Development of nested PCR protocol for the isolation of partial growth hormone gene fragment in marble goby

Abstract

A study was done to develop a Nested PCR protocol for the isolation of partial growth hormone (GH) gene fragment of Marble Goby. In this study, DNA from Marble Goby (Oxyeleotris marmorata) was extracted from the muscle tissue and used for PCR amplification. In the nested PCR protocol, amplification was done using two pairs of degenerate primers that were specific to the GH gene sequence. In this experiment, Nested PCR was used to confirm the PCR product as it allows discrimination between specific and nonspecific amplification signals. In this experiment, several optimizations were done. The optimizations involved four parameters that are annealing temperature, PCR cycle, concentration of Magnesium Chloride (MgCh) and polymerase enzymes. From the results obtained, the optimum annealing temperature was 32 ° C, the optimum PCR cycle was 35, the optimum MgCh concentration was 2mM and the optimum enzymes used was Finnzymes DyNAzyme EXT DNA polymerase.