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Optimized Real‐Time Quantitative PCR Measurement of Male Fetal DNA in Maternal Plasma
Author(s) -
ZIMMERMANN BERNHARD G.,
HOLZGREVE WOLFGANG,
AVENT NEIL,
HAHN SINUHE
Publication year - 2006
Publication title -
annals of the new york academy of sciences
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.712
H-Index - 248
eISSN - 1749-6632
pISSN - 0077-8923
DOI - 10.1196/annals.1368.047
Subject(s) - real time polymerase chain reaction , fetus , andrology , dna , obstetrics , computational biology , medicine , biology , pregnancy , genetics , gene
DNA of fetal origin is present in the plasma of pregnant women. The quantitative measurement of circulatory fetal DNA (cfDNA) by real‐time quantitative PCR (qPCR) has been applied to investigate a possible correlation between increased levels and pregnancy‐related disorders. However, as the levels of cfDNA are close to the detection limit (LOD) of the method used, the measurements may not be reliable. This is also problematic for the evaluation of preanalytical steps, such as DNA extraction and cfDNA enrichment by size separation. We optimized a protocol for the qPCR analysis of the multi‐copy sequence DYS14 on the Y chromosome. This was compared with an established assay for the single‐copy SRY gene. Probit regression analysis showed that the limit of detection (LOD) of the DYS14 assay, (0.4 genome equivalents (GE)) and limit of quantification (LOQ) were 10‐fold lower in comparison to SRY (4 GE). The levels of cfDNA obtained from the first trimester of pregnancy could be quantified with high precision by the DYS14 assay (CV below 25%) as opposed to the SRY measurements (26–140%). Additionally, fetal sex was correctly determined in all instances. The low copy numbers of fetal DNA in plasma of women in the first trimester of pregnancy can be measured reliably, targeting the DYS14 that is present in multiple copies per Y chromosome.