Flow cytometric investigation on degradation of macro-DNA by common laboratory manipulations  

作　　者：Hee-Bong Yoo Hyuk-Min Lim Inchul Yang Sook-Kyung Kim Sang-Ryoul Park 

机构地区：[1]Center for Bioanalysis, Korea Research Institute of Standards and Science, Daejeon 305-340, Korea

出　　处：《Journal of Biophysical Chemistry》2011年第2期102-111,共10页生物物理化学（英文）

摘　　要：The degree and characteristics of physical degradation of macro-DNA molecules by common laboratory manipulations are reported. With linearized lambda-phage viral DNA as the model DNA, fragmentation of macro-DNA by various indispensable laboratory manipulations were investigated using a high sensitivity flow cytometric setup. Investigated manipulations included pipetting, vortexing, rocking, freeze-thawing, ultrasonication and ultrafiltration. “Exhaustive counting” of the intact lambda DNA molecules following such manipulations enabled a quantitative assessment of the resulting fragmentation, which also revealed the type of degradation reflected in the fragmentation patterns. The use of high sensitivity flow cytometry was especially suited to investigate the degradation of dilute DNA solutions that may not be suitable for analysis using traditional methods. Notable findings of this study included: the boarderline-size of DNA chains in terms of susceptibility to shear stresses by such manipulations;discernable instability of nicked DNAs;shattering-fragmentation of DNAs by freeze-thawing or ultrasonication;effectiveness of some protection media;marked “self-protection effect” of concentrated DNA solutions. These findings support and refine our traditional knowledge on how to maintain the physical integrity of macro-DNA molecules against inevitable laboratory manipulations.The degree and characteristics of physical degradation of macro-DNA molecules by common laboratory manipulations are reported. With linearized lambda-phage viral DNA as the model DNA, fragmentation of macro-DNA by various indispensable laboratory manipulations were investigated using a high sensitivity flow cytometric setup. Investigated manipulations included pipetting, vortexing, rocking, freeze-thawing, ultrasonication and ultrafiltration. “Exhaustive counting” of the intact lambda DNA molecules following such manipulations enabled a quantitative assessment of the resulting fragmentation, which also revealed the type of degradation reflected in the fragmentation patterns. The use of high sensitivity flow cytometry was especially suited to investigate the degradation of dilute DNA solutions that may not be suitable for analysis using traditional methods. Notable findings of this study included: the boarderline-size of DNA chains in terms of susceptibility to shear stresses by such manipulations;discernable instability of nicked DNAs;shattering-fragmentation of DNAs by freeze-thawing or ultrasonication;effectiveness of some protection media;marked “self-protection effect” of concentrated DNA solutions. These findings support and refine our traditional knowledge on how to maintain the physical integrity of macro-DNA molecules against inevitable laboratory manipulations.

关 键 词：Physical DEGRADATION Of DNA LABORATORY MANIPULATIONS FLOW Cytometry Exhaustive Counting SELF-PROTECTION Effect 

分 类 号：R73[医药卫生—肿瘤]