DNA is found primarily in the cell nucleus, where it exists in a linear form, in protein complexes. To study this nuclear DNA, cells must be broken open, and the DNA purified. However, DNA also circulates in the bloodstream outside of cells, known as cell-free DNA (cfDNA). This cfDNA can be easily collected through plasma separation without the need for cell lysis.

Cell-free DNA originates from apoptotic (programmed cell death) processes occurring throughout the body. During apoptosis, genomic DNA is fragmented and released into the bloodstream. Various cell types, including fat cells (adipocytes), placental cells, tumor cells, and transplanted cells, contribute to cfDNA when they undergo apoptosis. Although “foreign” cfDNA makes up a small fraction of the total cfDNA in the blood, it can be analyzed to detect placental aneuploidy, cancer-specific variants, and conditions like graft-versus-host disease (GvHD).

This technique uses allele-specific probes for targeted detection of pathogenic variants, particularly those on the Y chromosome. It can also detect duplications or deletions through relative quantification.

ddPCR involves massive parallel partitioning a PCR mixture into thousands of tiny oil droplets, effectively diluting the starting material. This method offers high sensitivity and enables absolute quantification of cfDNA. It can distinguish between maternal and placental cfDNA and is used to detect point mutations, copy number variations (CNVs), loss of heterozygosity, and aneuploidy.

Shotgun sequencing
Shotgun sequencing, like ddPCR, can detect point mutations, CNVs, loss of heterozygosity, and aneuploidy in cfDNA. It also allows for whole-genome sequencing of the placental genome, making it a powerful tool for comprehensive analysis.

MALDI-TOF MS can be used to sequence point mutations in cfDNA. In this process, an oligonucleotide binds just before the site of the point mutation and is extended by a single nucleotide (single nucleotide sequencing). Differences in mass allow for differentiation between homozygous and heterozygous base pair positions.