Karen Sermon, M.D., PhD
Center for Medical Genetics,
University Hospital and Medical School of the Dutch-speaking Brussels Free
Laarbeeklaan 101, 1090 Brussels, Belgium
Materials and Methods
Preimplantation diagnosis (PGD) is a very early form of prenatal diagnosis:
embryos obtained in vitro from couples at risk of contracting genetic
disease are analyzed for the presence of this disease and only the embryos
which are shown to be free of the disease being tested for are transferred
to the uterus. The PCR* (Polymerase Chain Reaction) has been the only
tool available for diagnosing monogenetic diseases at the DNA level in
single cells. At the Center for Medical Genetics, we have developed PGD
protocols for numerous diseases, including Cystic Fibrosis (CF) where
primarily the DF508 mutation was analyzed, Duchenne's
As a source of single cells, lymphoblasts transformed with the Epstein-Barr
virus were used. The three cell populations used were from the following
individuals; a carrier of the DF508 mutation, a female heterozygous for
the CA repeat in intron 45 of the DMD gene, and an individual heterozygous
for the DM-repeat (5 and 12 repeats) respectively. The cells were washed
three times with PBS, after which individual cells were washed three times
in 2 l drops of Ca2+-free and Mg2+-free medium and transferred to
200 l PCR tubes (Eppendorf PCR tubes) containing 2.5 or 5 l
alkaline lysis buffer (ALB; 50 mM DTT and 200 mM KOH for CF and DMD or NaOH
Figure 1: PCR fragments for CF (DF508) from single cells analyzed
on a 2% agarose gel. Lanes 4, 8, 12, 16, 20, and 24 contain blanks;
lanes 5, 11, and 13 are not amplified. The smallest, brightest band
represents the PCR product (see arrow) obtained after two PCR rounds;
the larger products are from a combination of primers from the first
second PCR rounds.
The tubes were kept at -80 C before being processed further. The cells
were lysed by incubating them at 65 C for 10 min, after which the ALB
was neutralized with 2.5 or 5 l neutralization buffer (900 mM TrisHCl
pH 8.3, 300 mM KCl, 200 mM HCl) if KOH was used, and 2.5 l 200 mM
Tricine pH 4.9 if NaOH was used.
Primer sequences and PCR programs are summarized in Table 1. The DMD
sequence was amplified in a similar way to the CF sequence (Figure 3).
For the detection of DF508 on conventional ethidiumbromide-stained gels,
reaction mix was added to the cells to a final volume of 50 l and
final concentrations of 50 mM KCl, 100 mM TrisHCl pH 8.3, 2 mM MgCl 2,
0.1 mg/ml gelatin, 0.2 mM dNTP, 1 mM primers, and 1.25 U Taq polymerase.
Three l from the first PCR were taken as a template in the second
PCR. The reaction mix had the same final concentrations and volume as
the first PCR. The PCR products were run on a horizontal 2% agarose gel
to check for amplification and contamination (Figure 1), after which the
samples showing amplification were separated on a vertical 4% Metaphor
Agarose gel (Sanver Tech, Boechout,
Belgium), and run at 350 V for 90
min (Figure 2).
Figure 2: The same PCR products from Figure 1 analyzed on a 4% Metaphor
Agarose gel. The lowest band re p resents the mutated allele, the
middle band re p resents the healthy allele (three bp larger), and
the top band re p resents heteroduplexes. The larger bands are again
results from combinations of primers from the first and second PCR
rounds. The cell in lane 7 shows ADO, i.e. only the healthy allele
is amplified, such that this cell seems homozygous normal.
PCR for DM was carried out using the Extended
Long Template Kit (Boehringer Mannheim) in a total volume of 25 l
with final concentrations of 5% DMSO, 200 mM dNTPs, 1 x buffer 2 provided
by the manufacturer, 20 mM tricine pH 4.95, and 1.4 U of DNA polymerase
provided with the kit. Figure 4 shows a result of a PCR amplifying the triplet
repeat at the 3-end of the DM gene.
For the detection of the DF508 mutation with a conventional PCR, 23/26 (88%)
single lymphoblasts showed amplification (Figure 1 2). Of these, 4/23 (17%)
showed allele drop-out (ADO), i.e. one of both alleles in a heterozygous
cell was not amplified (Table 2). Similar results were obtained for the
detection of DF508 using fluorescent
PCR: 25/27 (92%) cells showed amplification using the Eppendorf Mastercycler
gradient (Figure 3), while 2/25 (8%) cells showed ADO. For Myotonic Dystrophy,
the results were also very good: 18/18 (100%) amplification and 0% ADO with
cycler (Figure Finally, the DMD-sequence also gave good results
(Figure 5): 18/ (100%) amplification and 2/18 (11%) ADO for the Mastercycler
Figure 3: Cells heterozygous for the DF508 mutation after fluorescent
PCR and analysis on an automated DNA sequencer. The cells in lanes
14 to 24 show two bands, while the cell in lane 27 only shows one
band (i.e. ADO). Lanes 1 and 39 are molecular weight markers representing
150, 200, 250, and 300 bp.
Figure 4: Single-cell fluorescent PCR for the 3 triplet repeat
in the DM gene in an individual heterozygous for 5 and 12 repeats.
Lanes 2, 3, 4, 6, 7, 8, and 10 show single cells, while lanes 5 and
9 re p resent blank samples.
Figure 5: The result of an amplification for intron 45 of the DMD
gene. Lanes 18, 19, 20, 24, 25, and 26 show heterozygous cells, while
lanes 21 and 27 show blanks. The high peaks on the left of the diagram
The aim of this small-scale study was to show the efficiency and accuracy
of the new Eppendorf Mastercycler gradient in
single-cell PCR. To achieve
this, we compared the results obtained with the Eppendorf Mastercycler with
our results previously obtained using Perkin-Elmer GeneAmp PCR systems.
None of the programs used for the Perkin-Elmer PCR machines had to be altered,
with regard to both the incubation temperatures and the incubation times.
Brook, J.D., et al. (1992) Molecular basis of myotonic dystrophy:
expansion of a trinucleotide (CTG) repeat at the 3-end of a transcript
encoding a protein kinase family member. Cell 68, 799-808.
Clemens, P.R., et al. (1991) Carrier detection and prenatal diagnosis
in Duchenne and Becker Muscular Dystrophy families, using dinucleotide
repeat polymorphisms. Am. J. Hum. Genet. 49, 951-960.
Liu, J., Lissens, W., Devroey, P., Van Steirteghem, A. and Liebaers,
I. (1992) Efficiency and accuracy of polymerase-chain-reaction assay for
cystic fibrosis allele DF508 in single cell. Lancet, 339, 1190-1192.
Sermon, K., Lissens, W., Joris, H., Desmyttere, S., Devroey, P., Van
Steirteghem, A. and Liebaers, I. (1997) Clinical application of preimplantation
diagnosis for myotonic dystrophy. Prenat. Diagn. 10, 925-932.
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