At the 3′ end of the positive-sense viral RNA, an adenine tail was directly synthetized using an Escherichia coli poly(A) polymerase, allowing subsequent hybridization of the oligo(dT) during cDNA synthesis. The incorporation of the poly(A) tail at the 5′ and 3′ ends of the dengue virus cDNA and RNA, respectively, allowed for successful primer hybridization, PCR amplification and direct sequencing. This approach can be used for completing dengue virus genomes obtained through direct and next-generation sequencing methods.

Comparative Effect of Ulvan and Biosynthesized Silver Nanoparticles on Different Cell Lines Cytotoxicity and Gene Expression

Until now, researchers searching for compounds that contribute for treating the most serious disease of this age, cancer. Researchers hope to contribute to this through this research. Activities of both Ulvan extracted from Ulva lactuca and silver nanoparticles (Ag-NPs) were examined against seven different cancer cell types called (Caco2 cells, A549 cells, Mcf7 cells, Hep2 cells, HepG2 cells, PC3 cells, HELA cells). Green synthesized Ag-NPs; via Ulva Lactuca; was characterized by UV-VIS, SEM and TEM. Cell viability assay was conducted to study cytotoxicity effects of both Ulvan and Ag-NPs treatments. RTPCR; using different sense and antisense primers; was carried out to study the expression of GAPDH, Bcl-2, Bax, P21 and P53 (apoptosis related genes) in treated cell lines. SEM and TEM showed that Ag-NPs shape is spherical with average size of 3.89-55 nm. Also, cell viability study showed that both Ulvan and Ag-NPs have cytotoxicity effects on different cancer cells. However, Ag-NPs are more effective than Ulvan. Gene expression data illustrated that both treatments induced down-regulation of Bcl2 and induced up-regulation of P53 genes. This work showed that both Ulvan and Ag-NPs have activities against the investigated cancer cell types.

Impact of Chemotherapy and Thermotherapy Treatments on the Presence of Potato Viruses PVY, PVX and PLRV in Tissue-Cultured Shoot Tip Meristem

Potato tubers (Solanum tuberosum L.) is considered as one of the most essential vegetable crops worldwide, The most communal viruses attacking potato all over the world are potato leaf roll virus (PLRV), potato virus Y (PVY) and potato virus X (PVX). Our current study aimed to eradicate potato viruses PVY, PVX and PLRV through shoot tip meristem via tissue culture combined with thermotherapy and chemotherapy. Sprouts of potato cultivars ex. Spunta, Selana, Diamond and Cara were used as biological material for investigating their performance using shoot tip meristem. During multiplication stage, the resulted plantlets were subjected to ribavirin (50 mg/L), while the untreated plantlets served as control. In general, the control treatment (0.0 mg/L) provided the best results, while ribavirin (50 mg/L) gave reverse effect on the estimated vegetative parameters (shoot length (cm), number of leaves and number of shoots). Detection of the presence and absence of PVY, PVX and PLRV using DAS-ELISA technique indicated that, thermotherapy and chemotherapy treatments for shoot tip meristem culturing minimized the occurrence percentage of PVX, PVY and PLRV in all tested potato cultivars compared to positive control. The results of RT-PCR revealed that, the amplicons of amplified fragments and primed by PVX-, PVY- and PLRV-specific sense and antisense primers were appeared only in the positive control (lane 1 at ~750, 801 and 548 bp), respectively, while, no amplicons appeared in the rested treatments. In conclusion, shoot tip meristem followed by thermotherapy and chemotherapy treatments enhanced the growth of potato plantlets and suppressed potato viruses.

