Polymerase Chain Reaction Cloning Research Articles

Evaluation of a Quantitative Taqman Real-Time PCR Assay to Measure Proviral load from Human Immunodeficiency Virus Type 1 individuals

Human Immunodeficiency Virus (HIV) is a virus belonging to the family Retroviridae. HIV – 1 is found to be predominant in India and many parts of Africa. The intention of this study was to quantify the HIV Proviral Deoxyribonucleic Acid (DNA) from newly infected HIV-1 individuals. Fifty patients who were tested positive for HIV were included in this study. Proviral Ribo Nucleic Acid (RNA) was extracted by QIAmp® RNA Mini Kit (QIAGEN, Germany) method. Complementary Deoxyribo Nucleic Acid (cDNA) was synthesized by using Invitrogen Superscript III cDNA synthesis Kit (USA). This cDNA was subjected to Polymerase Chain Reaction (PCR) and Gene cloning by transformation method. The quantification of Real time PCR was done by Applied Bio-System (ABI)-Prism 7700. A linear standard curve was obtained 10 copies to 106 copies per reaction. The assay had good analytic sensitivity and linear dynamic range greater than 6 logs. From the results obtained in this study, It was concluded that Taqman Real-Time PCR Assay plays a major role in monitoring the HIV infected patients in routine diagnostics and clinical practice.

Reliable Detection of T-Cell Clonality by Flow Cytometry in Mature T-Cell Neoplasms Using TRBC1: Implementation as a Reflex Test and Comparison with PCR-Based Clonality Testing.

The T-cell receptor β constant region 1 (TRBC1) antibody can identify T-cell clonality and distinguish pathological from normal T cells. This study aims to establish optimal cutpoints for establishing monotypia and validate the diagnostic abilities of the TRBC1 antibody when used as a reflex test in conjunction with an existing T-cell antibody panel. We used 46 normal peripheral blood specimens and examined 8 patients with reactive lymphoproliferations to determine the normal biological range of TRBC1 on CD4+ and CD8+ T cells. We also evaluated 43 patient specimens that were submitted for investigation of a lymphoproliferative disorder for CD2/CD3/CD4/CD5/CD7/CD8/CD16/CD26/CD45/CD56/TCR αβ/TCR γδ, along with TRBC1 expression. The results were compared to TCR gene rearrangement patterns using polymerase chain reaction (PCR) analysis. Statistical analysis established differing cutoff points for establishing monotypia dependent on restricted TRBC1 or TRBC2 usage. Direct comparison with molecular analysis indicated that no specimen identified with the restricted expression of TRBC1 was reported as polyclonal by PCR with a concordance rate of 97% between a clonal PCR result and monotypic TRBC1 expression. Incorporation of the TRBC1 antibody using statistically derived cutoff points in a reflex setting for the evaluation of a suspected T-cell neoplasm improves the identification of clonal T-cell populations by flow cytometry and correlates well with molecular methods.

Molecular Cloning of DARPins G3 in pET28b Expression Vector and Optimization of the Expression of This Protein in Escherichia Coli

Background: Human epidermal growth factor receptor 2 (HER2) is over-expressed in breast, ovarian, gastric, and prostate cancers and is used as a tumor marker in the diagnosis of cancer. Monoclonal antibodies have been used as a diagnostic and therapeutic tool against HER2. Because of the difficulties associated with the stability and complexity of the construct and the high cost of antibody production, we aimed to investigate, cloning, and expression of HER2- binding DARPins genes to identify, HER2-positive tumor markers, we aimed to investigate. Materials and Methods: After synthesis, the DARPins peptide gene was cloned into the M13 vector and sub-cloned into the TOP10 pet28b bacterial vector. After culturing the bacteria on an agarose plate containing antibiotics, the transfected bacteria expressing the DARPins gene were selected. To ensure gene cloning, we used enzymatic digestion and recombinant plasmid delivery for sequencing. Isopropyl β-d-1-thiogalactopyranosideIPTG was used for the induction of recombinant protein expression and the SDS-PAGE method and Western blot for expression confirmation. Results: The polymerase chain reaction (PCR) amplification product of DARPins was analyzed using agarose gel electrophoresis. Plasmid was purified from the positive clone by PCR cloning, sequenced and gene cloning was confirmed. After culturing from competent cells, protein expression was obtained from positive colonies. SDS- PAGE results showed the effect of different conditions including temperature, IPTG concentration, and time on the pET-DARPins expression. Conclusion: We were succeeded to express a new codon-optimized DARPins gene in Escherichia coli and HEK293t system.

