Extensive studies have demonstrated that DNA methylation has played a major role in many biological procedures, such as cellular proliferation and differentiation. Bisulfite genomic sequencing is regarded as the gold-standard technology for analyzing DNA methylation. This technology is based on the different consequences after cytosine and 5-methycytosine (5-mC) treated with sodium bisulfate. The cytosine in DNA will be converted to uracil residues and recognized as thymine in subsequent PCR amplification and sequencing. On the other hand, the 5-mC will remain the same so that it can be distinguished from cytosine.
One of the major concerns of bisulfite conversion is the degradation of DNA. Under known conditions, at least 80% (up to 98%) of the DNA is degraded due to the non-specific degradation under acidic conditions and more significant losses are found to occur during the subsequent removal of bisulfite and desulphonation steps. To obtain decent sequencing coverage in the whole genome, hundreds of nanograms (up to 2 μg) input DNA is needed. This results in huge problem when dealing with rare samples, such as blood sample and embryo. In addition, the bisulfite conversion usually takes 4~16 hours in traditional protocols and commercial available kits. The long incubation time results in high conversion rate, but also significantly affects the integrity of DNA. To reduce the amount of DNA needed, we developed a diffusion-based microfluidic chip to minimize sample loss by decreasing reaction time without compromising conversion rate. In addition, the diffusion strategy eliminates the need for column based DNA purification which improves the sensitivity of the assay. This strategy will be potentially used for reduced representative bisulfite sequencing (RRBS) and whole genome bisulfite sequencing (WGBS) with limited DNA samples.
In this work, we demonstrate a simple scheme for conducting multi-step DNA bisulfite conversion, including denaturation, sulphonation, desalting, desulphonation and elution. To the best of our knowledge, this is the first demonstration of DNA bisulfite conversion on a microfluidic platform. Our microfluidic device has a simple structure that includes a reaction chamber connected with two loading chambers on both sides. The connections between the reaction chamber and the two loading chambers could be cut off by closing two-layer valves. This simple microfluidic platform allows the replacement of small molecules inside the reaction chamber by diffusion from/into the loading chambers under the concentration gradient, while preserve the large DNAs inside throughout such process. This device eliminates the need of column-based purification which results in higher DNA recovery and better DNA integrity. The diffusion scheme also increases the concentration of bisulfite mix which shorten the reaction time from 4~16 hours to 2 hours. The single chamber (“one-pot”) design drastically minimizes the complexity of the microfluidic device and improves the performance of DNA bisulfite conversion.
See more of this Group/Topical: 2015 Annual Meeting of the AES Electrophoresis Society