Specific enrichment of prokaryotic DNA using a recombinant DNA-binding protein

• Published in: Analytical and bioanalytical chemistry Vol. 406; no. 15; pp. 3755 - 3762
• Main Authors: Sandetskaya, Natalia, Naumann, Andreas, Hennig, Katharina, Kuhlmeier, Dirk
• Format: Journal Article
• Language: English
• Published: Berlin/Heidelberg Springer Berlin Heidelberg 01.06.2014; Springer; Springer Nature B.V
• Subjects: Analytical Chemistry; Bacteria; Binding Sites; Biochemistry; Characterization and Evaluation of Materials; Chemistry; Chemistry and Materials Science; Chemistry Techniques, Analytical; Deoxyribonucleic acid; DNA - analysis; DNA - chemistry; DNA binding proteins; DNA Gyrase - chemistry; DNA, Bacterial - analysis; DNA-Binding Proteins - chemistry; E coli; Enrichment; Enzymes; Escherichia coli - metabolism; Food Science; Humans; Laboratory Medicine; Magnetics; Mathematical analysis; Monitoring/Environmental Analysis; Physiological aspects; Polymerase Chain Reaction; Protein Binding; Protein research; Protein-protein interactions; Proteins; Recombinant; Recombinant proteins; Recombinant Proteins - chemistry; Research Paper; Sepsis; Sepsis - diagnosis; Sepsis - microbiology; Staphylococcus aureus; Staphylococcus aureus - metabolism; Toxicology; Germany; Bacteria detection; Gyrase; Bacterial DNA; Target enrichment
• ISSN: 1618-2642; 1618-2650
• DOI: 10.1007/s00216-014-7787-7

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.