Cloning Polymerase Chain Reaction (PCR) Products: TA Cloning.

The nontemplate-dependent terminal transferase activity inherent in nonproofreading DNA polymerases such as Taq provides a highly efficient method to clone PCR products. The enzyme adds a single, unpaired residue-preferentially an adenosyl residue-to each 3' end of a double-stranded amplified product. The unpaired terminal (A) residues can pair with a linear T vector that carries an unpaired 3'-thymidyl residue at each end. The two chief advantages of TA cloning are speed and lack of reliance on restriction enzymes. The major disadvantage is an inability to clone directionally. For this reason, it is important to pick and analyze several transformed clones when a particular orientation of the amplified fragment is required.

Bioinformatic Analysis in Designing Mega-primer in Overlap Extension PCR Cloning (OEPC) Technique

Bioinformatics has developed into an application tool for basic and applied research in the biomedical and biotechnology field. Polymerase Chain Reaction (PCR) is a common technique in the molecular area that has always involved bioinformatics science. PCR cloning techniques such as TA cloning and PCR-mediated cloning exhibit complex processes with low success rates. One easy, effective, and practical solution is to use a mega-primer with the Overlap Extension PCR Cloning (OEPC) technique. The success of PCR cloning using the mega-primer design in the OEPC technique is strongly influenced by the characteristics of the mega-primer used. Knowledge of mega-primer characteristics is one of the important factors in the success of PCR cloning. The design process for the mega-primer str promoter was characterized based on the principle of a genetic algorithm using the web-based bioinformatics tools such as ClustalW, NetPrimer, and BLAST. The success of the mega-primer construction in producing recombinant pSB1C3 vector has been confirmed by the sequencing method and the function of the reporting protein (AmilCP). DNA analysis shows a 100 % homologous sequence on the str promoter, while E. coli colonies successfully express the purplish-blue color. Mega-primer characters can save costs and time of the research by maintaining the primer parameters that provide optimal values and increase the success value of PCR cloning via bioinformatics software. Hence, implications on biological problems, especially using DNA and amino acid sequences, could solve rapidly.

Cloning Polymerase Chain Reaction Products: Addition of Restriction Sites to the Termini of Amplified DNA.

To generate polymerase chain reaction (PCR) products that can be directionally cloned into a vector, different restriction sites are built into the forward and reverse primers that are used in the PCR. After PCR, the amplified product is purified, cleaved with the appropriate restriction enzymes, ligated into a vector with compatible cohesive ends, and used to transform E. coli.

A Novel Familial PHP1B Variant With Incomplete Loss of Methylation at GNAS-A/B and Enhanced Methylation at GNAS-AS2.

