Automated Capillary Electrophoresis Based Fragment Size Distribution Analysis of cfDNA: A DNA Microchip-based Method to Study Size Distribution of DNA Fragments Isolated from Biofluids

A dying cell releases fragments of cell-free or cfDNA outside the cell. To analyze the size distribution of cfDNA fragments, begin with a multiwell DNA chip - a miniature capillary electrophoretic system. Each well in the chip contains numerous microfluidic separation channels at its base.

Load the polysaccharide gel matrix and fluorescence dye mix in the designated well on the chip. Position the chip on the priming assembly equipped with an empty syringe. Close the priming assembly and press the plunger down to create downward pressure. This facilitates the entry of the viscous gel from the well into the respective separation channel.

Open the assembly and fill the remaining wells with cfDNA suspension and a DNA ladder of the known size range. Mount the chip onto the fragment analyzer. The strong voltage drives the entry of cfDNA suspension from sample wells into the separation channel pre-loaded with gel matrix, where the DNA gets stained with the dye.

As the DNA moves from the cathode towards the anode, smaller DNA fragments migrate faster through the gel while the larger fragments move slower. This leads to size-based separation of DNA fragments. The detector detects the relative fluorescence from the fragments, compares it with the DNA ladder, and generates an electropherogram - a graph indicating the size distribution of cfDNA.