Optimized real-time quantitative PCR measurement of male fetal DNA in maternal plasma.
BACKGROUND
Circulating fetal DNA (cfDNA) in maternal plasma has been measured to investigate its possible relationship with pregnancy-related disorders, including fetal trisomy 21 and preeclampsia. The circulating concentrations of single-copy fetal genes, however, are close to the detection limits of PCR methods.


METHODS
We optimized a protocol for the real-time quantitative PCR amplification of the multicopy sequence DYS14 on the Y-chromosome. This was compared with an established real-time PCR assay for the single-copy SRY gene.


RESULTS
By probit regression analysis, the measurements of male DNA by the DYS14 assay had a 10-fold lower detection limit (0.4 genome equivalents) than did measurements of SRY. For plasma samples from women in the first trimester of pregnancy, imprecision (CV) was 2%-22% when amplifying DYS14 compared with 26%-140% for SRY.


CONCLUSIONS
The low copy numbers of fetal DNA in plasma of women in the first trimester of pregnancy cannot be measured precisely when targeting single-copy sequences. Better results are obtained by amplifying a sequence that is present in multiple copies per male genome.
Zimmermann
Bernhard
B
University Women's Hospital/Department of Research, University Hospital Basel, Basel, Switzerland. Bernhard.Zimmermann@uwe.ac.uk
El-Sheikhah
Ahmad
A
Nicolaides
Kypros
K
Holzgreve
Wolfgang
W
Hahn
Sinuhe
S
D041322:Chromosomes, Human, Y / Q000235:genetics; D004247:DNA / Q000097:blood*; D005260:Female; D005333:Fetus / Q000378:metabolism*; D018628:Gene Dosage; D005865:Gestational Age; D006801:Humans; D008297:Male; D016133:Polymerase Chain Reaction; D011247:Pregnancy; D011296:Prenatal Diagnosis / Q000379:methods; D012680:Sensitivity and Specificity; D012732:Sex Determination Analysis
D004247:DNA