Pseudohypoparathyroidism type 1B (PHP1B), also referred to as inactivating PTH/PTHrP signaling disorder (iPPSD), is characterized by proximal renal tubular resistance to parathyroid hormone (PTH) leading to hypocalcemia, hyperphosphatemia, and elevated PTH values. Autosomal dominant PHP1B (AD-PHP1B) with loss of methylation at the maternal GNAS A/B:TSS-DMR (transcription start site-differentially methylated region) alone can be caused by maternal deletions involving STX16. Characterize a previously not reported AD-PHP1B family with loss of methylation at GNAS A/B:TSS-DMR, but without evidence for a STX16 deletion on the maternal allele and assess GNAS-AS2:TSS-DMR methylation. DNA from 24 patients and 10 controls were investigated. AD-PHP1B patients without STX16 deletion from a single family (n = 5), AD-PHP1B patients with STX16 deletion (n = 9), sporPHP1B (n = 10), unaffected controls (n = 10), patUPD20 (n = 1), and matUPD20 (n = 1). Methylation and copy number analyses were performed by pyrosequencing, methylation-sensitive multiplex ligation-dependent probe amplification, and multiplex ligation-dependent probe amplification. Molecular cloning of polymerase chain reaction-amplified, bisulfite-treated genomic DNA from healthy controls revealed evidence for 2 distinct GNAS-AS2:TSS-DMR subdomains, named AS2-1 and AS2-2, which showed 16.0 ± 2.3% and 31.0 ± 2.2% methylation, respectively. DNA from affected members of a previously not reported AD-PHP1B family without the known genetic defects revealed incomplete loss of methylation at GNAS A/B:TSS-DMR, normal methylation at the 3 well-established maternal and paternal DMRs, and, surprisingly, increased methylation at AS2-1 (32.9 ± 3.5%), but not at AS2-2 (30.5 ± 2.9%). The distinct methylation changes at the novel GNAS-AS2:TSS-DMR will help characterize further different PHP1B/iPPSD3 variants and will guide the search for underlying genetic defects, which may provide novel insights into the mechanisms underlying GNAS methylation.

Cloning short DNA into plasmids by one-step PCR.

BackgroundPlasmid construction of small fragments of interest (such as insertion of small fragment marker genes, expression of shRNA, siRNA, etc) is the basis of many biomolecular experiments. Here, we describe a method to clone short DNA into vectors by polymerase chain reaction (PCR), named one‐step PCR cloning. Our method uses PCR to amplify the entire circular plasmid. The PCR was performed by the primers containing the gene of short DNA with overlapping sequences between 10–15 bp. The PCR products were then transformed into E. coli and cyclized by homologous recombination in vivo.MethodsThe pEGFP‐N1‐HA plasmid was constructed by one‐step PCR and transformation. Cells were transfected with pEGFP‐N1‐HA and pEGFP‐N1 plasmid using TurboFect transfection reagent. Protein expression was detected by western blotting and the HA‐GFP fusion protein was detected by confocal microscopy.ResultsThe pEGFP‐N1‐HA plasmid was successfully constructed and HA expression in cells.ConclusionsFree from the limitations of restriction enzyme sites and omitting the ligation process, our method offers a flexible and economical option of plasmid construction.Key pointsSignificant findings of the studyA method to clone short DNA into plasmids was found.What this study addsOur study provides a flexible and economical option to clone short DNA into plasmids.

Increased Quinolone-Resistant Mutations of gyrA and parC Genes after Pouchitis Treatment with Ciprofloxacin

Background: Oral antibiotics, such as ciprofloxacin (CFX), are widely used for the treatment of acute and chronic pouchitis. Most bacterial mutations that confer quinolone resistance are at Ser-83 and Asp-87 in the gyrA gene and Ser-80 and Glu-84 in the parC gene. Methods: We obtained 51 stool samples from 43 patients who were diagnosed with ulcerative colitis and underwent ileal pouch-anal anastomosis. Patients were divided into 2 groups: 13 patients with CFX treatment of pouchitis and 30 patients without pouchitis. After extraction of fecal DNA, the amount of Escherichia coli 16S rRNA, gyrA, and parC gene DNA were measured using real-time polymerase chain reaction (PCR). Possible mutations at gyrA 83 and 87 and at parC 80 and 84 were investigated by PCR cloning and sequencing, and mutation rates were quantified by rapid PCR-restriction fragment length polymorphism. Results: Samples from both CFX-treated and -untreated patients had comparable levels of gyrA and parC gene DNA. Nucleic acid and amino acid mutations were identified at gyrA 83 and 87, and at parC 80 and 84. We successfully quantified mutation rates at gyrA 83 and 87, and at parC 84, all of which were significantly higher in samples from CFX-treated patients (70, 84, and 38%) than from CFX-untreated patients (13, 11, and 5%). Conclusion: E. coli in patient pouches may have mutations in their gyrA and parC genes that produce CFX resistance. Mutation rates of these genes were significantly higher in samples from CFX-treated patients. This study contributes to understanding the decrease and loss of CFX effectiveness against pouchitis.

