When transplant patients receive a new organ, it must be monitored for organ health, and the standard of care is typically an organ biopsy. Several studies have looked at using cfDNA biomarkers to monitor the health of the transplanted organ since a blood draw is less invasive than a biopsy; healthy organs release less cfDNA than failing organs and this can be measured by observing changes in the ratio of alleles at a site of genetic difference between the donor and host.
While cfDNA fragments tend to be 167 bp or multiples thereof (the size required to wrap around a histone), fragments from transplanted organs are smaller and are present within 80-120 bp fragment size range (Pedini et. al., 2023). Working with such small DNA fragments requires optimized methods to both capture and amplify the DNA. Traditional DNA extraction methods have a lower fragment size cutoff of ~50 bp, and yield is typically lower as fragment size gets closer to 50 bp. PCR reactions must also be optimized for smaller amplicon size in order to amply small size DNA.
This study will compare the recovery of different extraction methods in this size range. Differentiation of host and donor DNA can also be difficult; several studies have looked single nucleotide polymorphisms (SNPs) that vary across populations and may provide a site for differences in host and donor genomes. We utilized PCR assays designed by Kokelj et al. (2021) to evaluate the workflow's ability to capture and concentrate both spiked cfDNA and transplant patient cfDNA as a proof of concept for the detection of these biomarkers from donor organ plasma.
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