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Rapid detection of genomic,,,duplications and deletions using,,,the Agilent 2100 bioanalyzer

ier status. In this case the mutation to look for is already known, and the question is simply whether a given DNA sample has the same mutation. It is therefore not necessary to examine all 79 exons, but simply compare probes for exons within the potential rearrangement to those outside. To test whether the Agilent system would be suitable for this analysis we first had to determine the practical resolution of separation and the minimum concentration required for accurate analysis. An experiment was performed to separate fragments of a known length. It was found that having a probe separation of > 20 base pairs resulted in peaks that could readily be distinguished (figure 1). We routinely amplify the MAPH products with a 25 cycle PCR reaction. This gave sufficient product to be easily detected. A concentration of approximately 0.5 ng/l is necessary to give a peak of approximately 5 units in height, the default minimum set in the analysis program. We analyzed a total of 17 samples from females where the carrier status was known4. For each sample at least 1 exon was chosen within the deleted/duplicated region, and several exons outside. Changes in peak height correspond to changes in copy number, and in most cases the difference could be easily detected by eye (figure 2).

Exon specific peaks were all normalized against unlinked autosomal probes, and the normalized ratios of the exons within the rearrangement were compared with those from exons outside the rearrangement using Student's ttest (table 1).

In the 13 cases where the female was a carrier the differences were highly significant (p < 0.001), whereas the 4 non-carriers showed no significant difference (p > 0.1). To estimate the error rate of the assay the 99 % confidence intervals of the difference were
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