The complete genome of Banana streak GF virus Yunnan isolate infecting Cavendish Musa AAA group in China.

Banana streak virus (BSV) belongs to the members of the genus Badnavirus, family Caulimoviridae. At present, BSV contains nine species in the International Committee on Taxonomy of Viruses (ICTV) classification report (2018b release). Previous study indicated that the viral particles of Banana streak virus Acuminata Yunnan (BSV-Acum) were purified from banana (Cavendish Musa AAA group) leaves in Yunnan Province, China, and its complete genome was obtained. To further determine whether this sample infecting with Banana streak GF virus (BSGFV), the polymerase chain reaction (PCR) cloning and complete genome analysis of the Banana streak GF virus Yunnan isolate (BSGFV-YN) isolate were carried out in this study. The result showed that BSGFV-YN infecting Cavendish Musa AAA group was co-infecting this sample. Its genome contains a total of 7,325 bp in length with 42% GC content. This complete genome sequence was deposited in GenBank under accession number MN296502. Sequence analysis showed that the complete genome of BSGFV-YN was 98.14% sequence similarity to BSGFV Goldfinger, while it was 49.10–57.09% to other BSV species. Two phylogenetic trees based on the complete genome and ORFIII polyprotein indicated that BSGFV-YN and other BSV species clustered into a group, while it was the highest homology with BSGFV Goldfinger. Although BSGFV-YN and BSGFV Goldfinger were highly homologous, their cultivating bananas are different. The former cultivating banana was from Cavendish Musa AAA group, while the latter cultivating banana was from Goldfinger Musa AAAB group. Compared with BSGFV Goldfinger, the genome of BSGFV-YN has an extra multiple repetitive sequences in the intergenetic region between ORFIII and ORFI, suggesting that this region might be related to host selection. In summary, a BSGFV-YN distant from BSV-Acum was identified from the same sample, and its complete genome sequence was determined and analyzed. The study extends the polymorphism of BSVs in China and provides scientific clue for the evolutionary relationship with host selection of badnaviruses.

Graft versus host disease and microchimerism in a JAK3 deficient patient

BackgroundThe lymphohematopoietic cells originating from feto-maternal trafficking during pregnancy may cause microchimerism and lead to materno-fetal graft versus host disease (GVHD) in severe combined immunodeficiency (SCID) patients. However, definitive diagnosis between GVHD and Omenn’s syndrome is often difficult based on clinical and immunological phenotypes particularly in the patients with hypomorphic mutations.Case presentationA 3-year-old girl with a history of erythroderma and immunodeficiency was studied. The whole exome sequencing method was used to find the disease-causing variants, and T-A cloning and Quantitative Florescence Polymerase Chain Reaction (QF-PCR) methods were utilized to detect the presence of mosaicism or microchimerism. We identified a homozygous missense Janus Kinase 3 mutation (JAK3, c.2324G>A, p.R775H) as a new disease-causing variant in the patient, and the presence of microchimerism with maternal origin was proven as an underlying cause of her clinical presentation.ConclusionThe findings highlighted the importance of appropriate diagnostic approach in GVHD cases with hypomorphic JAK3 mutations. When analyzing the results of the next generation sequencing, the possibility of microchimerism should be considered based on the context of the disease.

Establishment of a Transgenic Zebrafish Expressing GFP in the Skeletal Muscle as an Ornamental Fish.

