Optimized Real-Time Quantitative PCR Measurement of Male Fetal DNA in Maternal Plasma

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 meth...

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Published in:Clinical chemistry (Baltimore, Md.) Md.), 2005-09, Vol.51 (9), p.1598-1604
Main Authors: Zimmermann, Bernhard, El-Sheikhah, Ahmad, Nicolaides, Kypros, Holzgreve, Wolfgang, Hahn, Sinuhe
Format: Article
Language:English
Subjects:
Analytical, structural and metabolic biochemistry
Biological and medical sciences
Chromosomes, Human, Y - genetics
DNA - blood
Female
Fetus - metabolism
Fundamental and applied biological sciences. Psychology
Gene Dosage
Genomes
Gestational Age
Humans
Investigative techniques, diagnostic techniques (general aspects)
Male
Medical sciences
Plasma
Polymerase Chain Reaction
Preeclampsia
Pregnancy
Prenatal Diagnosis - methods
Regression analysis
Sensitivity and Specificity
Sex Determination Analysis
Y chromosomes
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Summary: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. 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. 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. 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.
ISSN:0009-9147
1530-8561
DOI:10.1373/clinchem.2005.051235