Sense Primer Research Articles

Age-related promoter-switch regulates Runx1 expression in adult rat hearts

BackgroundRunt-related transcription factor-1 (RUNX1), a key member of the core-binding factor family of transcription factors, has emerged as a novel therapeutic target for cardiovascular disease. There is an urgent need to fully understand the expression pattern of Runx1 in the heart and the mechanisms by which it is controlled under normal conditions and in response to disease. The expression of Runx1 is regulated at the transcriptional level by two promoters designated P1 and P2. Alternative usage of these two promoters creates differential mRNA transcripts diversified in distribution and translational potential. While the significance of P1/P2 promoter-switch in the transcriptional control of Runx1 has been highlighted in the embryogenic process, very little is known about the level of P1- and P2-specific transcripts in adult hearts, and the underlying mechanisms controlling the promoter-switch.MethodsTo amplify P1/P2 specific sequences in the heart, we used two different sense primers complementary to either P1 or P2 5'-regions to monitor the expression of P1/P2 transcripts. DNA methylation levels were assessed at the Runx1 promoter regions. Rats were grouped by age.ResultsThe expression levels of both P1- and P2-derived Runx1 transcripts were decreased in older rats when compared with that in young adults, paralleled with an age-dependent decline in Runx1 protein level. Furthermore, older rats demonstrated a higher degree of DNA methylation at Runx1 promoter regions. Alternative promoter usage was observed in hearts with increased age, as reflected by altered P1:P2 mRNA ratio.ConclusionOur data demonstrate that the expression of Runx1 in the heart is age-dependent and underscore the importance of gene methylation in the promoter-mediated transcriptional control of Runx1, thereby providing new insights to the role of epigenetic regulation in the heart.

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.

Electroactive nanocarbon materials as signaling tags for electrochemical PCR

Electrochemical polymerase chain reaction (PCR) represents a valid alternative to the optical-based PCR due to reduced costs of signaling labels, use of simpler instrumentation, and possibility of miniaturization and portability of the systems, which can facilitate decentralized detection. The high intrinsic electroactivity and strong linear relationship between the material concentration and its redox signal suggest a possible use of oxidized nanocarbon materials as electroactive tags for PCR. Herein, we compared three different nanographene oxide materials namely nGO-1, nGO-2 and nGO-3 as signaling tags for the detection of genetically modified organisms (GMO) by electrochemical PCR. The three materials differ in size, chemical composition as well as type and amount of oxygen functionalities verified by extensive characterization with X-ray photoelectron spectroscopy (XPS), attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR), transmission electron microscopy (TEM) and electrochemical methods. A sense primer sequence belonging to the Cauliflower Mosaic Virus 35S promoter (a common genetic marker for GMO screening) was used to conjugate to the nanocarbon materials by carbodiimide chemistry before PCR amplification with a biotinylated antisense strand. Finally, the amplified electroactive PCR product was detected, where the reduction signal derived from the electrochemically reducible oxygenated functionalities on the nanocarbon material surface was directly correlated to the presence of GMO. Overall, we were able to correlate the different material characteristics with their performance as electroactive labels and identify the nanocarbon material that exhibits the highest potential to be used as innovative electroactive label for PCR in the amplification and detection of the selected target sequence.

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

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.

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.

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

Bindel-PCR: a novel and convenient method for identifying CRISPR/Cas9-induced biallelic mutants through modified PCR using Thermus aquaticus DNA polymerase

We developed a novel and convenient method for rapidly identifying CRISPR/Cas9-based genome-edited biallelic knockout (KO) cells/individuals carrying insertions or deletions of a few nucleotides (indels) by performing PCR on genomic DNA samples under stringent conditions and low MgCl2 concentrations. The biallelic KO samples can be judged as ‘negative’ under these conditions. The sense primer corresponds to the sequence recognised by guide RNA and subsequently cleaved by Cas9 immediately upstream of a target gene’s proto-spacer adjacent motif (PAM), and the reverse primer corresponds to the sequence ~200 bp downstream from the PAM. PCR performed using this primer set under standard MgCl2 concentrations (1.5–2.5 mM) should generate PCR products derived from both mutated and unedited alleles, whereas PCR performed using lower MgCl2 concentrations (0.8–2 mM) should yield products derived from unedited alleles. This enables high-throughput screening of biallelic mutants among cells/embryos having ≥1 indels at a region within 5 bp upstream of the PAM (where more than 94% of indels are known to appear). We performed proof-of-principle analyses of this novel approach using genome-edited Et1, Tyr, Ramp1, Ramp3, and Rosa26 mouse samples carrying various types of indels, and demonstrate that this new technique allows rapid identification of biallelic KO mutants among samples carrying various types of indels and mosaic mutations with 100% accuracy. We name this system detection of biallelic KO mutants harbouring indels using PCR (Bindel-PCR).

