DNA AND CELL BIOLOGY
Volume 25, Number 11, 2006
© Mary Ann Liebert, Inc.
Pp. 635–640
Quantification of Fetal and Total Circulatory DNA in
Maternal Plasma Samples Before and After
Size Fractionation by Agarose Gel Electrophoresis
I. HROMADNIKOVA,1 L. ZEJSKOVA,1 J. DOUCHA,2 and D. CODL1
ABSTRACT
Fetal extracellular DNA is mainly derived from apoptotic bodies of trophoblast. Recent studies have shown
size differences between fetal and maternal extracellular DNA. We have examined the quantification of fetal
(SRY gene) and total (GLO gene) extracellular DNA in maternal plasma in different fractions (100–300,
300–500, 500–700, 700–900, and 900 bp) after size fractionation by agarose gel electrophoresis. DNA was
extracted from maternal plasma samples from 11 pregnant women carrying male foetuses at the 16th week
of gestation. Fetal circulatory DNA was mainly detected in the 100–300 bp fraction with the median concentration being 14.4 GE/ml. A lower median amount of 4.9 GE/ml was also found in the 300–500 bp fraction.
Circulatory DNA extracted from the 100–300 bp fraction contained 4.2 times enriched fetal DNA when compared with unseparated DNA sample. Fetal DNA within the 300–500 bp fraction was 2.5 times enriched. Circulatory fetal DNA is predominantly present in a fraction with molecular size 500 bp, which can be used
for the detection of paternally inherited alleles. However, the usage of size-separated DNA is not suitable for
routine clinical applications because of risk of contamination.
INTRODUCTION
T
HE PRESENCE of stable placental-derived nucleic acids,
packed in apoptotic bodies, in maternal peripheral blood
during pregnancy has enabled risk-free noninvasive prenatal diagnosis of those paternally inherited alleles which are absent in
the maternal genome (Lo et al., 1997; Poon et al., 2000). As
such, it is already being put to use in the determination of fetal Rhesus D status in RhD negative mothers at risk of
haemolytic diseases of newborn babies, and for fetal sex determination in pregnancies at risk of X-linked disorders (Faas
et al., 1998; Lo et al., 1998a, 1998b; Hahn et al., 2000; Zhong
et al., 2000, 2001; Costa et al., 2001; Rijnders et al., 2001; Sekizawa et al., 2001; Honda et al., 2002).
Noninvasive cell-free fetal RHD genotyping has become a
routine part of prenatal care in several European countries.
However, the low quantity of fetal DNA in maternal circulation as well as interference from an excessive amount of maternal DNA has made detection of fetal point mutations difficult, and has thus increased the chance of false negative results.
Most interestingly, size fractionation of circulatory DNA extracted from maternal peripheral blood revealed that the major
proportion of circulatory fetal DNA fragments had an approximate molecular size of less than 300 bp (Li et al., 2004a; Chan
et al., 2004). It was also discovered that the enrichment of fetal DNA could be achieved by selecting the fragments with a
size of less than 300 bp. PCR analysis performed on that enriched fetal DNA might also enable the detection of fetal loci
involving single-point mutation. This approach was used to detect the fetal point mutation in fibroblast growth factor receptor 3 (FGFR 3) which causes achondroplasia (Li et al., 2004b)
or for the detection of four common beta-thalassemia point mutations: IVSI-1, IVSI-6, IVSI-110, and codon 39 (Li et al.,
2005). Currently, we examined the quantification of fetal and
total extracellular DNA in maternal plasma after size fractionation by agarose gel electrophoresis in pregnant women carrying male fetuses. We also investigated the distribution of fetaland maternal-derived DNA molecules and evaluated the grade
of selective circulatory fetal DNA enrichment in individual sizefractionated fragments.
1Cell Biology Laboratory, Paediatric Clinic, 2Clinic of Obstetrics and Gyneacology, 2nd Medical Faculty, Charles University, University Hospital Motol, Prague, Czech Republic.
