Effect of DNA target size on the efficiency of chimerism measurement in circulating free plasma DNA

Introduction. The analysis of free circulating DNA (cfDNA) holds promise for molecular diagnostics, but its fragmentation and low concentration can complicate PCR analysis.

Objective. To investigate the effect of target length on the amplification efficiency of Y-chromosome markers from cfDNA.

Material and methods. Fifty cfDNA samples were obtained from 39 patients: patients after liver transplantation (n=19), patients with acute leukemia after allogeneic hematopoietic stem cell transplantation (n=10), and pregnant women (n=10). In addition, we prepared 16 chimeric samples by sequential dilution of male cfDNA into female cfDNA from healthy donors. We determined the proportion of male cfDNA using the Y-chromosome marker S02, which is 211 bp in length as suggested by M. Alizadeh et al. We also modified Alizadeh's primer design to obtain a DNA target with a length of 138 bp. The proportion of male cfDNA was also determined by fragment analysis using the amelogenin Y marker (84 bp) from the COrDIS Plus kit (Gordiz LLC, Russia).

Results. In the three groups of patients, amplification of male cfDNA was more efficient when shorter DNA targets were used (p<0.05). In artificially created ‘chimeras’ with a known ratio of male to female cfDNA, analysis of a marker of 84 bp in length gave values closest to the real ones.

Conclusions. In the quantitative models tested so far, shorter PCR targets are preferred for the analysis of cfDNA.

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