Optimization of a Quantitative Real-Time RT-PCR Technique for Evaluation of Concentration of Genomic +RNA of Tick-Borne Encephalitis Virus

Background. The specific detection of genomic/template +RNA and replicative –RNA of tick-born encephalitis virus (TBEV) is necessary to study the mechanisms of viral replication in the cells of reservoir and accidental hosts. However, the current approaches of quantitative reverse transcription – polymerase chain reaction (qRT-PCR) are rather focused on the detection of total viral RNA load in the sample. Thus, the significant optimization is necessary both for RT-PCR and for RNA copy number standard preparation.Aims. To develop the set of standard samples of synthetic +RNA of TBEV and to optimize qRT-PCR for quantification of genomic +RNA of the virus.Materials and methods. Fragment of the genomic +RNA of TBEV was synthesized using pTZ57R-T\A plasmid vector with embedded T7 promoter and T7 RNA polymerase. The DNA contamination was removed using RNase-free DNase I treatment followed by additional RNA purification step. Reverse transcription was performed using specific antisense primer 11154R 5`- AGCGGGTGTTTTTCCG-3` and qPCR detection was used according to the modified procedure of M. Schwaiger and P. Cassinotti (2003).Results. As a result of the amplification of standard samples, the concentration of positive polarity ТBEV RNA, carried out in five independent repetitions on different days, the correlation coefficient R2 between the quantification cycle and the concentration of the standard sample was 0.99, and the efficiency of PCR was 100 %. The coefficient of variation in assessing the inter-test accuracy of determination averaged 2.8 %.Conclusions. Optimized qRT-PCR procedure and set of +RNA standards allow to determine the concentration of genomic +RNA of TBEV in routine laboratory practice.

Precise Construction and Tuning of an Aerolysin Single-Biomolecule Interface for Single-Molecule Sensing

Precise Construction and Tuning of an Aerolysin Single-Biomolecule Interface for Single-Molecule Sensing

Identification of Mouse and Human Antibody Repertoires by Next-Generation Sequencing.

The immense adaptability of antigen recognition by antibodies is the basis of the acquired immune system. Despite our understanding of the molecular mechanisms underlying the production of the vast repertoire of antibodies by the acquired immune systems, it has not yet been possible to arrive at a global view of a complete antibody repertoire. In particular, B cell repertoires have been regarded as a black box because of their astronomical number of antibody clones. However, next-generation sequencing technologies are enabling breakthroughs to increase our understanding of the B cell repertoire. In this report, we describe a simple and efficient method to visualize and analyze whole individual mouse and human antibody repertoires. From the immune organs, representatively from spleen in mice and peripheral blood mononuclear cells in humans, total RNA was prepared, reverse transcribed, and amplified using the 5'-RACE method. Using a universal forward primer and antisense primers for the antibody class-specific constant domains, antibody mRNAs were uniformly amplified in proportions reflecting their frequencies in the antibody populations. The amplicons were sequenced by next-generation sequencing (NGS), yielding more than 105 antibody sequences per immunological sample. We describe the protocols for antibody sequence analyses including V(D)J-gene-segment annotation, a bird's-eye view of the antibody repertoire, and our computational methods.

Identification of male-specific SNP markers and development of PCR-based genetic sex identification technique in crucifix crab (Charybdis feriatus) with implication of an XX/XY sex determination system

In this study, we first identified male-specific SNP markers using restriction site-associated DNA sequencing, and further developed a PCR-based sex identification technique for Charybdis feriatus. A total of 296.96 million clean reads were obtained, with 114.95 and 182.01 million from females and males. After assembly and alignment, 10 SNP markers were identified being heterozygous in males but homozygous in females. Five markers were further confirmed to be male-specific in a large number of individuals. Moreover, two male-specific sense primers and a common antisense primer were designed, using which, a PCR-based genetic sex identification method was successfully developed and used to identify the sex of 103 individuals, with a result of 49 females and 54 males. The presence of male-specific SNP markers suggests an XX/XY sex determination system for C. feriatus. These findings should be helpful for better understanding sex determination mechanism, and drafting artificial breeding program in crustaceans.

Developing a One-Step Multiplex PCR Assay for Rapid Detection of Four Stubby-Root Nematode Species, Paratrichodorus allius, P. minor, P. porosus, and Trichodorus obtusus.

