LOOXSTER
®
Enrichment Kit 11/2013
5
Principle and Applications
The enrichment effect of LOOXSTER
®
is achieved by means of the specific affinity of
the LOOXSTER
®
-protein, a derivative of the human CGBP-protein, for non-methylated
CpG-dinucleotides. Bacterial and fungal genomic DNA contains unmethylated CpG-
dinucleotides or shows only a low level of CpG methylation. On the other hand, it was
shown that in mammals between 60% and 90% of all CpG-dinucleotides are methylated.
For bacterial and fungal DNA enrichment, DNA-extracts containing a mixture of
methylated host-DNA and minute amounts of double stranded genomic DNA from
bacteria or fungi, were incubated under stringent buffer conditions with
LOOXSTER
®
-protein decorated paramagnetic particles. Under these conditions the
CXXC-domain of the LOOXSTER
®
-protein forms a stable complex with non-methylated
CpG-dinucleotides (Dissociation constant: Kd
≈
2.7µM
1
). Unbound methylated DNA can
be removed from the sample by means of stringent washing step. Subsequently, the
protein-DNA complex is solved in presence of an elution buffer and the enriched
bacterial or fungal DNA can be harvested from the LOOXSTER
®
-particles. After
performing a final solid-phase extraction step the desalted and concentrated bacterial or
fungal DNA can be applied to downstream analytical and preparative applications.
All components have been treated to reduce the risk of contaminations with bacterial
and fungal DNA.
Schematic representation of the
LOOXSTER
®
-principle:
Total-DNA
consisting of host DNA and minute
amounts of bacterial or fungal was
isolated from human whole-blood
and applied to LOOXSTER
®
.
The specific affinity of LOOXSTER
®
for DNA molecules originating from
bacteria and fungi leads to their
enrichment
relative
to
the
background of host DNA.
