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The use of the Mastercycler gradient for single-cell PCR and preimplantation ,,, diagnosis

Karen Sermon, M.D., PhD
Center for Medical Genetics,
University Hospital and Medical School of the Dutch-speaking Brussels Free University,
Laarbeeklaan 101, 1090 Brussels, Belgium

Introduction

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

Materials and Methods

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 for DM).

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; cells in
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 and
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 the
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. Results

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 the Mastercycler (Figure Finally, the DMD-sequence also gave good results (Figure 5): 18/ (100%) amplification and 2/18 (11%) ADO for the Mastercycler gradient. 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 represent
primer peaks. Ordering information Discussion

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.

References

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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