[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

Isolation, cloning and expression of recombinant human FVII in baculovirus expression system

Background and purpose: Factor VII is one of the important coagulation factors in extrinsic blood coagulation pathway, which can resolve the use of FVIII and FIX for hemophilia patients by activating FX. Recombinant expression of this factor can eliminate the potential problems in preparing those factors from plasma and the risk of transferring hematological diseases. Therefore, the present study intended to investigate the expression of recombinant FVII at a higher level using Gateway technology and TOPO cloning

Methods and Materials: In this experimental study, Factor VII cDNA was isolated from HepG2 cell line by PCR, and cloned to prokaryote TOPO vector by TOPO cloning reaction. The recombinant vector was extracted for bacterial colonies after screening, and was used in Gateway adapted Baculovirus DNA by LR recombination reaction. The recombinant virus was transfected onto insect cell line, and the expression of the protein was analyzed after necessary screening. Findings of the protein expression via ELISA were presented in triadic [Mean +/- SD]; the differences across the three groups were investigated using Student t-test

Results: Cloning and recombination reaction analysis by PCR determined cloning of rFVII in high accuracy [90%] in the vectors. High level expression of recombinant FVII was confirmed by SDS-PAGE, ELISA, and Western blot analysis [30g/ml]. The highest expression level was produced on the 7th day after transfection [1.960 +/- 0.076]. Determined by ELISA, this result was negatively significant in the transfected sample [P<0.001]

Conclusion: Findings of the analysis of the recombinant protein expression by Baculovirus expression system indicated its production in a larger scale than similar eukaryote and prokaryote expression systems