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

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,
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