635
636
HROMADNIKOVA ET AL.
MATERIALS AND METHODS
The Local Ethics Committee’s approval and informed consent was obtained for all patients in the study.
To minimize the risk of contamination, plasma preparation,
DNA extraction from maternal plasma, and preparation of realtime PCR reaction were performed in laminar air flow, and
aerosol-resistant tips were used.
DNA extraction from maternal plasma samples
Ten milliliters of maternal peripheral blood from 11 pregnant women carrying male fetuses at 16 weeks gestation were
collected into EDTA-containing tubes and processed within a
few hours (not longer than 24 h). In detail, blood samples were
centrifuged first at 1200 g for 10 min, then plasma samples
were recentrifuged again and the supernatants were collected
and stored at 80°C until further processing (Hahn et al., 2000;
Hromadnikova et al., 2003). DNA was extracted from 1 ml
plasma using QIAamp DSP Virus Kit (Qiagen, Hilden, Germany) according to the modified manufacturer’s instructions.
DNA extraction from maternal plasma samples by
using QIAamp DSP virus kit
A lower amount of QIAGEN Protease (Qiagen) (20 l) was
used for the degradation of the proteins. Residual contaminants
were removed by wash buffers AW1, AW2, and ethanol (600
l AW1, 750 l AW2, 750 l ethanol) and by using QIAvac
24 Plus vacuum system and vacuum pump (Qiagen). DNA was
eluted in 40 l Buffer AVE.
Size fractionation of extracellular DNA by agarose
gel electrophoresis
The extracted DNA was fractionated by 1% agarose gel electrophoresis (Serva, Heidelberg, Germany) containing 0.5 g/ml
ethidium bromide (Sigma-Aldrich, Steinheim, Germany) at 90
V (6.2 V/cm) for 25 min on Mini-Sub Cell GT (Bio-Rad, Hercules, CA) (Li et al., 2004a). Five microliters of 100 bp DNA
Ladder (Fermentas, Burlington, Canada) and 4 l of Orange
Loading Dye Solution (Fermentas, Burlington, Canada) were
added to each sample before size fractionation.
Each trace containing DNA fragments was sliced using a
sterile scalpel blade into five fractions with an approximate size
of 100–300, 300–500, 500–700, 700–900, or 900 bp according to the 100 bp DNA Ladder after the exposition on UV light
transilluminator (Ultra-Lum, Claremont, CA).
DNA extraction from agarose sections
DNA was eluted from the agarose section using QIAEX II
Gel Extraction kit (Qiagen) according to the manufacturer’s in-
TABLE 1.
PRIMERS
AND
structions, (Li et al., 2004a). DNA was eluted by 45 l of lowsalt solution (pH 7.5). To prevent contamination we cleaned the
electrophoretic equipment using DNA Remover (Minerva Biolabs, Berlin, Germany), we used fresh buffers for each electrophoresis and also made a parallel examination of blank gel
slices in each analysis. In all cases no amplification of SRY
gene occurred.
