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 Hsp...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.展开更多
文摘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.
