Molecular characterisation of SGCE-associated myoclonus-dystonia ...

Patients, material and methods 33
PCR reaction according to the manufacturer’s protocol (Fig 5). Amplification products were
separated by polyacrylamide gel electrophoresis (PAGE, see 2.3.3.2) and visualized on an
automated sequencing machine. For all samples, a specific pattern of bands, varying in size
and intensity, was observed. Each of these bands represented one amplified pair of probes
from the kit. The MLPA gel images were analyzed with the help of an image processing
program (TotalLab), employing a “nearest neighbour” model (Djarmati et al., 2007), which
allows calculating the relative initial concentration of the specific DNA fragment. Normalized
value ratios between 0.8 and 1.2 were considered normal, a heterozygous deletion was
expected at a ratio between 0.3 and 0.7, and a heterozygous duplication between 1.3 and 1.7.
MLPA was performed for all exons of SGCE except the rare splicing variant, exon 10, in the
M-D approach (Fig 9).
Hybridisation sequence A’ and A’’
Primer X
Stuffer sequence A
Primer Y
Hybridisation sequence B’ and B’’
Primer X
Primer Y
Stuffer sequence B
1. Hybridisation
3’ 5’
Target A
3’ 5’
Target B
2. Ligation
3’ 5’
Target A
3’ 5’
Target B
3. Amplification
Fig 5 Principle of the MLPA analysis. For each target a specific pair of probes is needed. The included primer
sequences X and Y are constant whereas the stuffer sequence varies for each pair of probes. Two parts of each
probe hybridise to adjacent target sequences and are ligated by a thermostable ligase. In a next step all probe
ligation products are amplified by PCR using only one primer pair. If one of the two probes can not bind to their
target because of a mutation or a lack of target sequence a ligation is abolished and no amplification will occur.
The amount of probes binding to a target can be quantified after the amplification (Schouten et al., 2002).
2.3.3 Gel electrophoresis
Three different types of electrophoresis were applied here: agarose gel electrophoresis and
polyacrylamide gel electrophoresis in a denaturing as well as a non-denaturing form.