Real-time PCR analysis
The real-time PCR analysis was performed using 7300 realtime PCR system (Applied Biosystem, Branchburg, NJ). Primer
and probe sequences are shown in Table 1 (Lo et al., 1997,
1998a). TaqMan amplification reactions were set up in a reaction volume of 50 l using the TaqMan Universal PCR Master Mix (Applied Biosystems), optimized concentration of
primers (300 nM), and TaqMan probes (200 nM) and 18 l of
DNA template. PCR was carried out in eight-well reaction optical tubes/stripes (Applied Biosystem). The TaqMan PCR conditions were used as described in TaqMan guidelines using 50
cycles of 95°C for 15 sec and 60°C for 1 min with 2-min preincubation at 50°C required for optimal AmpErase UNG activity, and 10-min preincubation at 95°C required for activation
of AmpliTaq Gold DNA polymerase. Each DNA sample eluted
from the agarose section was analyzed in two settings (one tube
amplification of SRY gene, and one tube amplification of GLO
gene). Quantification of fetal and total extracellular DNA present in maternal plasma was also performed on unseparated DNA
extracted from another maternal plasma aliquot. A patient’s
specimen was considered positive if amplification signal occurred on threshold cycle 40. The calibration curves were run
in parallel with each analysis (Figs. 1 and 2). The concentration of fetal and total extracellular DNA in unsepareted DNA,
and in each section after size fractionation, expressed in
genome-equivalents per milliliter of maternal plasma was calculated by the use of the following equation:
[total volume of DNA after extraction (ml)/volume of DNA
used for PCR (ml)] [target quantity determined by
sequence detector in PCR (copies)/volume of plasma for
DNA extraction (ml)].
RESULTS
Size-separated and unseparated plasma DNA samples from
11 pregnant women at the 16th week of gestation carrying male
fetuses were analyzed for the quantification of fetal and total
circulatory DNA based on the amplification of SRY locus on
Y chromosome and the ubiquitous GLO gene. The data are
shown in Table 2.
TAQMAN PROBES
FOR
SRY
AND
GLO REAL-TIME PCR
Gene
Primer sequences
Probe sequences
References
SRY
5-TGG CGA TTA AGT CAA ATT CGC-3
5-CCC CCT AGT ACC CTG ACA ATG TAT T-3
5-GTG CAC CTG ACT CCT GAG GAG A-3
5-CCT TGA TAC CAA CCT GCC CAG-3
5–(FAM) AGC AGT AGA GCA GTC
AGG GAG GCA GA (TAMRA)-3
5–(FAM) AAG GTG AAC GTG GAT
GAA GTT GGT GG (TAMRA)-3
Lo et al.,
1997, 1998
Lo et al.,
1997, 1998
GLO
QUANTIFICATION OF CIRCULATORY DNA AFTER SIZE FRACTIONATION
637
FIG. 1. Standard curve for SRY gene (in logarithmic scale) plotting the threshold cycle (Ct) against known concentrations of
