Application of bacterial magnetic particles for highly selective mRNA recovery system

Summary

A novel mRNA detection system was developed using DNA immobilized on bacterial magnetic particles isolated from the magnetic bacterium Magnetospirillum sp. AMB-1 (Matsunaga et al., 1991). Immobilization of oligonucleotides was carried out using N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP). As a model system, we have attempted to detect bonito growth hormone mRNA from the bonito pituitary gland. Oligonucleotides corresponding to the 5'end of bonito GH mRNA was covalently immobilized onto bacterial magnetic particles. The particles were then incubated in the pituitary gland lysate, and recovered by Sm-Co magnet, followed by washing. cDNA synthesis was carried out using reverse transcriptase from mRNA recovered using a bacterial magnetic particles. The cDNA synthesized was analyzed by PCR. As a result, a unique band of bonito GH gene was detected. Furthermore, using recombinant Escherichia coli containing cloned bonito GH cDNA under downstream of lac promoter, the transcriptional level of cloned bGH gene was analyzed.