Multiplex PCR optimization for coamplification of int-2 and y-IFN genes

Multiplex PCR optimization for coamplification of int-2 and -IFN genes Atefeh Arbabi, Zohreh Zahraei Cell and Molecular Biology Department, Faculty of Chemistry, University of Kashan, Kashan, IRAN Fibroblast growth factor 3 (FGF3), is encoded by the int-2 gene which is involved in cellular functions such as: proliferation, angiogenesis and metastasis. This gene is amplified in some cancer patients and may be associated with advanced tumor progression. In this study, for evaluation the amplification of int-2 gene in patient’s blood samples, Multiplex PCR (Polymerase Chain Reaction) technique was used. After extracting genomic DNA from human blood by salting out procedure, amplification of int-2 gene was determined by optimizing coamplification of single-copy gene, -IFN and the target gene (int-2) in PCR reaction. The 25 µl reaction mixture contained 10mM Mgcl2, 0.2 mM dNTP, 0.7 µM of primer int-2, 0.5 µM of primer -IFN and 100 ng of genomic DNA in the presence of 2 U of Taq DNA polymerase. Further optimization of PCR conditions were achieved, when the melting point of the primers set the upper limit on annealing temperature (Touchdown PCR). For the first phase (5 cycles), the annealing temperature was reduced 10C per cycle, so that at the second phase (25 cycles) the annealing temperature was adjusted at 580C.

Keywords:

Multiplex PCR , Optimization , int-2 gene