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Detection and Protein Modelling for Consensus Region of hsp70 Gene Family in Egyptian Arundo donax

Journal of Life Sciences 2014年第7期8卷 593-602页

作者： Mohamed A. Ezz Mona I. Salah Abdullah I. Ammoura GEBR1 Bioinformatics Department Sadat University P.O. Box 79/22857 Egypt College of Bioteehnology Misr University for Scienceand Technology 6th of October City October 12451 Egypt Molecular Genetics Lab College of Biotechnology Misr University for Scienceand Technology 6th of October City October12451 Egypt

Degenerate primers are particularly useful in amplifying homologous genes from different organisms. This paper describes a method for designing degenerate primers for a given multiple alignment of DNA sequences of hsp70 gene family using clustalw algorithm and detect the consensus region in gene family of hsp70 in a plant species which have not any recorded information about hsp70 gene family in the Genbank of National Centre of Biotechnology Information （NCBI） like Arando donax .The sequenced consensus sequence ofArando donax is considered a gene marker in building the genome map sequence. The BLASTn program is used to find a homology between more than one accession numbers of DNA sequences, （X67711.2） was for Oryza sativa （hsp70）, （AY372071. l） was for Nicotiana tabacum （hsp70） and （L41253.2） was for Lycopersicon esculentum （Hsc70）. In silco PCR module was performed to detect the melting temperatures （Tm） and predicted the PCR product size （783 bp）.The result of designed degenerate primers showed that there was a homology founded among the designed primers and the DNA templates of the recorded sequences （AY372071.1, X67711.2 and L41253.2） with at least 80% identity. The designed degenerate primers were used to isolate a consensus region ofhsp70 gene family ofArando donax at the expected molecular weight （783 bp）. The isolated PCR product, （783 bp） ofArando donax was sequenced and submitted to the Database of Japan （DDBJ） with accession number AB819871. The ORF finder tool translated the accession number AB819871 and gave a selected frame which used to build 3D structure model. In conclusion, this study focused on the importance of designing the degenerate primers to isolate the gene family and predict the 3D structure of gene family depending on the ORF finder tool of Genebank.

关键词： clustalw tool degenerate PCR different melting temperatures in silico PCR SWISS-MODEL workspace.

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Molecular Studies on Heat Shock Protein 70 Gene （Hsp70） Using clustalw Multiple Sequence Alignment Analysis

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Journal of Life Sciences 2010年第6期4卷 1-8页

作者： Mohamed A. Ezz Mohamed H. Soliman Abd Alkader Y. Gamal El-Din Aly Z.E. Abdelsalam Molecular Genetics Lab Biotechnology College Misr University for Science and Technology 6th of October City October 12451 Egypt Department of Genetics Faculty of Agriculture Cairo University Giza 12613 Egypt A in Shams Center for Genetics Engineering and Biotechnology Faculty of Agriculture Ain Shams University Cairo 11241 Egypt

Degenerate primers are particularly useful in amplifying homologous genes from different organisms. This paper describes a method for designing degenerate primers for a given multiple alignment of DNA sequences of Hsp70 gene family using clustalw algorithm. The authors used an in silico approach to find a homology between more than one accession numbers of DNA sequences, X67711.2 was for Oryza sativa Hsp70, AY372071.1 was for Nicotiana tabacum Hsp70 and L41253.2 was for Lycopersicon esculentum Hsc70. The three accession numbers which were retrieved by the BLASTn program depend on their expected value （E-value）. Multiple sequence alignment was performed by clustalw algorithm to produce a conserved blocks and determine the consensus region which had been used to produce the forward and reverse primer by the primer select module of DNAStar Lasergene V7 and In-Silco PCR module of FASTPCR program ver.4.0.8 was performed to detect the melting temperatures （Tm） and predict the PCR product size, The results of designed degenerate primer showed that there was a homology found between the designed primers and the DNA templates for the three accession numbers with at least 80% identity. The result of degenerate PCR showed that the three bands of the amplified PCR products of the three accession numbers were detected at the same molecular weight of marker （400 bp） with a difference about 15 pb compared to the in silco PCR product （385 pb）. In conclusion, this study focused on the importance of using the clustalw algorithm for designing the degenerate primer.

关键词： clustalw tool degenerate PCR different melting temperatures In-Silico PCR.

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