Abstract
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.
Highlights
Deoxyribonucleic acid (DNA) is an essential component of modern biology
‘Exhaustive counting’ refers to counting all DNA particles in the sample volume, which results in high reproducibility as the counting results are unaffected by variations in the operation conditions of the measurement system
Using a home-made flow cytometry system capable of counting individual DNA particles and providing information concerning DNA size, the fragmentation of linearized lambda phage DNA by common laboratory manipulations was examined in a quantitative manner
Summary
Deoxyribonucleic acid (DNA) is an essential component of modern biology. Its size spans from several 10s of bp (base-pairs) of synthetic oligonucleotides to a few million bp of double-stranded DNA. DNA is known to have a robust chemical structure that can stably retain genetic information, large DNA molecules can be degraded by unattended physical experiments [1]. This could be an important concern when one has to maintain the integrity of DNA as a critical agent such as in the area of gene-therapeutics [2]. Multiple gene therapy strategies have recently began to use artificial chromosomes in the mega-base size range [3] The handling of such large DNAs with minimal impairment of macromolecular integrity is of paramount importance in this field
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