Schlipf et al., supplement, page 1
220 patients with autosomal dominant spastic paraplegia do not display mutations
in the SLC33A1-gene (SPG42)
Nina A. Schlipf, Christian Beetz, Rebecca Schüle, Giovanni Stevanin, Anne Kjersti
Erichsen, Sylvie Forlani, Cécile Zaros, Kathrin Karle, Stephan Klebe, Sven Klimpe,
Alexandra Durr, Susanne Otto, Chantal ME Tallaksen, Olaf Riess, Alexis Brice, Peter
Bauer and Ludger Schöls
Supplementary Material
SPG42 amplification and high resolution melting (HRM) analysis
The primers used to screen the SPG42 gene SLC33A1 (ENSG00000169359) were
designed to flank the coding regions of SPG42 exons 1-6 and to amplify fragments of
an average size of 259 bp (ranging from 206 to 298). All primers were 5´tailed with
universal M13 or revM13 sequences to facilitate sequencing. Primer sequences are
available in supplementary table1.
The PCR and HRM was performed in a single run on a LightCycler®480 instrument
(Roche Diagnostics, Mannheim, Germany) with a total reaction volume of 10 µl in a
384-well microtiter plate. The reaction mixture containing 20ng of genomic DNA, 2.5
mM MgCl2 (Roche Diagnostics, Mannheim, Germany), 10 pmol of each primer, 5 µl 2x
LightCycler®480 High Resolution Master Mix (Roche Diagnostics, Mannheim,
Germany) and PCR grade water in a volume of 10 µl.
The PCR cycling conditions included an activation step at 95°C for 10 minutes followed
by 45 cycles of 95°C for 10 seconds, a touch down of 68°C to 58°C for 15 seconds
(1°C/cycle) and 72°C for 20 seconds. The amplicons were denaturated at 95°C for 1
Schlipf et al., supplement, page 2
minute, renaturated at 40°C for 1 minute. Afterwards the HRM was executed over the
range from 65°C to 95°C with 25 signal acquisitions per degree.
The HRM analysis was performed using Gene Scanning Software Version 1.5.0 (Roche
Diagnostics, Mannheim, Germany), which allows clustering the samples into groups on
the basis of difference plot obtained analyzing the differences in melting curve shapes.
To guarantee a high detection sensitivity, HRM analysis sensitivity settings were 0.7
(high sensitivity).
Schlipf et al., supplement, page 3
Supplementary table 1: SPG42 HRM amplification and sequencing primers
Exon
SPG42 Exon01
Fragment
Lenght
primer
sequence 5´->3
250 bp
1-1F
1-1R
259 bp
1-2F
1-2R
277 bp
1-3F
1-3R
239 bp
2-1F
2-1R
246 bp
2-2F
2-2R
M13-CGGATCCAAAACGCTCAG
revM13GGCTTTTAAGAAGTCGCCAGT
M13-AGGCGGTTGGGATGACAGT
revM13GGGCCAAAAGACAAAACTGA
M13-AAGCATCCCACTCATTTTGC
revM13CACACACTCTTTCAAAAGGATAAA
T
M13-GCATGAGCACCTGTACTTGG
revM13TGTGATCCCTTGTGTTTCTTCTT
M13-ATTGGTTGCCCTTCTGAAAA
revM13TGGTAGTGGAAAGGGACCTG
M13TGCATGGAGGAAAATATTTCATAA
revM13TGTTCCTCATTTTACCCTTTCTT
M13GCTAAAATGGAAGTCAACTCAATG
revM13TCTTCAGTGTCAGATTCATGCTT
M13TTTCAGGTTTATGACTTTGATGTTC
revM13-TTTCCTCCCAGATTGGACAC
M13CAATGCAAAGGTTAGTGATCCA
revM13CCAGAACCTATGGACAGATATGA
M13TTTCATTTTGAAATTGTGGAGTATT
revM13CCTTGCTAGAATGTCCAGTAGCA
SPG42 Exon02
3F
SPG42 Exon03
298 bp
3R
4F
SPG42 Exon04
277 bp
4R
5-1F
206 bp
5-1R
5-2F
SPG42 Exon05
294 bp
5-2R
6F
SPG42 Exon06
244 bp
6R
Schlipf et al., supplement, page 4
Direct sequencing
To re-amplify the amplicons, we used the same primers as for HRM (see supplementary
table 1). PCR was performed in 20 µl volumes. Amplification reactions included 10ng
genomic DNA, 10 pmol of each primers, 10 mM of dNTPs (Fermentas GmbH, St.
Leon-Rot, Germany), 5x PCR buffer (including 25 mM MgCl2) (Promega GmbH,
Mannheim, Germany), 0.5U Go Taq (Promega GmbH, Mannheim, Germany) DNA
Polymerase and H2O to a volume of 20 µl. All 10 Amplicons were amplified using a
touch down protocol. The cycling conditions which were run on a DNA Engine Dyad
thermal cycler (Bio-Rad Laboratories, München, Germany), consisted of an initial
denaturation step at 94°C for 3 minutes, followed by 8 cycles of denaturation at 94°C
for 30 seconds, annealing starting at 68°C for 30 seconds (decreasing 1°C/cycle) and
extension at 72°C for 60 seconds. This step was followed by 27 additional cycles of
denaturation at 94°C for 30 seconds, annealing at 60°C for 30 seconds and extension at
72°C for further 90 seconds. Final extension was carried out at 72°C for 2 minutes.
The PCR products were purified using ethanol precipitation according to the
instructions of the manufacturers. The purified PCR products were eluted in 15 µl
volume and then used as template in cycle sequencing using Big Dye Terminator Cycle
Sequencing Ready Reaction Kit (Applied Biosystems Inc, Foster City, CA, USA). The
reaction mix consisted of 2 µl ABI BigDye 3.1, 10 pmol primer, 1 µl ABI BigDye
Buffer and approx. 20ng purified PCR template in a 10 µl total volume. The sequencing
reactions were run on a DNA Engine Dyad thermal cycler (Bio-Rad Laboratories,
München, Germany) according to the following protocol; one cycle of 94°C for 1
minute; 30 cycles of 94°C for 10 seconds, 50°C annealing temperature for 5 seconds,
60°C for 4 minutes. The sequencing reaction was purified using ethanol precipitation
Schlipf et al., supplement, page 5
and run on an ABI3100 Genetic Analyzer (Applied Biosystems Inc, Foster City, CA,
USA). All sequences were examined using SequencePilot Software (JSI medical
systems GmbH, Kippenheim, Germany).