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

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 Master
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