Shear‐Induced Degradation of Plasmid DNA

Lengsfeld, Corinne S.; Anchordoquy, Thomas J.

doi:10.1002/jps.10140

Cited by 96 publications

(88 citation statements)

References 54 publications

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“…It has to be understood that the shear stresses induced varies depending on the size of the DNA. For a given physical agitation, the imparted tensional force is pro-(b) (a) portional to the 1st to 2nd power of the molecular weight of a DNA particle [2]. For linear DNA chains, the exponent was found to be 1.2 by Bowman and Davidson [5].…”

Section: Shear Degradation By Pipetting Vortexingmentioning

confidence: 99%

“…We confirmed this expectation by observing no substantial degradation of X-bal-digested lambda DNA (24.5 kbp) following application of the aforementioned manipulations (data not shown). In mass production environment, smaller DNA (5 kbp plasmid) is found to be insensitive to shear-induced degradation [2,3]. On the other hand, DNA particles substantially larger than lambda DNA would be readily fragmented by these manipulations.…”

Section: Shear Degradation By Pipetting Vortexingmentioning

confidence: 99%

“…Although DNA is known to have a robust chemical structure that can stably retain genetic information, large DNA molecules can easily 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].…”

Section: Introductionmentioning

confidence: 99%

“…Various influencing parameters were methodically investigated and theoretically inferred. As reviewed by Lengsfeld and Anchordoquy [2], the most important parameters that influence shear-induced breaks of macro-DNA are strain rate, ionic strength, gas-liquid interface and turbulence, in addition to DNA molecular weight, size, and flexibility. Recently, concerns about DNA degradation have been raised in relation to the mass production of high quality plasmids for use in DNA therapeutic strategies [3,10,11,14].…”

Section: Introductionmentioning

confidence: 99%

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

Yoo¹,

Lim²,

Yang³

et al. 2011

JBPC

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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, freezethawing, 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 freezethawing 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.

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Section: Shear Degradation By Pipetting Vortexingmentioning

confidence: 99%

Section: Shear Degradation By Pipetting Vortexingmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

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

Yoo¹,

Lim²,

Yang³

et al. 2011

JBPC

View full text Add to dashboard Cite

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“…Subsequent lysis of E. coli cells are carried out using a modified version of previously reported methods (Ciccolini et al 2002;Meacle et al 2004;Clemson and Kelly 2010). Modifications to this step are especially important due to the potential for pDNA damage from caustic solvents and shear stresses (Lengsfeld and Anchordoquy 2001;Freitas et al 2007). Briefly, the cell pellet is resuspended to 150 g/L cell in resuspension buffer (50 mM Tris-HCl, 10 mM EDTA, pH 8.0).…”

Section: Recoverymentioning

confidence: 99%

Rapid, Large-Scale Production of Full-Length, Human-Like Monoclonal Antibodies

Warner¹

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Current recombinant protein production systems require several months to develop.Existing systems fail to provide timely, flexible, and cost-effective therapies to protect against emergency mass-casualty infections or poisonings. As the identity of many new biological threat agents are unlikely to be known in advance, pre-emptive manufacturing and stockpiling of countermeasures cannot be performed. Preparedness for a biological catastrophe requires a radical solution to replace the current slow scale-up and manufacture of lifesaving medical countermeasures. Subunit vaccines and antibody fragments may be produced in bacteria, yeast, plant or insect cells. However, generation of full-length, human like glycosylated antibodies requires mammalian cell culture due to the cell's ability to carry out complex assembly and processing. Although commercial cell culture methods for antibody production from stable gene expression have been substantially improved over the last 30 years, the time required to achieve full scale production for a new product is too long for a rapid, emergency

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