Background: Transgenic animals have a critical role in the advancement of our knowledge in different fields of life sciences. Along with recent advances in genome engineering technologies, a wide spectrum of techniques have been applied to produce transgenic animals. Tol2 transposase method is one of the most popular approaches that were used to generate transgenic animals. The current study was set out to produce an ornamental fish, which express enhanced green fluorescent protein (EGFP) under control of mylpfa promoter by using Tol2 transposase method. Materials and Methods: Polymerase chain reaction (PCR) cloning method was performed to insert zebrafish myosin promoter (mylpfa) into Tol2-EGFP plasmid at the upstream of EGFP. In vitro transcription method was used to prepare the transposase mRNA. The Tol2-EGFP plasmid and transposase mRNA were then co-injected into the one-cell stage of zebrafish zygotes. After two days, the fluorescent microscopic analysis was used to select transgenic zebrafishes. Results: Our data showed that the optimum concentration for recombinant Tol2 vector and transposase mRNA were 50 ng/ul and 100 ng/ul, respectively. The results also revealed that the quality of embryos and quantity of injected construct had the important effects on Tol2 transposase method efficiency. Conclusion: Data showed that Tol2 transposase is an appropriate method to generate zebrafish transgene. Our finding also showed that mylpfa promoter is a strong promoter that can be used as a selected promoter in the ornamental fish industry.

Establishment of a Transgenic Zebrafish Expressing GFP in the Skeletal Muscle as an Ornamental Fish

Background: Transgenic animals have a critical role in the advancement of our knowledge in different fields of life sciences. Along with recent advances in genome engineering technologies, a wide spectrum of techniques have been applied to produce transgenic animals. Tol2 transposase method is one of the most popular approaches that were used to generate transgenic animals. The current study was set out to produce an ornamental fish, which express enhanced green fluorescent protein (EGFP) under control of mylpfa promoter by using Tol2 transposase method. Materials and Methods: Polymerase chain reaction (PCR) cloning method was performed to insert zebrafish myosin promoter (mylpfa) into Tol2-EGFP plasmid at the upstream of EGFP. In vitro transcription method was used to prepare the transposase mRNA. The Tol2-EGFP plasmid and transposase mRNA were then co-injected into the one-cell stage of zebrafish zygotes. After two days, the fluorescent microscopic analysis was used to select transgenic zebrafishes. Result: Our data showed that the optimum concentration for recombinant Tol2 vector and transposase mRNA were 50 ng/ul and 100 ng/ul, respectively. The results also revealed that the quality of embryos and quantity of injected construct had the important effects on Tol2 transposase method efficiency. Conclusion: Data showed that Tol2 transposase is an appropriate method to generate zebrafish transgene. Our finding also showed that mylpfa promoter is a strong promoter that can be used as a selected promoter in the ornamental fish industry. [GMJ. 2019;8:e1068]

Identifying the inserted locus of randomly integrated expression plasmids by whole-genome sequencing of Aspergillus strains.

Whole-genome sequencing was conducted on two Aspergillus oryzae strains used for the manufacturing of food enzymes, Acrylaway® and Shearzyme®, with the aim of identifying the inserted locus of randomly integrated expression plasmid and obtaining flanking sequences for safety assessment. Illumina paired-end sequencing was employed, and the obtained reads were mapped to two references: the public genome sequence of Aspergillus oryzae RIB40 and the in-house sequence of the used expression plasmid. Introducing the concept of linking-reads, one locus for each was successfully identified as the integrated site. In the case of Acrylaway®, the obtained sequences suggested that the expression plasmid had been integrated as multiple copies in tandem form. In the case of Shearzyme®, however, information on one edge of the insert was missing, which required extra polymerase chain reaction (PCR) cloning for safety assessment. A 4-kb deletion was detected at the integrated site. There was also evidence of rearrangement occurring in Shearzyme® strain.

Geographical Distribution and Diversity of Gut Microbial NADH:Ubiquinone Oxidoreductase Sequence Associated with Alzheimer's Disease.

