Expression of green fluorescent protein [GFP] using in vitro translation cell free system

One of the major concerns about recombinant protein production is its possible toxicity for the organism. Purification of the recombinant protein is another challenge in this respect. Recently In Vitro translation cell free system that provides a coupled transcription-translation reaction for protein synthesis to overcome the above mentioned problems has been emerged. The aim of this study was expression of GFP as a marker for gene expression and protein in In Vitro translation system. pIVEX2.3-GFP plasmid was cloned to E. coli and the plasmid DNA extracted. In Vitro translation was performed with RTS 100 E. coli Hy kit according to manufacture's instructions. Expression of recombinant fusion protein, His- GFP, was determined by SDS-PAGE, ELISA and western blot analysis. Expected size of recombinant protein was detected in SDS-PAGE and further confirmed by western blot analysis and ELISA. Results showed that In Vitro translation is suitable for expression of recombinant protein and fusion of the recombinant protein with His-tag facilitates the purification

[Quantitative real-time PCR technique for rapid diagnosis of trisomy 21]

Prenatal diagnosis and prevention of live-born children with Down syndrome is a principle priority of Iran's ministry of health. The aim of this study was the rapid diagnosis of Down syndrome by quantitative real-time PCR technique as a new method for prenatal diagnosis. In this experimental study, two milliliters of peripheral blood was obtained from each patient and normal control. Then genomic DNA was extracted using salting out method. DYRK1A2 gene as target gene and PMP22 gene as reference gene were analyzed by quantitative real-time PCR technique. DYRK1A2/PMP22 gene ratio was 1.68 +/- 0.13 and 1.00 +/- 0.09 in Down syndrome and normal samples, respectively [p<0.001], demonstrating 3 copies of target [DYRK1A2] gene in trisomy 21 syndrome and 2 copies in normal individuals. DYRK1A2/PMP22 gene ratio is significantly higher in patients with Down syndrome compared with normal individuals. So, quantitative real-time PCR technique can be used as a sensitive, accurate and reliable technique for rapid diagnosis of trisomy 21 syndrome