This article describes a method for analyzing the size distribution of cell-free DNA (cfDNA) fragments released by dying cells using a multiwell DNA chip. The process involves loading a gel matrix and cfDNA samples into the chip, followed by electrophoretic separation based on fragment size.
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.
To analyze the size of the DNA fragments, first, secure the base plate to the chip-priming station and adjust the clip at the lowest position. Place a new High Sensitivity DNA chip onto the chip priming station and add 9 microliters of gel-dye mix to the bottom of the 'G' chip well. Position the plunger at 1 milliliter and close the chip priming station. Close the lock of the latch until it clicks, set the timer to 60 seconds, and press down the plunger until it is held by the clip.
After exactly 60 seconds, use the clip-release mechanism to release the plunger. When the plunger retreats to at least the 300-microliter mark, wait for 5 seconds before slowly retracting the plunger to the 1-milliliter position. Open the chip-priming station to remove 9 microliters of the gel-dye mix and transfer the gel to the bottom of the High Sensitivity DNA chip 'G' well.
To load the DNA marker, dispense 5 microliters of DNA marker into the ladder well and to the 11 sample wells. To load the ladder and samples, add 1 microliter of the DNA ladder to the DNA ladder well, 1 microliter of sample to the used sample wells, and 1 microliter of marker to the unused sample wells. Place the High Sensitivity DNA chip horizontally in the adapter for 60 seconds of vortexing at 2,400 revolutions per minute, taking care that the bulge that fixes the High Sensitivity DNA chip is not damaged.
After vortexing, confirm that the electrode cartridge is properly inserted and that the chip selector is positioned to double-stranded High Sensitivity DNA. Carefully mount the High Sensitivity DNA chip into the receptacle and confirm that the electrode cartridge is fit exactly into the wells of the chip before closing the lid. The fragment analyzer software screen will display a chip icon to indicate that the chip has been inserted and the lid has been closed.
To initiate the analysis, open the assay menu and select the double-stranded DNA High Sensitivity Assay. Enter the sample names into the table and click "Start" to initiate the chip run. At the end of the run, immediately remove and discard the chip according to good laboratory practice.
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