Four trichodorid species, Paratrichodorus allius, P. minor, P. porosus, and Trichodorus obtusus, were found in multiple states in the United States. Traditional diagnosis based on morphology and morphometrics is laborious and requires an experienced taxonomist. Additionally, end-point diagnosis using PCR was only available for P. allius. To increase diagnostic efficiency and reduce costs, a one-step multiplex PCR assay was developed to simultaneously identify these four species using one PCR reaction. Available sequences of 18S ribosomal DNA and internal transcribed spacer 1 (ITS1) region of these species were aligned and five primers were designed. The conserved forward primer located in the 18S region, in combination with the species-specific antisense primer in the ITS1 region, amplified a single distinctive PCR fragment for each species (421/425 bp for P. allius, 190 bp for P. minor, 513 bp for P. porosus, and 353 bp for T. obtusus). In silico analysis with 10 other trichodorid species and experimental analysis using samples with these four species, 20 other plant-parasitic and three non-plant-parasitic nematodes demonstrated high specificity with the primers designed. The multiplex PCR amplified desirable fragments using a set of artificially mixed templates containing one, two, three, or four targeted species. The reliability of multiplex PCR results was demonstrated by using nematode populations isolated from infested fields from diverse geographic regions in eight states. The multiplex PCR-based tool developed in this study for the first time provides a simple, rapid, and cost-friendly assay for accurate diagnosis of the four major trichodorid nematodes in the United States.

Genetic code restoration by artificial RNA editing of Ochre stop codon with ADAR1 deaminase

Site directed mutagenesis is a very effective approach to recode genetic information. Proper linking of the catalytic domain of the RNA editing enzyme adenosine deaminase acting on RNA (ADAR) to an antisense guide RNA can convert specific adenosines (As) to inosines (Is), with the latter recognized as guanosines (Gs) during the translation process. Efforts have been made to engineer the deaminase domain of ADAR1 and the MS2 system to target specific A residues to restore G→A mutations. The target consisted of an ochre (TAA) stop codon, generated from the TGG codon encoding amino acid 58 (Trp) of enhanced green fluorescent protein (EGFP). This system had the ability to convert the stop codon (TAA) to a readable codon (TGG), thereby restoring fluorescence in a cellular system, as shown by JuLi fluorescence and LSM confocal microscopy. The specificity of the editing was confirmed by polymerase chain reaction-restriction fragment length polymorphism, as the restored EGFP mRNA could be cleaved into fragments of 160 and 100 base pairs. Direct sequencing analysis with both sense and antisense primers showed that the restoration rate was higher for the 5' than for the 3'A. This system may be very useful for treating genetic diseases that result from G→A point mutations. Successful artificial editing of RNA in vivo can accelerate research in this field, and pioneer genetic code restoration therapy, including stop codon read-through therapy, for various genetic diseases.

