Rapid detection of genomic,,,duplications and deletions using,,,the Agilent 2100 bioanalyzer
d.The DNA is labeled with eitherradioactive or fluorescent moleculesduring the PCR step and theseparation has been performed byelectrophoresis either on a polyacrylamidegel or capillary system.We have tried the possibility ofanalyzing the MLPA and MAPHproducts using the Agilent 2100bioanalyzer in combination withthe DNA 500 chip. Parametersunder investigation
d.
The DNA is labeled with either
radioactive or fluorescent molecules
during the PCR step, and the
separation has been performed by
electrophoresis, either on a polyacrylamide
gel or capillary system.
We have tried the possibility of
analyzing the MLPA and MAPH
products using the Agilent 2100
bioanalyzer in combination with
the DNA 500 chip. Parameters
under investigation included resolution,
reproducibility and the
accuracy of quantitation.
Methods
MAPH
A total of 17 female DNA samples
were examined, of whom 13 were
known to be carriers for mutations
in the DMD gene and 4 were
non-carriers. The MAPH protocol
has been described previously3,
and is also available on http://www.dmd.nl/DMD_MAPH.html
For each hybridization several
DMD gene exon probes were chosen,
both within and outside the
suspected breakpoints. At least
2 control autosomal probes were
also included in the probe mix.
MLPA
Full details regarding the MLPA
protocol are in reference 2,
and on the website
http://www.mrc-holland.nl/
The MLPA reaction was performed
on male and female DNA,
as well as DNA from a cell line
49,XXXXY. Bioanalyzer analysis
was performed according to the
manufacturer's instructions.
The peak patterns were visually
compared using the Bio Sizing
Data Evaluation program
(Agilent Technologies).
Results and discussion
We have previously published
work on the analysis of deletions
and duplications in the DMD geneusing MAPH3. While this concentrated
on the analysis of a large
group of samples, it is often necessary
or sufficient to screen only
one or a small number of samples
with a few probes. This is particularly
the case when a relative of a
patient wishes to be screened for
carr
'"/>Source:
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