Optimized short digestion protocol for free fetal DNA detection using methylation-dependent markers

Abstract Background: Circulating free DNA (cfDNA) digestion with methylation sensitive restriction enzymes constitutes an important diagnostic tool for differentiating methylated from non-methylated DNA sequences. In the context of pregnancy, this is used to differentiate fetal from maternal DNA. Current protocols are of long duration and use multiple enzymes with different incubation and inactivating temperatures. We describe a short protocol for the digestion of circulating free DNA focused on its future adaptation to miniaturized microfluidic devices based on lab-on-a-chip technology. Methods: cfDNA was extracted from plasma samples of pregnant and non-pregnant women with chemagic Viral NA/gDNA and QIAamp circulating nucleic acids kits. For digestion protocol optimization, different methylation sensitive and insensitive restriction enzymes were used. Detection of RASSF1A, SRY and (β-actin) ACTB sequences was performed by real time polymerase chain reaction (PCR). Results: The digestion protocol is optimized to a 3.5 h one-step protocol using the enzymes BstUI, BstY1 and HhaI resulting in a complete digestion of the hypomethylated maternal RASSF1A with a limit of digestion of 3.65E10 gene copies. Conclusions: This work provides a digestion protocol for cfDNA samples with a combination of temperatures (37 °C and 60 °C) and a protocol length (<4 h) which facilitates its adaptation to miniaturized microfluidic devices based on lab-on-a-chip technology. In this technology, the shorter the duration of the protocol, the greater the rate of success and the less sample evaporation.