Sod1 Genotyping based on 118G>A Missense Mutation
Genomic DNA Extraction
gDNA was isolated from 100 µl of whole blood sample using a commercial kit (DNeasy®Blood and Tissue,
Qiagen, UK) following manufacturer’s recommendations. Canine anti-coagulated blood was mixed with
proteinase K (600mAU/ml/reaction) and lysis buffer containing guanidine hydrochloride and incubated at 56 °C
for 10 minutes. Following the addition of 100% ethanol the sample was centrifuged through a spin column,
washed twice using buffers containing ethanol and guanidine hydrochloride and eluted in 200 μl elution buffer
(10 mM Tris-hydrochloric acid and 0.5 mM EDTA, pH 9.0). For spleen samples, 10 mg of spleen was incubated
for two hours in the presence of lysis buffer and proteinase K. Following lysis, the procedure was as for blood
DNA extraction as described above. All gDNA were qualitatively assessed by electrophoresis.
Primer Design and PCR
The restriction enzyme site, HpyAV present in wild type is abolished in the mutant Sod1gene. The forward (5-/
GCC TGT TGT GGT ATC AGG AAC CA-3/) and reverse (5/-AGA GTC AAA AAC CGG C TT TGT GGA3/) Sod1 primers (Eurofins, Germany) generate a 236 base pair (bp) DNA fragment encompassing the point
mutation and the HpyAV restriction site.
A 25 μl PCR reaction was prepared, comprising 12.5μl RedTaq® DNA Reaction (Sigma-Aldrich Co, UK),
5pmol of each primer and 200 ng gDNA. Amplification conditions were (94 °C /five minutes, 94 °C /one
minute, 58 °C /one minute, 72 °C /one minute) for 32 cycles followed by an extension cycle (72 °C for 10
minutes). The PCR products were quantified against the 100 bp mass ladder for DNA (Quick-Load, New
England Biolabs, UK) using Image J software (NIH). Purification of PCR products was performed according to
manufacturer’s recommendations using the QIAquick® PCR purification kit (Qiagen, UK) and eluted in 30μl of
elution buffer.
HpyAV Digestion
100ng of purified PCR products were digested with HpyAV (2 U/µl) in a 25 µl at 37 °C for 30 minutes and heat
inactivated at 65 °C for 15 minutes. The digestion products were analysed by 2.5% agarose gel electrophoresis
in 1X Tris-borate EDTA. Gel images were captured using UV image capture system (Syngene, UK).
Supplementary 1. We have demonstrated that the RLFP technique could be used to differentiate three Sod1
genotypes; wild type (WT), heterozygous (het) and homozygous (homo). Mixtures of wild type and
homozygous PCR products were generated to represent each of the genotypes: wild type (100% WT: 0%
homozygous), heterozygous (50% WT: 50% homozygous), and homozygous (0% WT: 100% homozygous).
Intermediate ratios were also included; 75% WT: 25% homozygous and 25% WT: 75% homozygous. The
heterozygous genotype displayed two bands at equal intensities (236 bp and 204 bp) in a mixture containing
50% wild type and 50% homozygous. In 100% wild type mixture, a prominent band was observed at 204bp,
suggesting DNA fragments are completely digested whereas in 100% homozygous sample, a single band was
observed at 236bp, which is comparable with undigested (UD) PCR product.
Supplementary 2. Sod1 genotyping of canine spleen and blood-derived DNA. HpyAV digestion for 30 minutes
in spleen and blood-derived DNA are depicted below. Partial digestion observed in control sample from spleen
(C1 and C2), a single band (204 bp) consistent with wild type profile was detected in B1 blood sample,
indicating a complete digestion had occurred with 100 ng of PCR product. The heterozygous profile was
detected in B3 blood sample, demonstrating two bands at almost equal intensities (236bp and 204bp) and
therefore showing a clear-cut differentiation between partially digested DNA products as observed in spleenderived DNA. Two blood samples (B2 and B4) and a spleen sample demonstrated a homozygous profile, which
represented a single band with size corresponding to undigested (UD) sample (236bp).