title: High-Speed Atomic Force Microscopy Revealing Contamination in DNA Purification Systems.
abstract: Motivated by reports of low-level DNA contamination in popular commercial DNA purification kits, we employed a novel high-speed atomic force microscopy (HS-AFM) method to detect and characterize particulate and polymeric contaminants in four such systems: Qiagen MinElute PCR Purification, Zymo Research DNA Clean and Concentrator-5, Invitrogen ChargeSwitch-Pro PCR Purification, and Beckman Coulter AMPure XP. HS-AFM avoids amplification artifacts present in PCR or in the sequencing of amplified products, and it requires no chemical labels and easily achieves near-single-molecule sensitivity. Using this technique, we found trace levels of filamentous contamination, similar in appearance to dsDNA, in eluates from the Zymo, Qiagen, and ChargeSwitch kits. Conversely, we detected no contaminants in magnetic bead-based AMPure XP solutions. Eluates from the Zymo kits also tested positive for DNA in fluorescent intercalator dye and whole genome amplification (WGA) assays. Qiagen kits tested positive in the fluorescence assay but negative in the WGA assay. Both ChargeSwitch and AMPure XP tested negative in the fluorescence assay while the WGA results for these two kits were ambiguous. Taken together, our findings suggest AMPure XP would be the best choice for analyses requiring very high analytical stringency. While HS-AFM alone does not provide chemical specificity, it is a potentially valuable tool for characterizing and quantifying trace contaminants in molecular biology reagents and instruments in cases where conventional techniques fail.
authors.affiliation: Department of Physics, Virginia Commonwealth University , Richmond, Virginia 23284, United States.
authors.forename: Andrey
authors.initials: A
authors.lastname: Mikheikin
authors.affiliation: Department of Physics, Virginia Commonwealth University , Richmond, Virginia 23284, United States.
authors.forename: Anita
authors.initials: A
authors.lastname: Olsen
authors.affiliation: Interface Analysis Centre, H. H. Wills Physics Laboratory, University of Bristol , Tyndall Avenue, Bristol, BS8 1TL, United Kingdom.
authors.forename: Loren
authors.initials: L
authors.lastname: Picco
authors.affiliation: Interface Analysis Centre, H. H. Wills Physics Laboratory, University of Bristol , Tyndall Avenue, Bristol, BS8 1TL, United Kingdom.
authors.forename: Oliver
authors.initials: O
authors.lastname: Payton
authors.affiliation: Departments of Computer Science and Mathematics, Courant Institute of Mathematical Sciences, New York University , New York, New York 10012, United States.
authors.forename: Bud
authors.initials: B
authors.lastname: Mishra
authors.affiliation: Department of Chemistry and Biochemistry, UCLA , Los Angeles, California 90095, United States.|California NanoSystems Institute (CNSI) at the University of California, Los Angeles , Los Angeles, California 90095, United States.
authors.forename: James K
authors.initials: JK
authors.lastname: Gimzewski
authors.affiliation: Department of Physics, Virginia Commonwealth University , Richmond, Virginia 23284, United States.|VCU Massey Cancer Center , Richmond, Virginia 23298, United States.
authors.forename: Jason
authors.initials: J
authors.lastname: Reed
mesh_terms: D004247:DNA / Q000032:analysis*; D059018:DNA Contamination; D018625:Microscopy, Atomic Force / Q000379:methods
chemical_list: D004247:DNA