Earlier we reported induction of neurotoxicity and neurodegeneration by tryptophan metabolites that link the metabolic alterations to Alzheimer's disease (AD). Tryptophan is a product of Shikimate pathway (SP). Human cells lack SP, which is found in human gut bacteria exclusively using SP to produce aromatic amino acids (AAA). This study is a first attempt toward gene-targeted analysis of human gut microbiota in AD fecal samples. The oligonucleotide primers newly-designed for this work target SP-AAA in environmental bacteria associated with human activity. Using polymerase chain reaction (PCR), we found unique gut bacterial sequence in most AD patients (18 of 20), albeit rarely in controls (1 of 13). Cloning and sequencing AD-associated PCR products (ADPP) enables identification of Na(+)-transporting NADH: Ubiquinone reductase (NQR) in Clostridium sp. The ADPP of unrelated AD patients possess near identical sequences. NQR substrate, ubiquinone is a SP product and human neuroprotectant. A deficit in ubiquinone has been determined in a number of neuromuscular and neurodegenerative disorders. Antibacterial therapy prompted an ADPP reduction in an ADPP-positive control person who was later diagnosed with AD-dementia. We explored the gut microbiome databases and uncovered a sequence similarity (up to 97%) between ADPP and some healthy individuals from different geographical locations. Importantly, our main finding of the significant difference in the gut microbial genotypes between the AD and control human populations is a breakthrough.

Analysis of Reverse Transcribed mRNA Using PCR and Polyacrylamide Gel Electrophoresis.

The patterns of gene expression in the fission yeast Schizosaccharomyces pombe under various experimental conditions form the basis of any transcriptomic study. We describe a method involving reverse transcription of the mRNA, Polymerase Chain Reaction (PCR), and the subsequent separation of the products onto Urea-Polyacrylamide gel that can be used to study the gene expression patterns in the fission yeast. The method described is cost effective and reproducible with satisfactory resolution of expressed transcripts in the gel. The method has the following essential steps: total RNA isolation and purification, cDNA synthesis from mRNAs, PCR amplification of cDNAs, visualization of PCR products, re-amplification and cloning of the differentially expressed PCR products, sequencing the confirmed clones, and finally cDNA library screening to isolate the genes of interest. The technique is also popularly known as Differential Display Reverse Transcription (DDRT-PCR). After its invention in 1992, a number of modifications have been introduced to optimize the technique and specifically to reduce the major problem of "false positives." Since understanding of specific gene expression patterns that regulate developmental and stress responses is a major concern of biology, DDRT-PCR has become a very popular molecular technique during the past two decades.

Isolation and Tissue Distribution of an Insulin-Like Androgenic Gland Hormone (IAG) of the Male Red Deep-Sea Crab, Chaceon quinquedens.

The insulin-like androgenic gland hormone (IAG) found in decapod crustaceans is known to regulate sexual development in males. IAG is produced in the male-specific endocrine tissue, the androgenic gland (AG); however, IAG expression has been also observed in other tissues of decapod crustacean species including Callinectes sapidus and Scylla paramamosain. This study aimed to isolate the full-length cDNA sequence of IAG from the AG of male red deep-sea crabs, Chaceon quinquedens (ChqIAG), and to examine its tissue distribution. To this end, we employed polymerase chain reaction cloning with degenerate primers and 5′ and 3′ rapid amplification of cDNA ends (RACE). The full-length ChqIAG cDNA sequence (1555 nt) includes a 366 nt 5′ untranslated region a 453 nt open reading frame encoding 151 amino acids, and a relatively long 3′ UTR of 733 nt. The ORF consists of a 19 aa signal peptide, 32 aa B chain, 56 aa C chain, and 44 aa A chain. The putative ChqIAG amino acid sequence is most similar to those found in other crab species, including C. sapidus and S. paramamosain, which are clustered together phylogenetically.

PCR-DGGE Analysis: Unravelling Complex Mixtures of Badnavirus Sequences Present in Yam Germplasm.

Badnaviruses (family Caulimoviridae, genus Badnavirus) have emerged as serious pathogens especially affecting the cultivation of tropical crops. Badnavirus sequences can be integrated in host genomes, complicating the detection of episomal infections and the assessment of viral genetic diversity in samples containing a complex mixture of sequences. Yam (Dioscorea spp.) plants are hosts to a diverse range of badnavirus species, and recent findings have suggested that mixed infections occur frequently in West African yam germplasm. Historically, the determination of the diversity of badnaviruses present in yam breeding lines has been achieved by cloning and sequencing of polymerase chain reaction (PCR) products. In this study, the molecular diversity of partial reverse transcriptase (RT)-ribonuclease H (RNaseH) sequences from yam badnaviruses was analysed using PCR-dependent denaturing gradient gel electrophoresis (PCR-DGGE). This resulted in the identification of complex ‘fingerprints’ composed of multiple sequences of Dioscorea bacilliform viruses (DBVs). Many of these sequences show high nucleotide identities to endogenous DBV (eDBV) sequences deposited in GenBank, and fall into six monophyletic species groups. Our findings highlight PCR-DGGE as a powerful tool in badnavirus diversity studies enabling a rapid indication of sequence diversity as well as potential candidate integrated sequences revealed by their conserved nature across germplasm.

