An Improved Homologous Recombination Method for Rapid Cloning of the Antibody Heavy Chain Gene and Its Comparison with the Homologous Recombination and Traditional Cloning Methods

Background: The homologous recombination (HR) is one of the conventional cloning methods for the production of recombinant DNA. It is a quick method for in vivo DNA cloning without using the high cost restriction enzymes. A few modifications in the cloning procedure can increase the efficiency of this method.Materials and Methods: In this study, effect of heating on the rate of the IgG1 heavy chain gene cloning was investigated in the HR method and then it was compared with HR method without heating and traditional cloning method. For doing this comparison, three cloning methods including HR, HR with the heat treatment, and traditional cloning were used to clone the human IgG1 heavy chain into the pcDNA3.1(+) plasmid.Results: The results showed that adding heat in the HR method converts insert and vector from the double strand DNA to the single strand DNA. This allows them to copulate with each other better and faster than the two other methods. The heat addition also helps the cell enzyme system for a faster and easier recombination and moreover it increases the cloning efficiency of the HR method in case of in vitro processing.Conclusion: The results showed that giving heat in the HR method increases cloning rate 7.5% and this increase reaches 10% in comparison with the traditional method.

Correlation of Antibiotic Resistance with SHV, CTX-M and TEM Extended-Spectrum Beta Lactamases Genes among Klebsiella pneumoniae Isolates from Patients in Kermanshah Hospitals

Background & objectives: One of the main problems in the control of nosocomial infections due to Klebsiella pneumoniae is increase of antimicrobial resistance and prevalence of extended spectrum β-lactamase (ESBLs) producing isolates. The aim of this study was to investigate the correlation of antibiotics resistance with SHV, CTX-M and TEM extended-spectrum beta lactamases genes among Klebsiella pneumoniae isolates isolated from the patients in Kermanshah hospital. Methods: The clinical isolates of Klebsiella pneumoniae were collected during the spring from Kermanshah hospitals, and identification of Klebsiella pneumoniae strains was performed using standard microbiological and biochemical tests. Antibiotic resistance of Klebsiella pneumoniae isolates was determined using disk diffusion method. Then, the presence of CTX-M, SHV, and TEM was investigated using multiplex-PCR method. Finally, the relationship between variables was analyzed by SPSS-22 software using logistic regression and chi-square. Results: A total of 98 isolates out of 112 samples were identified as Klebsiella pneumonia. Also, 82.8% of isolates were resistant to cefotaxime, 40.2% to ceftriaxone, 62.88% to ceftazidime, 3.9% to imipenem, 39.17% to cefepime, 64.94% to cefixime and 26.8% to amikacin. Further, 35.55% of isolates had CTX-M gene, 63.91% of isolates had SHV gene and 9.27% of samples had TEM gene. Conclusion: The presence of CTX-M, SHV and TEM genes along with high antibiotic resistance are very concerning, indicating the importance of rational use of antibiotic for the treatment of infectious diseases. &nbsp

A 15;15 Translocation in a couple with Repeated Abortions: Case report

Carriers of structural chromosomal rearrangements such as Robertsonian or reciprocal translocations have an increased risk of spontaneous abortion and producing offspring with genetic abnormalities. Robertsonian translocations are present in 0.1% of the general population and 1% of the infertile population. Two types of Robertsonian translocations occur more frequently than all others, being 45,XX,rob(13;14)(q10;q10) and 45,XX,rob(14;21)(q10;q10) respectively. The history of repeated abortions could be the outcome of unbalanced gametes (either monosomy or trisomy) resulting during the meiotic segregation of the balanced heterozygote female carrier. In the present report, uncommon Robertsonian translocation in a couple with spontaneous repeated abortions is reported. Cytogenetic analysis of a couple revealed the presence of 45, XY, t (15; 15) (10q; 10q) chromosomal constitution in the male partner. The cytogenetic analysis of couples with repeated abortions is obligatory to identify any probable chromosomal aberrations. As far as we know this is the first instance reported in Iran

Molecular genetic study of glutaric aciduria, type I: Identification of a novel mutation

Glutaric acidemia type I (GA-1) is an inborn error of metabolism due to deficiency of glutaryl-CoA dehydrogenase (GCDH), which catalyzes the conversion of glutaryl-CoA to crotonyl-CoA. GA-1 occurs in about 1 in 100 000 infants worldwide. The GCDH gene is on human chromosome 19p13.2, spans about 7 kb and comprises 11 exons and 10 introns. Tandem mass spectrometry (MS/MS) was used for clinical diagnosis in a proband from Iran with GA-1. Sanger sequencing was performed using primers specific for coding exons and exon-intron flanking regions of the GCDH gene in the proband. Cosegregation analysis and in silico assessment were performed to confirm the pathogenicity of the candidate variant. A novel homozygous missense variant c.1147C > A (p.Arg383Ser) in exon 11 of GCDH was identified. Examination of variant through in silico software tools determines its deleterious effect on protein in terms of function and stability. The variant cosegregates with the disease in family. In this study, the clinical and molecular aspects of GA-1 were investigated, which showed one novel mutation in the GCDH gene in an Iranian patient. The variant is categorized as pathogenic according to the the guideline of the American College of Medical Genetics and Genomics (ACMG) for variant interpretation. This mutation c.1147C > A (p.Arg383Ser) may also be prevalent among Iranian populations

Molecular genetic study of glutaric aciduria, type I: Identification of a novel mutation

Glutaric acidemia type I (GA-1) is an inborn error of metabolism due to deficiency of glutaryl-CoA dehydrogenase (GCDH), which catalyzes the conversion of glutaryl-CoA to crotonyl-CoA. GA-1 occurs in about 1 in 100 000 infants worldwide. The GCDH gene is on human chromosome 19p13.2, spans about 7 kb and comprises 11 exons and 10 introns. Tandem mass spectrometry (MS/MS) was used for clinical diagnosis in a proband from Iran with GA-1. Sanger sequencing was performed using primers specific for coding exons and exon-intron flanking regions of the GCDH gene in the proband. Cosegregation analysis and in silico assessment were performed to confirm the pathogenicity of the candidate variant. A novel homozygous missense variant c.1147C > A (p.Arg383Ser) in exon 11 of GCDH was identified. Examination of variant through in silico software tools determines its deleterious effect on protein in terms of function and stability. The variant cosegregates with the disease in family. In this study, the clinical and molecular aspects of GA-1 were investigated, which showed one novel mutation in the GCDH gene in an Iranian patient. The variant is categorized as pathogenic according to the the guideline of the American College of Medical Genetics and Genomics (ACMG) for variant interpretation. This mutation c.1147C > A (p.Arg383Ser) may also be prevalent among Iranian populations