Rapid detection of genomic,,,duplications and deletions using,,,the Agilent 2100 bioanalyzer
ier status. In this case themutation to look for is alreadyknown and the question is simplywhether a given DNA sample hasthe same mutation. It is thereforenot necessary to examine all 79exons but simply compare probesfor exons within the potentialrearrangement to those outside.To test whether the Agilent systemwould be suitable for this analysiswe first had to de
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
'"/>Source:
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