생쥐의 조직 특이적 발현 유전자의 분리를 위한 차감 방법

Abstract

A subtractive hybridization method was developed to facilitate the isolation of novel tissue-specific genes. The phenol emulsion reassociation technique was exploited to increase the efficiency of subtraction. A large excess amount of driver cDNA (liver) with NotI-cleaved cohesive ends was hybridized with target cDNA (brain) with EcoRI-cleaved cohesive ends. Cloning of the reassociation products into an EcoRI site of the vector plasmid effectively selected the DNA fragments with EcoRI-cohesive ends derived from the target cDNA strands. Subtracted clones were tested for their tissue-specificity by Northern blot analysis. Among 20 clones analyzed, 17 were identified to be the same as confirmed by restriction digestion patterns. The redundancy of a single clone sequence suggests that the gene for this sequence is abundantly transcribed in the brain. Thus, the subtraction described here can be widely applied for isolation of abundantly transcribed tissue-specific genes.