Maryam M. Matin; David P. Hornby
Abstract
We have developed a rapid, quantitative method for analysing the outcome of the first strand synthesis step in cDNA library preparation, yield and molecular weight range of the final cDNA products are determined after size fractionation. This method involves conventional cDNA library ...
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We have developed a rapid, quantitative method for analysing the outcome of the first strand synthesis step in cDNA library preparation, yield and molecular weight range of the final cDNA products are determined after size fractionation. This method involves conventional cDNA library construction including all enzymatic steps usually required, but replaces radioactive labelling of nucleic acids with fluorescence detection. The separation and quantification steps all involve ion-pair reversed-phase high performance liquid chromatography (IP RP HPLC). This quantitative method replaces the use of autoradiography and size exclusion chromatography with combined ion-pair reversed-phase high performance liquid chromatography and in line fluorescence detection. The result of this approach is combination of speed with the generation of reproducible, high quality cDNA libraries.
Ehsan Nasr; Mohammad Pourkazemi; Hrachia Hovhannisyan
Abstract
Administering growth hormone increases growth rate of cultured fish. The aim of this study was isolation and synthesize of Persian sturgeon GH cDNA. The total RNA was extracted from pituitary gland of Persian sturgeon and cDNA was synthesized. The full-length cDNA sequence of Persian sturgeon contains ...
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Administering growth hormone increases growth rate of cultured fish. The aim of this study was isolation and synthesize of Persian sturgeon GH cDNA. The total RNA was extracted from pituitary gland of Persian sturgeon and cDNA was synthesized. The full-length cDNA sequence of Persian sturgeon contains a 645 nucleotide open reading frame, which encoding a 214 amino acid residues. The position of the signal peptide cleavage site was predicted to be at position 72. After cleaving of a signal peptide of 24 amino acid resi dues, a mature peptide of 190 aa formed. The blast observed that the PS pre GH have highest nucleotide sequence similarity with Acipenseridae family and mammals GH. Secondary structure and tertiary structure of Persian sturgeon growth hormone gene were prediction by online software. The secondary structure of the GH was revealed the predomination of a- helix (> 55%), the domains of high conservation across the vertebrate GH protein. The predicted 3D structure of Persian sturgeon growth hormone attribute to the typical 4-a-helix bundle protein conformation, the characteristic 3-D confirmation of growth hormones.
