Analysis of DNA Methylation in BRCA2 Gene Using Electrode Biochips

Overview

Background:
Since DNA methylation results in gene silencing, it plays an important role in cell proliferation and differentiation. Abnormal DNA methylation is often associated with carcinogenesis, and therefore its analysis could be an interesting option in early diagnostics of cancer, determination of tumour invasiveness, progression or metastatic potential. One of the methods for DNA analysis could be also electrochemistry, which offers inexpensive instrumentation, short measurement times and parallel detection of samples.

Material and Methods:
We have analyzed methylation status of a DNA site within BRCA2 gene promoter sequence, which is associated with breast cancer. We hybridized this sequence at magnetic beads and then we incubated them with methylation-sensitive restriction endonuclease BstUI, which specifically cleaves non-methylated restriction site CGCG, but not methylated one. After addition of labeled DNA probe and peroxidase, we monitored enzymatic reaction at the electrode chip.

Results:
First, we tested suitability of designed DNA probes and effectiveness of BstUI cleavage using gel electrophoresis. Afterwards, we applied these probes and the enzyme to the magnetic beads, with the final detection at the electrode chip. We show good correlation of electrochemical and electrophoresis results, i.e. that labelled DNA probe was removed only when DNA containing non-methylated restriction site was incubated with BstUI. When this restriction site was methylated, no cleavage occurred and the resulting signal was high. We also show good reproducibility of the measurement.

Conclusion:
Electrochemical detection, in combination with magnetic beads and restriction enzymes, could be a potentially useful tool for determination of methylation status of a specific DNA sequence.

Key words:
DNA methylation – DNA restriction enzymes – electrochemistry – nucleic acid hybridization

This work was supported by MEYS – NPS I – LO1413.

The authors declare they have no potential conflicts of interest concerning drugs, products, or services used in the study.

The Editorial Board declares that the manuscript met the ICMJE recommendation for biomedical papers.

Submitted:
6. 3. 2017

Accepted:
26. 3. 2017