Specific enrichment of prokaryotic DNA using a recombinant DNA-binding protein.
Targeted enrichment of DNA is often necessary for its detection and characterization in complex samples. We describe the development and application of the novel molecular tool for the specific enrichment of prokaryotic DNA. A fused protein comprising the DNA-binding subunit of the bacterial topoisomerase II, gyrase, was expressed, purified, and immobilized on magnetic particles. We demonstrated the specific affinity of the immobilized protein towards bacterial DNA and investigated its efficiency in the samples with high background of eukaryotic DNA. The reported approach allowed for the selective isolation and further detection of as few as 5 pg Staphylococcus aureus DNA from the sample with 4 × 10(6)-fold surplus of human DNA. This method is a promising approach for the preparation of such type of samples, for example in molecular diagnostics of sepsis.
Nanotechnology Unit, Fraunhofer Institute for Cell Therapy and Immunology IZI, Perlickstrasse 1, 04103, Leipzig, Germany, natalia.sandetskaya@izi.fraunhofer.de.
Natalia
N
Sandetskaya
Andreas
A
Naumann
Katharina
K
Hennig
Dirk
D
Kuhlmeier
D001665:Binding Sites; D002623:Chemistry Techniques, Analytical; D004247:DNA / Q000032:analysis* / Q000737:chemistry; D027081:DNA Gyrase / Q000737:chemistry; D004269:DNA, Bacterial / Q000032:analysis*; D004268:DNA-Binding Proteins / Q000737:chemistry*; D004926:Escherichia coli / Q000378:metabolism; D006801:Humans; D008280:Magnetics; D016133:Polymerase Chain Reaction; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry; D018805:Sepsis / Q000175:diagnosis / Q000382:microbiology; D013211:Staphylococcus aureus / Q000378:metabolism*
D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D011994:Recombinant Proteins; D004247:DNA; D027081:DNA Gyrase