Intra- and inter-isolate variation of ribosomal and protein-coding genes in Pleurotus: implications for molecular identification and phylogeny on fungal groups

BackgroundThe internal transcribed spacer (ITS), RNA polymerase II second largest subunit (RPB2), and elongation factor 1-alpha (EF1α) are often used in fungal taxonomy and phylogenetic analysis. As we know, an ideal molecular marker used in molecular identification and phylogenetic studies is homogeneous within species, and interspecific variation exceeds intraspecific variation. However, during our process of performing ITS, RPB2, and EF1α sequencing on the Pleurotus spp., we found that intra-isolate sequence polymorphism might be present in these genes because direct sequencing of PCR products failed in some isolates. Therefore, we detected intra- and inter-isolate variation of the three genes in Pleurotus by polymerase chain reaction amplification and cloning in this study.ResultsResults showed that intra-isolate variation of ITS was not uncommon but the polymorphic level in each isolate was relatively low in Pleurotus; intra-isolate variations of EF1α and RPB2 sequences were present in an unexpectedly high amount. The polymorphism level differed significantly between ITS, RPB2, and EF1α in the same individual, and the intra-isolate heterogeneity level of each gene varied between isolates within the same species. Intra-isolate and intraspecific variation of ITS in the tested isolates was less than interspecific variation, and intra-isolate and intraspecific variation of RPB2 was probably equal with interspecific divergence. Meanwhile, intra-isolate and intraspecific variation of EF1α could exceed interspecific divergence. These findings suggested that RPB2 and EF1α are not desirable barcoding candidates for Pleurotus. We also discussed the reason why rDNA and protein-coding genes showed variants within a single isolate in Pleurotus, but must be addressed in further research.ConclusionsOur study demonstrated that intra-isolate variation of ribosomal and protein-coding genes are likely widespread in fungi. This has implications for studies on fungal evolution, taxonomy, phylogenetics, and population genetics. More extensive sampling of these genes and other candidates will be required to ensure reliability as phylogenetic markers and DNA barcodes.

Infant sudden death: Mutations responsible for impaired Nav1.5 channel trafficking and function.

Two genetic variants in SCN5A, encoding the Nav1.5 Na+ channel α-subunit, were found in a 5-month-old girl who died suddenly in her sleep. The first variant is a missense mutation, resulting in an amino acid change (Q1832E), which has been described (but not characterized) in a patient with Brugada syndrome. The second is a nonsense mutation that produces a premature stop codon and a C-terminal truncation (R1944Δ). To investigate their functional relevance with patch clamp experiments in transfected HEK-293 cells. The Q1832E mutation drastically reduced Nav1.5 current density. The R1944Δ C-terminal truncation had negligible effects on Nav1.5 current density. Neither of the mutations affected the voltage dependence of steady activation and inactivation or influenced the late Na+ current or the recovery from inactivation. Biochemical and immunofluorescent approaches demonstrated that the Q1832E mutation caused severe trafficking defects. Polymerase chain reaction cloning and sequencing the victim's genomic DNA allowed us to determine that the two variants were in trans. We investigated the functional consequences by coexpressing Nav1.5(Q1832E) and Nav1.5(R1944Δ), which led to a significantly reduced current amplitude relative to wild-type. These sudden infant death syndrome (SIDS)-related variants caused a severely dysfunctional Nav1.5 channel, which was mainly due to trafficking defects caused by the Q1832E mutation. The decreased current density is likely to be a major contributing factor to arrhythmogenesis in Brugada syndrome and the sudden death of this SIDS victim.