Prenatal diagnosis of genetic disorders has traditionally relied on invasive procedures such as amniocentesis and chorionic villus sampling (CVS). As these procedures carry a small but significant risk of pregnancy loss, a convenient noninvasive prenatal diagnostic has long been sought. Although fetal cells circulating in the maternal blood have been used for prenatal screening, because of their rarity they require ∼104- to 105-fold enrichment to be detectable (1). Recently, extracellular fetal DNA has been detected in maternal circulation, suggesting that maternal blood might be a useful source of material for noninvasive prenatal diagnosis (2)(3).
To quantify fetal DNA in maternal circulation, we established a quantitative, homogeneous TaqMan PCR assay based on fluorogenic probes (4). This technique involves continuous monitoring of the progress of amplification and permits target quantification. Such analytical characteristics as precision, sensitivity, and dynamic range were analyzed using human genomic DNA as template. The reproducibility of DNA extraction and PCR amplification was also determined using as target cell-free DNA extracted from pooled plasma. This assay was then tested by determining the genders of adult donors by measuring cell-free DNA in their plasma. Finally, using the male-specific SRY gene as a marker for male fetuses, we successfully determined the gender of all fetuses tested.
We collected blood samples from 38 healthy adult volunteers in EDTA. For the fetal DNA study, following approval by the Perinatal Research Committee and the Research Subjects Review Board at the University of Rochester, 30 pregnant patients were enrolled in a prospective trial with masking of those performing all tests. Maternal blood samples were collected before clinically indicated amniocentesis or CVS. Plasma was isolated immediately by centrifugation twice at 1600g for 10 min, and then stored at −80 °C. Fetal gender was known from the karyotype, which was withheld from the molecular laboratory.
We processed 2 mL of plasma from healthy adult donors and 5 mL of plasma from pregnant women. Free circulating DNA was extracted by use of the QIAamp DNA Blood Midi method (Qiagen Inc.). DNA was eluted from the column into 250 μL of AE buffer. Triplicate 5-μL aliquots were amplified in each PCR reaction.
Amplification was monitored in real-time on a PE ABI 7700 Sequence Detector using the primers and probes below (3):
SRY forward primer: 5′-TGGCGATTAAGTCAAATTCGC-3′
SRY reverse primer: 5′-CCCCCTAGTACCCTGACAATGTATT-3′
SRY TaqMan probe: 5′-(VIC)TCTGCCTCCCTGACTGCTCTACTGCT (TAMRA)-3′
β-actin forward primer: 5′-TCACCCACACTGTGCCCATCTACGA-3′
β-actin reverse primer: 5′-CAGCGGAACCGCTCATTGCCAATGG-3′
β-actin TaqMan probe: 5′-(FAM)ATGCCC-X(TAMRA)-CCCCCATGCCATCCTGCGT-3′
VIC™ is a proprietary fluor developed by P.E. Corporation, TAMRA is 6-carboxytetramethylrhodamine, FAM is 6-carboxyfluorescein, and X represents the base to which TAMRA is linked.
A typical amplification reaction occurred in a volume of 50 μL containing 5 μL of 10× reaction buffer, 300 nM each of the forward and reverse primers, 200 nM TaqMan probe, 3.5 mM MgCl2, 200 μM each of dATP, dGTP and dCTP, 400 μM dUTP, 0.5 U of AmpErase uracil N-glycosylase, and 1.25 U of AmpliTaq Gold. Thermal cycling was initiated with a hold at 50 °C for 2 min and a hold at 95 °C for 10 min, followed by 45 cycles of 95 °C for 15 s and 60 °C for 60 s.
We subjected 36 replicates of each of two unknown samples, differing twofold in concentration, along with human male genomic DNA to PCR amplification with the β-actin primer/probe set. The resolution of such real-time monitoring was one PCR cycle, which produced an approximate doubling of amplicon concentration. Both samples exhibited a CV of ∼1% for the threshold cycle (Ct). Because of the logarithmic relationship between Ct and copy number, CV values for copy number were higher than those for Ct. The assay distinguished twofold concentration differences (P <0.025, Student’s t).
To determine assay sensitivity, human male genomic DNA was serially diluted and tested for the β-actin and SRY genes. Both assays produced log-linear calibration curves (R2 = 0.98 and 0.99, respectively). The numbers of PCR cycles required to detect one copy of target, as indicated by the y-intercepts, were 36.1 and 38.4 for β-actin and SRY, respectively. That it took 2.3 more cycles to detect one copy of SRY than β-actin may be attributable not only to differences in PCR efficiency but also to the existence of multiple detectable β-actin pseudogenes, each making a separate contribution to the signal. To determine dynamic range, a synthetic oligonucleotide was used as target because it could be varied over a much greater concentration range than human genomic DNA. The resulting calibration line was linear over seven orders of magnitude.