Corrigendum

Journal of NeurochemistryVolume 146, Issue 2 p. 198-198 CorrigendumFree Access Corrigendum This article corrects the following: Pre-treatment with the synthetic antioxidant T-butyl bisphenol protects cerebral tissues from experimental ischemia reperfusion injury Thi Thuy Hong Duong, Belal Chami, Aisling C. McMahon, Genevieve M. Fong, Joanne M. Dennis, Saul B. Freedman, Paul K. Witting, Volume 130Issue 6Journal of Neurochemistry pages: 733-747 First Published online: June 6, 2014 First published: 30 July 2018 https://doi.org/10.1111/jnc.14343AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat The following published article from the Journal of Neurochemistry, entitled “Pre-treatment with the synthetic antioxidant T-butyl bisphenol protects cerebral tissues from experimental ischemia reperfusion injury” Thi Thuy Hong Duong, Belal Chami, Aisling C. McMahon, Genevieve M. Fong, Joanne M. Dennis, Saul B. Freedman, Paul K. Witting (J. Neurochem. 2014 Sep;130(6):733-47; https://doi.org/10.1111/jnc.12747; PMID: 24766199) is being corrected. The corresponding author notified the Editorial Office that in Table 1, incorrect PCR primer sequences for β-actin and hemeoxygenase-1 (HO-1) were reported. The cited primers amplify the corresponding human cDNA, not rat cDNA as incorrectly stated in the publication. New validation experiments (listed below) have been performed that demonstrate that the correct primers were used in the original research: an in silico analysis of the primer sequences; review of the original qPCR data, specifically the unique thermal melting points of the PCR products; a repeat of the original experiment using the correct and validated rat PCR primers. The corrected Table 1 is shown below. Corrections are highlighted in green: Table 1. Sequences of primers used for mRNA detection by RT-PCR Gene Sense primer Anti-sense primer Anneal temp °C β-actin 5’-AGCCATGTACGTAGCCATCC-3’ 5’-CTCTCAGCTGTGGTGGTGAA-3’ 62 HIF-1α 5’-TCAAGTCAGCAACGTGGAAG-3’ 5’-TATCGAGGCTGTGTCGACT-3’ 57 HO-1 5’-CACGCATATACCCGCTACCT-3’ 5’-AAGGCGGTCTTAGCCTCTTC-3’ 55 SOD-1 5’-CCACTGCAGGACCTCATTTT-3’ 5’-CACCTTTGCCCAAGTCATCT-3’ 60 Glut-1 5’-GCCCTGGATGTCCTATCTGA-3’ 5’-CCCACGATGAAGTTTGAGGT-3’ 60 GPx-1 5’-TGAGAAGTGCGAGGTGAATG-3’ 5'AACACCGTCTGGACCTACCA-3’ 60 Primers were designed using a BLAST search of protein databases and converting sequence data to the primary DNA sequence. Primers were designed using this consensus sequence with specific fragments between 100 and 200 bp chosen for each of the genes of interest. Reference Duong T. T. H., Chami B., McMahon A. C., Fong G. M., Dennis J. M., Freedman S. B. and Witting P. K. (2014) Pre?treatment with the synthetic antioxidant T?butyl bisphenol protects cerebral tissues from experimental ischemia reperfusion injury. Journal of Neurochemistry 130, 733– 747. Volume146, Issue2July 2018Pages 198-198 ReferencesRelatedInformation

Exclusion of pituitary homeobox 2 gene polymorphism in vertical mandibular asymmetry patients: a preliminary study

Pituitary Homeobox 2 (PITX2), is an active gene as a paired-related homeobox gene that encodes multiple isoforms. Its Nodal pathway in determination of left-right patterning during embryogenesis has been reported in satellite cells and expressed in adult human skeletal muscle. PITX2A and PITX2B are produced by alternative splicing and used of different promoters. PITX2C uses an alternative promoter located upstream of exon 4. PITX2D is produced by PITX2C alternative promoter and differential splicing. The 5’-primers and 3’- antisense primer were unique for each isoforms. Variability measurement in vertical dimension showed stronger genetic component than sagittal. This study aims to obtain the genotype marker of vertical mandibular asymmetry related to PITX2A and PITX2D isoform by visualization of the amplified product on stained gel to allele specific oligonucleotide between the case and control with Restriction Fragment Length Polymorphism (RFLP). Determination of vertical mandibular asymmetry based on condylar height asymmetry index of pre-treatment panoramic radiograph using Kjellberg’s technique whilst vertical mandibular growth pattern using lateral cephalogram. The differences of condylar height asymmetry in case-control based on vertical growth pattern was compared using Pearson’s chi-squared test. DNA extraction of 129 out-coming orthodontic patients in Universitas Sumatera Utara Dental Hospital were obtained from Buccal swab. Then DNA samples were amplified by Polymerase chain reaction (PCR) and digested with NciI restriction enzyme prior to electrophoresis visualization. There was no significant statistical difference in vertical mandibular asymmetry compared to vertical mandibular growth pattern. The RFLP analysis did not show any polymorphism for PITX2A and PITX2D isoform. All of the samples showed wild type homozygote. Further analysis method, except RFLP, were required to understand the genetic factor in the variance of vertical mandibular asymmetry.