ارزيابي روش Touchdown Nested PCR براي جلوگيري از اتصالات غير اختصاصي و افزايش اختصاصيت در تكثير ژني ويروس هاي نقص ايمني انسان و هپاتيت جي

Evaluation of Touchdown Nested PCR to Circumvent Spurious Priming and Increase Specificity during HIV and GBV-C Gene Amplification

Introduction & Objective: Primer-Template hybridization temperature is one of the important parameters in Nested PCR optimization. Unlike instant temperature for sequence amplification in routine PCR process, Touchdown PCR is a modified form of standard PCR that employs a range of annealing temperature. This study intended to develop a Touchdown Nested PCR in order to circumvent spurious priming and enhancing specify during gene amplification.Materials & Methods: This is an experimental study conducted at Tarbiat Modarres University of Tehran during 2008-2009. Study samples were collected from Digestive Diseases Research Centre-at Shariʹati Hospital and HIV research center - Imam Khomeini Hospital. After extracting the nucleic acid, primer designing for HIV and GBV-C and c-DNA synthesis; Nested PCR was performed on negative and positive samples using standard and touchdown protocolsResults: The intended band was observed in all positive samples. No band was observed in any human and viral negative control samples. After electrophoresis of PCR products, non specific band were seen in HIV and GBV-C samples during standard PCR. Using the touchdown protocol, undesirable bands were omitted or significantly decreased.Conclusion: In the present study, despite the formation of uncalled bands in standard reaction; using the touchdown method led to omission of non-specific bands without any significant effect on the final products. As for its simplicity, cost and time saving, it seems that using this method is a rational and economical way for fast optimization of PCR reactions.