[PCR amplification of nerve growth factor (beta-NGF) gene in human brain]

Nerve growth factor, an important nutrition factor for neuronal survival, growth, development and maintenance of neuronal functions, consists of three kinds of subunit (alpha 2, beta and gamma 2), but only the subunit beta-NGF has the biological activity. beta-NGF coding sequence is located in a single exon of NGF gene. In our study, a pair of specific primers (29 mers) has been designed and synthesized with ABI 318 A DNA synthesizer. The upstream primer includes an initiation codon and the 1st to 20th base of the exon. The downstream primer includes the complementary sequence of 334 to 354 base of the exon and a stop codon. The full-length DNA fragment of beta-NGF gene has been successfully obtained from human brain cDNA library in lambda ZAPII/EcoRI and human brain genomic DNA respectively by using polymerase chain reaction. The reaction program consists of denaturing at 93 degrees C for 1 min, annealing at 55 degrees C for 1 min, and extending at 72 degrees C for 2 min. Both amplified products of 350 bp in length coincide with the exon coding sequence and are enough for the analysis of restriction mapping, DNA sequencing and construction of expression vector.