Detection of cell‐free fetal DNA in maternal plasma using two types of compound markers

Abstract With the discovery of circulating cell‐free fetal DNA (cffDNA) in maternal plasma, noninvasive prenatal testing became possible. However, analysis of low‐level cffDNA against high background maternal DNA remains complicated and challenging. To circumvent this limitation, selective amplification of cffDNA was used in this study. Two kinds of compound markers (namely DIP‐STR and SNP‐STR), both based on selective amplification, were used here for targeting fetal DNA. By designing two allele‐specific forward primers for DIP‐STR and SNP‐STR, DNA fragments with different DIP/SNP alleles can be selectively amplified. When analyzing maternal plasma DNA, these markers can selectively target paternally inherited fetal alleles whose DIP/SNP allele was not shared with the mother. In this study, 21 families were studied with six DIP‐STRs and 11 SNP‐STRs. Fetal DNA was successfully detected across plasma samples for at least one marker. Detection rate varied between DIP‐STR and SNP‐STR markers, and DIP‐STR outperforms SNP‐STR. Fetal alleles obtained from maternal plasma were double confirmed by genotyping paternal genomic DNA and fetal genomic DNA from amniocentesis. This study demonstrated that selective amplification strategy can be used to target cffDNA in maternal plasma, which will be a promising method for noninvasive prenatal paternity testing.