Detection of increased amounts of cell-free fetal DNA with short PCR amplicons.
OBJECTIVE
A digital PCR approach has recently been suggested to detect greater amounts of cell-free fetal DNA in maternal plasma than conventional real-time quantitative PCR (qPCR). Because the digital qPCR approach uses shorter PCR amplicons than the real-time qPCR assay, we investigated whether a real-time qPCR assay appropriately modified for such short amplicons would improve the detection of cell-free fetal DNA.


METHODS
We developed a novel universal-template (UT) real-time qPCR assay that was specific for the DYS14 sequence on Y chromosome and had a short amplicon size of 50 bp. We examined this "short" assay with 50 maternal plasma samples and compared the results with those for a conventional real-time qPCR assay of the same locus but with a longer amplicon (84 bp).


RESULTS
Qualitatively, both assays detected male cell-free fetal DNA with the same specificity and detection capability. Quantitatively, however, the new UT real-time qPCR assay for shorter amplicons detected, on average, almost 1.6-fold more cell-free fetal DNA than the conventional real-time qPCR assay with longer amplicons.


CONCLUSIONS
The use of short PCR amplicons improves the detection of cell-free fetal DNA. This feature may prove useful in attempts to detect cell-free fetal DNA under conditions in which the amount of template is low, such as in samples obtained early in pregnancy.
Sikora
Aleksandra
A
Department of Biomedicine, University Women's Hospital, Basel, Switzerland.
Zimmermann
Bernhard G
BG
Rusterholz
Corinne
C
Birri
Daniella
D
Kolla
Varaprasad
V
Lapaire
Olav
O
Hoesli
Irene
I
Kiefer
Vivian
V
Jackson
Laird
L
Hahn
Sinuhe
S
D004247:DNA / Q000097:blood*; D005260:Female; D005333:Fetus; D006801:Humans; D008297:Male; D008431:Maternal-Fetal Exchange; D016133:Polymerase Chain Reaction / Q000379:methods*; D011247:Pregnancy; D012189:Retrospective Studies
D004247:DNA