serially diluted DNA quantification of fetal circulatory DNA in maternal plasma.
FIG. 2. Standard curve for GLO gene (in logarithmic scale) plotting the threshold cycle (Ct) against known concentrations of
serially diluted DNA quantification of total circulatory DNA in maternal plasma.
638
TABLE 2.
HROMADNIKOVA ET AL.
QUANTIFICATION OF FETAL AND TOTAL EXTRACELLULAR DNA IN MATERNAL PLASMA (GENOME-EQUIVALENTS/ML)
BEFORE AND AFTER SIZE FRACTIONATION BY AGAROSE GEL ELECTROPHORESIS
Unique
patient
number
1395
1047
1098
1319
1522
1446
1560
1511
1547
1161
1640
SRY
Range
Median
GLO
Range
Median
% fetal DNA
Range
Median
Fetal DNA
Enrichment
Range
Median
Unseparated
DNA
Fraction
100–300 bp
Fraction
300–500 bp
Fraction
500–700 bp
SRY: 11.8
GLO: 5863.3
0.2%
SRY: 2.8
GLO: 4785.3
0.06%
SRY: 4.9
GLO: 4891.3
0.1%
SRY: 57.55
GLO: 3227.2
1.78%
SRY: 14.8
GLO: 3356.1
0.4%
SRY: 41.6
GLO: 9519.7
0.44%
SRY: 79.3
GLO: 5400.7
1.47%
SRY: 7.2
GLO: 4480.2
0.16%
SRY: 56.87
GLO: 4597.1
1.24%
SRY: 41.51
GLO: 13198.1
0.32%
SRY: 12.51
GLO: 9087.76
0.14%
SRY: 13.1
GLO: 1261.0
1%
SRY: 14.4
GLO: 1455.2
1%
SRY: 15.3
GLO: 1210.5
1.3%
SRY: 83.2
GLO: 1045
8.0%
SRY: 15.2
GLO: 934.6
1.6%
SRY: 11.3
GLO: 2755.6
0.41%
SRY: 13.7
GLO: 681.1
2.01%
SRY: 5.1
GLO: 1276.6
0.4%
SRY: 20.0
GLO: 1916.9
1.04%
SRY: 17.3
GLO: 1301.0
1.33%
SRY: 4.98
GLO: 698.2
0.71%
SRY: 0
GLO: 672.9
0%
SRY: 1.95
GLO: 472.8
0.4%
SRY: 1.1
GLO: 733.0
0.15%
SRY: 10.8
GLO: 344
3.14%
SRY: 4.9
GLO: 496.8
1%
SRY: 3.3
GLO: 1417.0
0.23%
SRY: 21.4
GLO: 404.2
5.3%
SRY: 6.0
GLO: 524.4
1.14%
SRY: 3.0
GLO: 1049.0
0.29%
SRY: 14.3
GLO: 664.5
2.15%
SRY: 6.1
GLO: 534.15
1.14%
SRY: 0
GLO: 423.2
0%
SRY: 0
GLO: 246.9
0%
SRY: 0
GLO: 336.5
0%
SRY: 0
GLO: 179.6
0%
SRY: 2.5
GLO: 217.2
1.2%
SRY: 0
GLO: 341.4
0%
SRY: 0
GLO: 107.2
0%
SRY: 0
GLO: 176.9
0%
SRY: 0
GLO: 270.2
0%
SRY: 0
GLO: 239.1
0%
SRY: 1.78
GLO: 228.9
0.78%
SRY:
GLO:
0%
SRY:
GLO:
0%
SRY:
GLO:
0%
SRY:
GLO:
0%
SRY:
GLO:
0%
SRY:
GLO:
0%
SRY:
GLO:
0%
SRY:
GLO:
0%
SRY:
GLO:
0%
SRY:
GLO:
0.8%
SRY:
GLO:
0%
2.8–79.3
14.8
4.98–83.2
14.4
0–21.4
4.9
0–2.5
0
0–2.7
0
0
0
3227.2–13198.1
4891.3
681.1–2755.6
1261.0
344.0–1417.0
534.15
107.2–423.2
239.1
90.3–336.5
134.9
85.6–794.4
202.3
0.06–1.78
0.32
0.4–8.0
1.04
0–5.3
1.0
0–1.2
0
0–0.8
0
0
0
0.8–16.7
4.2
0–8.1
2.5
Concerning unseparated DNA, we detected 4891.3 (range
3227.2–13198.1) GE of total extracellular DNA and 14.8 (range
2.8–79.3) GE of fetal extracellular DNA in 1 ml of maternal
plasma, which means that 0.32% (range 0.06–1.78) of the total circulatory DNA in maternal plasma was detected to be of
fetal origin.
