Ethidium bromide에 의한 high-resolution banding에 관한 연구

Abstract

중기염색체는 염색체가 가장 많이 응축된 상태로 염색체팔이 짧아 band수가 많지 않으므로 현재의 banding방법으로는 염색체 구조상의 재배열을 확실히 감지하기 어렵다. 근래에 임상적 유전진단에 있어서 미세한 염색체 변화를 알아내고, 여러 유전자좌위의 정확한 위치를 알아내기 위한 발전된 banding방법의 필요성이 주장되었다. 이어 염색체가 응축되는 초기단계에서 염색체응축 저지작용을 하는 물질을 이용하여 신장된 염색체를 얻어 염색체상에 많은 band가 나타나도록하는 high-resolution banding방법이 연구 발표되었다. 본 실험에서 colcemid (0.1 ㎍/㎖)처리 30분 경과후에 ethidium bromide를 처리하여 세포분열계수 변화와 분열세포상을 비교 분석한 결과는 다음과 같다. 1 세포분열계수는 ethidium bromide농도가 증가함에 따라 유의하게 감소되었다. 2. 분열세포상은 ethidium bromide증가에 따라 midmetaphase는 유의하게 감소하고, early metaphase는 25 ㎍/㎖에서는 6.05±0.60 50㎍/㎖에서는 18.05±0.47, 100㎍/㎖ 에서는 2.90±0.72이며 Pro metaphase는 각각 4.95±0.51, 22.80±0.61, 0.9±0.23이며 200㎍/㎖처리시는 분열세포가 관찰되지 않았다. 3. 분열기에 따른 염색체 band는 midmetaphase에서는 320bands 이상, early metaphase와 Prometaphase에서는 550bands,850bands 에 가깝게 나타났다. 이상의 결과를 종합하면, 50㎍/㎖의 ethidium bromide 처리시 high-resolution을 위한 염색체 신장효과가 매우 높았다.;At present, many small structural rearrangements may remain undetected because the resolving power of current banding techniques is inadequate. The importance of high-resolution chromosome study in clinical cytogenetic diagnosis and the need for improved chromosome banding resolution to aid in accurate regional assignment of various gene loci are well documented. High-resolution banding methods for the demonstration of an increased number of bands along human chromosomes in earlier stage of contraction have been described. This study was undertaken in order to determine ethidium bromide concentration to obtain elongated chromosome for high-resolution banding by compared mitotic index and mitotic figures in cells treated with ethidium bromide. In this study, concentration used of ethidium bromide were 0ug/ml, 25ug/ml, 50ug/ml, lOOug/ml and 200ug/ml. The results obtained were as follows: 1. Mitotic index were significantly decreased as concentration of ethidium bromide increased. 2. In analysis of mitotic figures, midmetaphase chromosome was significantly decreased as concentration of ethidium bromide increased. A number of early metaphase chromosome and prometaphase chromosome were observed most at concentration of 50ug/ml and no cells were observed at 200ug/ml. 3. A number of chromosome bands were more 320bands in midmetaphase, 550bands in early metaphase, and 850bands in prometaphase, approximately. It is, therefore, adequate that concentration of ethidium bromide to obtain elongated chromosome is 50ug/ml.