Magnetic bead-based solid phase for selective extraction of genomic DNA.
Magnetic bead-based solid phases are widely used for the separation of nucleic acids from complex mixtures. The challenge to selectively separate specific DNA molecules (via complementary hybridization) in a single step is the selection of a linker between the capture probe and the solid support that can be exposed to high temperatures in the presence of a high salt media. This article presents a general platform for the fabrication of a magnetic bead-based selective solid phase that can be used for subtractive hybridization or sequence capture applications. Phosphorus dendrimers are used for the first time as linkers in a magnetic bead-based selective solid phase for capture of genomic DNA. Aside from providing a high loading capacity, they render a stable bond between the capture probe and the surface under the high temperature and salt conditions required for denaturation and capture to proceed in a single step. The thermal stability of the solid phase under these conditions is first demonstrated by hybridizing a Cy3-labeled target. The selective capture of DNA targets in a single step is then demonstrated by subtractive hybridization of fragmented human genomic DNA. The specificity and selectivity of the solid phase are demonstrated by the recovery of adenovirus serotype 4 DNA spiked into the human DNA target. The effect of steric and electrostatic constraints was also investigated by using dendrimers of different generations that vary in their size and the number of branches. The results demonstrate that this platform can be used for single-step subtractive hybridization applications with better performance over the conventional two-step method using streptavidin-coated magnetic beads.
Archer
Marie J
MJ
Center for Biomolecular Science and Engineering, Naval Research Laboratory, Washington, DC 20375, USA.
Lin
Baochuan
B
Wang
Zheng
Z
Stenger
David A
DA
D000256:Adenoviridae / Q000235:genetics; D002232:Carbocyanines / Q000737:chemistry; D004247:DNA / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel; D005456:Fluorescent Dyes; D005821:Genetic Techniques; D016678:Genome; D006801:Humans; D008280:Magnetics; D009691:Nucleic Acid Denaturation; D009693:Nucleic Acid Hybridization; D016133:Polymerase Chain Reaction; D012492:Salts / Q000494:pharmacology; D017422:Sequence Analysis, DNA; D012703:Serotyping / Q000379:methods; D019809:Streptavidin / Q000737:chemistry; D013696:Temperature
D002232:Carbocyanines; D005456:Fluorescent Dyes; D012492:Salts; C089187:cyanine dye 3; D004247:DNA; D019809:Streptavidin