Y chromosome detection by Real Time PCR and pyrophosphorolysis-activated polymerisation using free fetal DNA isolated from maternal plasma.
OBJECTIVE
To validate the use of Real Time PCR, a widely used technique that can detect very low levels of Y chromosomal sequence, and to assess the use of a highly sensitive PCR technique, pyrophosphorolysis-activated polymerisation (PAP), for fetal sex determination using free fetal DNA (ffDNA).


METHODS
The fetal sex was determined by Real Time PCR in 58 pregnancies using ffDNA isolated from maternal plasma. In parallel with the Real Time PCR experiments, the presence of Y chromosome sequence was also determined using PAP on 54 isolated ffDNA samples.


RESULTS
Both techniques detected Y chromosome sequence at very low levels with 98% specificity and 100% sensitivity (Real Time n = 44, PAP n = 54). Furthermore, the PAP technique was shown to be more robust than the Real Time PCR as none of the samples tested failed to meet the acceptance criteria. Combining the two techniques for male fetal sex detection from maternal blood plasma increases the sensitivity and specificity to 100% in this series.


CONCLUSIONS
This study shows that both Real Time PCR and PAP can be used for Y chromosome detection on ffDNA. Furthermore, by using PAP in combination with Real Time PCR more reliable early prenatal sexing can be performed using ffDNA.
Boon
Elles M J
EM
Leiden University Medical Center, Center for Human and Clinical Genetics, Leiden, The Netherlands.
Schlecht
Hélène B
HB
Martin
Peter
P
Daniels
Geoff
G
Vossen
Rolf H A M
RH
den Dunnen
Johan T
JT
Bakker
Bert
B
Elles
Rob
R
D041322:Chromosomes, Human, Y / Q000235:genetics*; D004247:DNA / Q000097:blood*; D017931:DNA Primers; D005260:Female; D005333:Fetus / Q000737:chemistry*; D006801:Humans; D007231:Infant, Newborn; D008297:Male; D008431:Maternal-Fetal Exchange; D016133:Polymerase Chain Reaction / Q000379:methods; D011237:Predictive Value of Tests; D011247:Pregnancy; D011296:Prenatal Diagnosis; D012680:Sensitivity and Specificity; D012732:Sex Determination Analysis / Q000379:methods*
D017931:DNA Primers; D004247:DNA