Finally, the construct was cloned in PUC57 in SnapGene 3.2.0.1 and PCR was simulated on hybrid vector using designed primers.įindings: Analysis confirmed that conserved regions for each gene were located on hybrid vector, and simulation of PCR proved the accuracy of designed primers.Ĭonclusion: A genetic simulator hybrid construct with the types of primers required to identify the four factors Francisla, Varivola, Borghuleria and Yersinia has been designed in silico so that such factors could be identified under positive control in emergencies. Specific primers were designed for each region using Oligo7, BioEdit, OligoAnalyzer tool as well as NCBI database. The sequence of these genes were obtained from NCBI in FASTA format and were aligned in BioEdit 7.0.5.3 software for finding conserve region of each gene, then some purposeful changes were applied in the sequence of each gene and the sequences were placed next to each other and the construct was designed. The exact position of genetic elements is shown on the map (termination codons included). This vector is designed for cloning and generation of ExoIII deletions. pUC57 MCS contains 6 restriction sites with protruding 3’-ends, which are resistant to E.coli exonuclease III. Materials & Methods: In this study, fopA, caf1, 16srRNA and HA genes were chosen to be located on the vector, to respectively represent Francisella, Yersinia, Burkholderia and Variolla. pUC57 pUC57 Plasmid pUC57, 2710 bp in length, is a derivative of pUC19. In this research we designed an in silico hybrid vector and relevant primers for detection of Francisella, Variola, Burkholderia and Yersinia. We can design specific primers for each region and use the hybrid vector as positive control sample in PCR. The idea of using hybrid vectors, containing genes of different pathogens, can overcome this limitation. Dehghan Esmatabadi 2, M Zeinoddini 2, N Pourmahdi 2ġ- Faculty of Chemistry & Chemical Engineering, Malek Ashtar University of Technology, Iran, Faculty of Chemistry & Chemical Engineering, Malek Ashtar University of Technology, IranĪims: Today, one of the most important problems in detection of human pathogens, is lack of positive control. This can be supplied in any format, including WORD doc or SnapGene file. We can use these hybrid vectors as positive control, without any concern.In Silico Design of a Hybrid Structure as Positive Control for Simultaneous Detection of 4 Pathogenic Agents by PCR Method Subcloning Services: The default is blunt end cloning into pUC57 with either. Results: Analysis confirmed that conserved region for each gene is located on hybrid vector for each pathogen, and simulation of PCR proved the accuracy of designed primers.Ĭonclusion: Hybrid vectors design contain similar sequence of pathogens genome but they are none-pathogenic. Efficient cleavage requires at least two copies of the BpmI recognition sequence. After cleavage, BsrFI can remain bound to DNA and alter its electrophoretic mobility. Efficient cleavage requires at least two copies of the BsrFI recognition sequence. Finally, the construct cloned in PUC57 in SnapGene and PCRsimulated on hybrid vector using designed primers. For full activity, add fresh S-adenosylmethionine (SAM). Specific primers designed for each region using Oligo7, BioEdit, GeneRunner softwares, Oligo analyzer website and NCBI database. The sequence of these genes obtained from NCBI in FASTA format and aligned in BioEdit software for finding conserve region of each gene, then some purposeful changes were applied in the sequence of each gene and the sequences were placed next to each other and the construct was designed. Materials and Methods: In this study 16srRNA and HA genes were chosen to be located on the vector, to represent of Burkholderia and Variolla, in respectively. In this research we designed a hybrid vector and relevant primers for detection of Variolla and Burkholderia. Elena Caro Bernats lab is published in Unpublished This plasmid is available through Addgene. Background: Todays, one of the most important problems in detection of human pathogens, is lack of positive control. Plasmid MoClo adapted pUC57-Amp Level 1 position 7 from Dr.
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