RESUMEN
OBJECTIVE: The generally accepted method of quantifying hypermethylated DNA by qPCR using methylation-specific primers has the risk of underestimating DNA methylation and requires data normalization. This makes the analysis complicated and less reliable. METHODS: The end-point PCR method, called qDMA-HP (for quantitative DNA Melting Analysis with hybridization probes), which excludes the normalization procedure, is multiplexed and quantitative, has been proposed. qDMA-HP is characterized by the following features: (i) asymmetric PCR with methylation-independent primers; (ii) fluorescent dual-labeled, self-quenched probes (commonly known as TaqMan probes) covering several interrogated CpGs; (iii) post-PCR melting analysis of amplicon/probe hybrids; (iv) quantitation of unmethylated and methylated DNA alleles by measuring the areas under the corresponding melt peaks. RESULTS: qDMA-HP was tested in liquid biopsy of colorectal cancer by evaluating SEPT9 and HIST1H4F methylations simultaneously in the single-tube reaction. Differences in the methylation levels in healthy donors versus cancer patients were statistically significant (p < 0.0001), AUCROC values were 0.795-0.921 for various marker combinations. CONCLUSIONS: This proof-of-concept study shows that qDMA-HP is a simple, normalization-independent, quantitative, multiplex and "closed tube" method easily adapted to clinical settings. It is demonstrated, for the first time, that HIST1H4F is a perspective marker for liquid biopsy of colorectal cancer.