Effective, scalable chemistry for purification of circulating, cell-free DNA (ccfDNA) from plasma with less quantitation method bias

The interest in the use of circulating cell-free DNA (ccfDNA) as a tool in molecular oncology research is driving efforts to create purification methods that are efficient, rapid, and reproducible. The diluted, highly... [ view full abstract ]

The interest in the use of circulating cell-free DNA (ccfDNA) as a tool in molecular oncology research is driving efforts to create purification methods that are efficient, rapid, and reproducible. The diluted, highly fragmented, and transient nature of ccfDNA can pose challenges to researchers. Furthermore, co-purification of full-length genomic DNA contamination increases the wild-type DNA in the eluate, making it more difficult to detect the mutations of interest. To address these challenges we developed a novel purification method utilizing a cellulose-based surface and a new DNA binding chemistry. The chemistry was optimized to reduce binding of longer fragments while increasing affinity for small DNA fragments, thus significantly reducing the gDNA contamination of the isolated ccfDNA.  We have also developed a multiplexed qPCR assay designed to include small and large amplicon sizes (75and 300bp); quantitative differences between the amplicon sizes were calculated as a ratio to determine the level of gDNA contamination of ccfDNA samples. Since gDNA is expected to be larger in size than the expected 170bp population, the ratio of 75bp to 300bp targets can help predict the ratio of ccfDNA to gDNA.  ccfDNA samples with low 75/300bp ratios are indicative of gDNA contamination.  Testing of ccfDNA samples showed high 75/300bp ratios when the 170bp population was prevalent.  For NGS library preparation of ccfDNA samples, reducing losses of already limited DNA input levels is critical.  For that purpose, we have also developed a magnetic bead-based size-selection reagent set that significantly improves DNA recovery from cleanup or size-selection steps while increasing reproducibility and reducing pipetting errors due to current method buffer viscosity issues.