Fetal circulatory DNA was mainly detected in sections with
a size of 100–300 bp with the concentration of 14.4 GE/ml
Fraction
700–900 bp
0
191.3
0
122.04
0
101.6
0
90.25
0
134.9
0
179.5
0
92.2
0
100.2
0
236.7
2.7
336.5
0
222.13
Fraction
900 bp
SRY:
GLO:
0%
SRY:
GLO:
0%
SRY:
GLO:
0%
SRY:
GLO:
0%
SRY:
GLO:
0%
SRY:
GLO:
0%
SRY:
GLO:
0%
SRY:
GLO:
0%
SRY:
GLO:
0%
SRY:
GLO:
0%
SRY:
GLO:
0%
0
281.4
0
110.8
0
190.1
0
164.4
0
237.5
0
202.3
0
122.0
0
85.6
0
379.7
0
794.4
0
426.13
(range 4.98–83.2). Lower amount of fetal circulatory DNA 4.9
GE/ml (range 0–21.4) was also found in a fraction with a size
of 300–500 bp. Fetal DNA was detected rarely in fractions with
approximate sizes of 500–700, 700–900, and 900 bp. Circulatory DNA extracted from the gel fragment with a molecular
size 300 bp contained 4.2 times (range 0.8–16.7) enriched fetal DNA when compared with initial unseparated DNA sample. The median percentages of fetal-derived DNA with a size
QUANTIFICATION OF CIRCULATORY DNA AFTER SIZE FRACTIONATION
639
FIG. 3. Quantification of fetal and total circulatory DNA in maternal plasma before and after size fractionation by agarose gel
electrophoresis (patient UPN 1395): no. 1—total unseparated DNA; no. 2—total DNA in fraction 100–300 bp; no. 3—total DNA
in fraction 300–500 bp; no. 4—total DNA in fraction 500–700 bp; no. 5—total DNA in fraction 900 bp; no. 6—total DNA in
fraction 700–900 bp; no. 7—fetal DNA in fraction 100–300 bp; no. 8—fetal DNA in unseparated DNA.
of 100–300 bp was 1.04% (range 0.4–8.0). The fraction with a
size of 300–500 bp contained 1% (range 0–5.3) of fetal DNA.
Fetal DNA within the fraction with a size of 300–500 bp was
2.5 times (range 0–8.1) enriched (Fig. 3).
The size distribution analysis of total maternal plasma DNA
showed that the highest concentrations of plasma DNA occurred
within fractions with a size of 100–300 bp (median 1261.0
GE/ml) and 300–500 bp (median 534.15 GE/ml).
DISCUSSION
In summary, our findings showed that circulatory fetal DNA
is predominantly present in a fraction with molecular size 500
bp. However, the usage of that enriched size-fractionated fetal
DNA is not suitable for routine clinical applications due to the
finickiness of a multiple-step procedure which can be accompanied by the risk of contamination.
ACKNOWLEDGMENTS
This work was supported by grant projects MSM
0021620806, MZO 00064203, and SAFE (Special Non-Invasive Advances in Foetal and Neonatal Evaluation Network, no.
503243).
REFERENCES
CHAN, K.C., ZHANG, J., HUI, A.B., WONG, N., LAU, T.K., LEUNG, T.N., LO, K.W., HUANG, D.W., and LO, Y.M. (2004). Size
distributions of maternal and fetal DNA in maternal plasma. Clin.
Chem. 50, 88–92.
COSTA, J.M., BENACHI, A., GAUTIER, E., JOUANNIC, J.M., ERNAULT, P., and DUMEZ, Y. (2001). First-trimester fetal sex determination in maternal serum using real-time PCR. Prenat. Diagn. 21,
1070–1074.
FAAS, B.H., BEULING, E.A, CHRISTIAENS, G.C., VON DEM
BORNE, A.E., and VAN DER SCHOOT, C.E. (1998). Detection of
fetal RhD–specific sequences in maternal plasma. Lancet 352, 1196.
HAHN, S., ZHONG, X.Y., BURK, M.R., TROEGER, C., and HOLZGREVE, W. (2000). Multiplex and real-time quantitative PCR on fetal DNA in maternal plasma: A comparison with fetal cells isolated
from maternal blood. Ann. N. Y. Acad. Sci. 906, 148–152.
HONDA, H., MIHARU, N., OHASHI, Y., SAMURA, O., KINUTAMI,
M., HARA, T., and OHAMA, K. (2002). Fetal gender determination
in early pregnancy through qualitative and quantitative analysis of
fetal DNA in maternal serum. Hum. Genet. 110, 75–79.
HROMADNIKOVA, I., HOUBOVA, B., HRIDELOVA, D., VOSLAROVA, S., KOFER, J., KOMRSKA, V., and HABART, D.