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.

Phylloseptin-PBa1, -PBa2, -PBa3: Three novel antimicrobial peptides from the skin secretion of Burmeister's leaf frog (Phyllomedusa burmeisteri)

Skin secretions are known as a highly-complex mixture of abundant and diverse bioactive molecules and its study has attracted increasing attention over recent years. Phylloseptin is a unique family of antimicrobial peptides which have been only isolated from frogs of the Phyllomedusinae subfamily. Here, three novel peptide precursors were successfully cloned from a cDNA library, which was constructed from the skin secretion of Phyllomedusa burmeisteri, as pair of primers (one nested universal primer and a designed degenerate sense primer) were employed for “shotgun” cloning. The encoded mature peptides were validated by MS/MS sequencing, and subsequently termed as Phylloseptin-PBa1, -PBa2 and -PBa3. Phylloseptin-PBa1 and -PBa2 were demonstrated to possess potent antimicrobial activities against Gram-positive bacteria and yeast, as well as broad-spectrum anticancer activities, while they possess varying haemolytic activity at the effective concentration. In contrast, Phylloseptin-PBa3 was found to exhibit a strong haemolytic activity even though it was only found to possess a weak antimicrobial activity and inconspicuous anticancer activity.

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

A modified strategy of multiplex RT-PCR for simultaneous detection of H5, H7, and H9 subtypes of avian influenza virus based on common forward oligo

Avian influenza virus subtypes H5, H7, and H9 related epidemics are accountable for huge losses to the poultry and avian industry in Pakistan. Well-timed and accurate diagnosis of subtype(s) concomitant with a specific epidemic allows a margin of prophylaxis to farmers through requisite vaccination. The current study was designed to develop and validate a rapid, accurate, and efficient diagnostic technique based on a modified strategy in reverse transcription PCR (RT-PCR) for simultaneous detection of avian influenza virus subtypes H5, H7, and H9. Instead of 3 pairs of primers, a single common forward sense primer and 3 negative sense primers specific for 3 subtypes were used. Genotyping on 2% agarose gel sorted out specific products of desired sizes for individual and mixture samples of three subtypes.

Molecular Identification of Mucor and Lichtheimia Species in Pure Cultures of Zygomycetes.

BackgroundThe Mucorales are an important opportunistic fungi that can cause mucormycosis in immunocompromised patients. The fast and precise diagnosis of mucormycosis is very important because, if the diagnosis is not made early enough, dissemination often occurs. It is now well established that molecular methods such as polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) are feasible and reliable tools for the early and accurate diagnosis of mucormycosis agents.ObjectivesThe present study was conducted to evaluate the validity of PCR-RFLP for the identification of Mucorales and some important Mucor and Lichtheimia species in pure cultures of Zygomycetes.Materials and MethodsSpecific sense and anti-sense primers were used to amplify the Mucorales, Mucor, and Lichtheimia DNA. The PCR products were digested by AfIII, XmnI, and AcII restriction enzymes, and the resultant restriction pattern was analyzed.ResultsOn the basis of the molecular and morphological data, we identified Mucor plumbeus (10.83%), M. circinelloides (9.17%), Lichtheimia corymbifera (9.17%), M. racemosus (5.83%), M. ramosissimus (3.33%), and L. blakesleeana (0.83%).ConclusionsIt seems that PCR-RFLP is a suitable technique for the identification of Mucorales at the species level.

Sensitive allele-specific real-time PCR test for mutations in BRAF codon V600 in skin melanoma.

Mutations at BRAF codon V600 are used as predictive biomarkers for targeted therapy of skin melanoma. Here, a simple sensitive test to detect mutations of BRAF-V600 was developed using real-time PCR with allele-specific primers and TaqMan probes. Two versions of the test using sense and antisense allele-specific primers were designed and evaluated. The test detected 1% mutant allele V600E/K in 10 ng DNA standard made from wild-type human DNA spiked with BRAF-V600E or the V600K plasmid. The test was validated on clinical formalin-fixed paraffin-embedded samples of skin melanoma using pyrosequencing as a reference method. In the clinical samples, we detected the common mutation V600E, as well as the rare mutations V600K, V600E2 (codon GAA), V600E2 K601del, V600D-K601del, and V600R. In comparison with pyrosequencing, both versions of the test had 100% specificity with sensitivities of 97 and 86% for sense and antisense allele-specific primers, respectively. Using the PCR test with sense allele-specific primers, mutations in V600 were found in 33 of 51 Russian patients (64.7%) with cutaneous melanoma. This closed-tube real-time PCR test can be used as a simple and sensitive assay for mutations of BRAF-V600 in cutaneous melanoma.