Single Cell DNA Methylation Analysis via On-Chip Whole Genome Amplification and Bisulfite Treatment

H.C. Tian, J.J. Benitez, H.G. Craighead
Cornell University,
United States

Keywords: Single Cell, Microfluidics, Epigenetics, Methylation

Summary:

Single cell analysis allows for the study of genetic and epigenetic aberrations and their relation to human disease. Such information is lost when taking the average over a heterogeneous population of cells using bulk analysis methods. Here, we describe the use of a polydimethylsiloxane (PDMS) microfluidic device for single cell capture, lysis, DNA extraction, and on-chip chemistries including DNA amplification and sodium bisulfite treatment. A multitude of cells are first introduced into the microfluidic device via hydrodynamic flow. Within the device, separate channels each containing a single capture site physically entraps one cell while excess cells are flowed through the device. Cells are lysed within the capture region and the genomic DNA is subsequently trapped downstream upon an array of micropillars while RNA and cellular debris is washed away. The entangled DNA is then isothermally amplified with molecular displacement amplification and bisulfite converted via sodium bisulfite treatment. The original template DNA is not consumed or destroyed through the DNA amplification process thereby allowing for multiply rounds of amplification. Using qPCR, various gene loci are then amplified off-chip and the methylation profile of the genes are analyzed through sequencing. By comparing the DNA sequence of the single cells from their pre-bisulfite treated state versus their post-bisulfite treated state, single base resolution DNA methylation information can be derived. This technique is immediately applicable towards studying methylation within tumor cell heterogeneity. Therefore, the described microfluidic device is capable of trapping single cells, lysing the cells, capturing the DNA, multiple rounds of on-chip isothermal amplification, and sodium bisulfite treatment for applications in epigenetic analysis of cancer cells.