Pooled plasma from 38 adult donors was divided into 18 aliquots of 2 mL each. Free circulating DNA in the pooled plasma was independently extracted from the 18 aliquots with a Qiagen reagent set. Each extract was subdivided into 4 replicates for a total of 72 PCR reactions. β-actin was amplified in each reaction. A two-way ANOVA for extraction and amplification showed that these two processes produced a total CV for Ct of 2.2%. Of this, extraction contributed 1.7% (60% of total variance) and amplification 1.4% (40% of total variance). Data analyzed in terms of copy number exhibited higher CV values; however, each process contributed approximately the same “relative” fraction of total variance as when analyzed in relation to Ct. Mean copy number was 50 copies per well, equivalent to ∼1250 copies of free circulating DNA per mL of plasma.
Three sets of serially diluted human male genomic DNA were amplified for β-actin or SRY, either singly or together in multiplexed mode. Calibration lines were virtually identical irrespective as to whether SRY was amplified alone or with β-actin in the PCR, and vice versa, indicating that the two targets do not mutually interfere. We nonetheless determined fetal gender from SRY gene amplified alone.
To assess the concentrations of free circulating DNA in plasma of adults as a test of the technique, 38 donors were recruited, and their plasma DNA was analyzed for the presence of the SRY gene. Cell-free DNA was isolated from 2 mL of plasma from each donor and eluted into a total of 250 μL of buffer. A 5-μL portion of this extract was then coamplified for β-actin and SRY. β-actin was used as an endogenous positive control. Donor gender was assessed based on the SRY signal. Genders of all 38 donors comprising 16 males and 22 females were correctly identified. The odds of doing so by random chance were 4.4 × 10−11. The concentrations of free circulating β-actin DNA were 220-3070 copies/mL of plasma (mean ∼1000 copies/mL). There was no particular correlation between number of copies of the β-actin and SRY genes (Fig. 1⇓ ). This may be attributable to differing amplification efficiencies but also because β-actin contains variable numbers of pseudogenes, which although not expressed do give detectable PCR signals.
We next recruited 30 pregnant women (age range, 21–42 years; median, 36.5 years; Table 1⇓ ). Gestational age ranged from 9.8 to 29.3 weeks (median, 16.9 weeks). Twenty-five patients underwent amniocentesis and 5 underwent CVS. Plasma (5 mL) from each patient was processed, and the DNA was resuspended in 250 μL of buffer. β-actin and SRY were amplified separately from 5-μL aliquots of extract. β-actin was derived from both mother and fetus, whereas SRY was from the fetus alone. Of the 30 patients, 19 were carrying a male fetus and 11 a female. Genders of all were correctly determined. The odds of doing so by chance were 1.8 × 10−8. The 95% confidence interval for sensitivity was 0.82–1.00. The amount of β-actin present in plasma samples varied from 400 to 2990 copies/mL (median, 835 copies/mL; Table 1⇓ ). The amount of SRY varied from 13.8 to 291 copies/mL (median, 94.4 copies/mL). The percentage of fetal DNA present in plasma samples in the 19 male pregnancies was 1.28–47.7% (median, 11.8%). Of the two cases with the greatest amounts of fetal DNA, one (29.2% fetal DNA) was noted at amniocentesis to involve trisomy 21. This increased amount of fetal DNA relative to the other samples and relative to the amount of fetal DNA reported previously (5) is interesting.
Reliable detection and quantification of free circulating fetal DNA from maternal plasma has potential implications for prenatal diagnosis. The greatest practical challenge in doing so, however, is to differentiate fetal from maternal DNA. At present, only paternally inherited genetic traits can be identified by analyzing fetal DNA from maternal plasma. These include gender, Y-chromosome-linked disorders, the RhD gene in RhD− women, and paternally inherited autosomal dominant disorders. To detect autosomal recessive genetic disorders involving different mutations (such as compound heterozygotes in cystic fibrosis), fetal DNA in maternal circulation could serve as a first line of noninvasive screening. Detection of a paternally inherited mutation could warrant an invasive procedure to isolate fetal cells for further testing. Finally, inclusion of free circulating fetal DNA with other maternal serum markers as variables in an algorithm might enable patient-specific risk evaluation for conditions such as preeclampsia and fetal Down syndrome.
This work was supported by the Corporate Office of Science and Technology, Johnson & Johnson, New Brunswick, NJ.
- © 2001 The American Association for Clinical Chemistry