Separation of methylated CFDNA fraction with methyl miner method

Introduction: During the progression of colorectal cancer the circulating cell free DNA (cfDNA) level is increasing in the plasma from 10–20ng/ml to 100–200ng/ml or more. The examination of the plasma cfDNA can help us to understand the alteration of methylation profile like genereal hypomethylation and local hypermethylation in colorectal cancer.

Aims: Our aim has been separation, quantification and examination of the methylated and non-methylated cfDNA fractions from peripheral blood of cancer patients. Materials and methods: We have been isolating the cfDNA from plasma with QIAamp Circulating Nucleic Acid Kit (Qiagen) and performed optimised fragmentation with DNaseI nuclease enzyme. For DNA capturing methyl-CpG binding domain of human MBD2 protein was used, which is coupled to paramagnetic beads M-280 Streptavidin via a biotin linker (MethylMiner™ Methylated DNA Enrichment Kit, Invitrogen). Then the methylated fragments can be eluted based on the degree of methylation by increasing NaCl concentration. For the examination of the fractions PicoGreen and real-time PCR were applied by means of methylated and non-methylated artificial DNA control.

Results: We have used different DNaseI enzyme concentration and timescale to optimise DNA-fragmentation to get the ideal size (200–1000bp). The best result was shown with 50ng input DNA and 1.66U/µl Dnase enzym treatment. With real-time PCR we have established that elution with 750–850 mM NaCl concentration is the ideal to extract the methylated fraction. We have assessed that the 12.165±5% of cfDNA is methylated in patients with colorectal cancer. Conclusion: General hypomethylation and local hypermethylation is characteristic is tumours. To detect this changes from peripheral blood can be important step to understand the molecular communication and in the future can obtain diagnostic significance as well.