(2003). Replicate real-time PCR testing of DNA in maternal plasma
increases the sensitivity of non-invasive fetal sex determination. Prenat. Diagn. 23, 235–238.
LI, Y., ZIMMERMANN, B., RUSTERHOLZ, C., KANG, A., HOLZGREVE, W., and HAHN, S. (2004a). Size separation of circulatory
DNA in maternal plasma permits ready detection of fetal DNA polymorphisms. Clin. Chem. 50, 1002–1011.
LI, Y., HOLZGREVE, W., PAGE-CHRISTIAENS, G.C., GILLE, J.J.,
and HAHN, S. (2004b). Improved prenatal detection of a fetal point
mutation for achondroplasia by the use of size-fractionated circulatory DNA in maternal plasma—Case report. Prenat. Diagn. 24,
896–898.
LI, Y., DI NARO, E., VITUCCI, A., ZIMMERMANN, B., HOLZGREVE, W., and HAHN, S. (2005). Detection of paternally inherited fetal point mutations for beta-thalassemia using size-fractionated
cell-free DNA in maternal plasma. JAMA 293, 843–849.
640
LO, Y.M., CORBETTA, N., CHAMBERLAIN, P.F., RAI, V., SARGENT, I.L., REDMAN, C.W., and WAINSCOAT, J.S. (1997). Presence of fetal DNA in maternal plasma and serum. Lancet 350,
485–487.
LO, Y.M., TEIN, M.S., LAU, T.K., HAINES, C.J., LEUNG, T.N.,
POON, P.M., WAINSCOAT, J.S., JOHNSON, P.J., CHANG, A.M.,
and HJELM, N.M. (1998a). Quantitative analysis of fetal DNA in
maternal plasma and serum: Implications for noninvasive prenatal
diagnosis. Am. J. Hum. Genet. 62, 768–775.
LO, Y.M., HJELM, N.M., FIDLER, C., SARGENT, I.L., MURPHY,
M.F., CHAMBERLAIN, P.F., POON, P.M., REDMAN, C.W., and
WAINSCOAT, J.S (1998b). Prenatal diagnosis of fetal RhD status
by molecular analysis of maternal plasma. N. Engl. J. Med. 339,
1734–1738.
POON, L.L., LEUNG, T.N., LAU, T.K., and LO, Y.M. (2000).
Presence of fetal RNA in maternal plasma. Clin. Chem. 46,
1832–1834.
RIJNDERS, R.J., VAN DER SCHOOT, C.E., BOSSERS, B., DE
VROEDE, M.A., and CHRISTIAENS, G.C. (2001). Fetal sex determination from maternal plasma in pregnancies at risk for congenital
adrenal hyperplasia. Obstet. Gynecol. 98, 374–378.
SEKIZAWA, A., KONDO, T., IWASAKI, M., WATANABE, A.,
JIMBO, M., SAITO, H., and OKAI, T. (2001). Accuracy of fetal
HROMADNIKOVA ET AL.
gender determination by analysis of DNA in maternal plasma. Clin.
Chem. 47, 1856–1858.
ZHONG, X.Y., HOLZGREVE, W., and HAHN, S. (2000). Detection
of fetal rhesus D and sex using fetal DNA from maternal plasma by
multiplex PCR. Br. J. Obstet. Gynaecol. 107, 766–769.
ZHONG, X.Y., HAHN, S., and HOLZGREVE, W. (2001). Prenatal
identification of fetal genetic traits. Lancet 357, 310–311.
Address reprint requests to:
Ilona Hromadnikova, Ph.D.
Cell Biology Laboratory
Paediatric Clinic
2nd Medical Faculty
Charles University
University Hospital Motol
V Uvalu 84
150 06 Prague 5, Czech Republic
E-mail: [email protected]
Received for publication July 27, 2006; received in revised form
September 15, 2006; accepted September 25, 2006.