Assay deveolment for the detection of single-base mismatches in target DNA sequences via capture probe-target hybridization
ctDNA - a potential biomarker for liquid biposy
Circulating tumor DNA (ctDNA) is fragmented tumor-derived DNA released into the blood. As ctDNA reflects the entire tumor genome, it is a potential biomarker for liquid biopsy enabling the detection and monitoring of cancer from blood samples. The aim of this work is the development of an electrochemical POC testing system for detection of tumor-associated point mutations in circulating tumor DNA.
Design of an assay for detection of point mutations
Using microarray technology, a hybridization-based assay for detection of single-base mismatches was developed. Immobilization of capture probes on gold surfaces as well as subsequent mutation-specific probe-target hybridization were optimized. Furthermore, the effect of the mismatch position within the probe and target DNA sequences on mutation-specific hybridization was investigated. The hybridization signal is expressed as ratio of the signal intesities of perfect match (PM) and mismatch (MM) hybridizations.
Detection of point mutations
With the designed microarray assay, highly stringent hybridization conditions could be defined that allow the successful detetection of three selected point mutations in the PIK3CA gene. Mutation detection is based on a significant reduction of the signal intensity of probe-target hybridization containing a mismatch compared to perfect match hybridizations.
The mutation specificity of probe-target hybridization is influenced by the mismatch position on the probe and target DNA sequences. Specificity is increased, if the mismatch position is shifted from the center of the 19-mer probe sequence to the 5‘ end. Furthermore, positioning of the mismatch at the 5‘ end of the target DNA sequence lead to higher specificity of probe-target hybridization.