Supplementary Material and methods
PCR protocol
In Ghent primers were designed to cover the full coding sequence, 3’ and 5’ UTR and intronexon boundaries encompassing all 9 exons of RAD51C. RAD51C primers used in Montreal
were as described by Vas et al., except exon 2 and 6 (both were redesigned) (Table S1A). The
Ensembl genomic sequence database was used to verify the absence of SNPs in the primers.
The specificity of the designed primers was evaluated using the University of California Santa
Cruz in silico PCR program. In Ghent all primers were fused with universal M13-tails (forward
CACGACGTTGTAAAACGAC and reverse CAGGAAACAGCTATGACC) to simplify the sequencing
process afterwards (Table S1B).
PCR was performed in the presence of the double-stranded DNA dye LCGreen. The
amplification mixture included 50 ng of genomic DNA as template, 200 µM of each
deoxynucleotide triphosphate (dNTP), 0.65U of Platinum Taq Polymerase (Invitrogen,
Merelbeke, Belgium), 1× PCR Buffer (Invitrogen, Merelbeke, Belgium), 1.5mM MgCl 2
(Invitrogen, Merelbeke, Belgium), 30µM of each primer and 1× LCGreen TM Plus (Idaho
Technology Inc., Salt Lake City, Utah). Three percent of dimethyl sulfoxide (DMSO, VWR
International) was used in each amplification mixture, except for the amplification of exon 5
(no DMSO) and exon 6 (6% DMSO).
PCR reactions were carried out in a total volume of 10 µL with the following amplification
protocol: denaturation at 95°C for 5 minutes, followed by 35 cycles of denaturation at 95°C
for 30 seconds, annealing at 55°C (exon 6) or 58°C (other exons) for 30 seconds, and
extension at 72°C for 30 seconds. This PCR was followed by 7 minutes at 72°C, and finished
by a final step at 15°C for 10 minutes.
Four Canadian samples were assayed by HRM. The reagents for HRM were purchased from
Idaho Technologies (Salt Lake City, Utah, USA). The PCR reactions were performed in 5µl
total volume. The amplification mixture consisted out of 2 µl mastermix, 0.5µl LCGreen,
0.5µl forward primer (2.5µM stock), 0.5µl reverse primer (2.5uM stock), 0.5µl H2O and 1µl
DNA (25ng). The PCR program was as follows: 95°C during 2minutes, 7 cycles of 95°C for 45
sec, 76°C for 45 sec with -1°C increment at every cycle. 35 cycles of 95°C for 45 sec and 69°C
for 45 sec, followed by 95°C for 45 seconds and 28°C 45 for seconds. Exon 1 was amplified at
65°C and 1µl Q solution was added.
High-Resolution Melting
Melting acquisition was performed in 96 or 384-well plates suitable for HRM analysis. To
prevent evaporation during heating, PCR products were covered with a mineral oil layer.
Plates were then heated in the 96 or 384-well LightScanner (Idaho Technology Inc. Salt Lake
City, Utah) from 65°C up to 98°C with a heating rate of 0.1°C/s. Analysis of the melting curves
was accomplished with the standard LightScanner software (version 2.0).
PCR and HRM were performed by pooling samples from the same laboratory, because
pooling samples from different laboratories caused abnormalities in the melting curves. This
might be caused by differences in DNA-extraction methods.
DNA sequencing
In Ghent, samples which expressed an aberrant melting curve were sequenced using the
Bigdye v3.1 ET terminator cycle sequencing kit from Applied Biosystems. Sequencing
reactions were loaded on an Applied Biosystems Prism 3730 Genetic Analyzer. Sequences
were analyzed with SeqScape v2.5 (Applied Biosystems, Foster City, CA).
In Montréal, the PCR reactions used as a template for direct sequencing were carried out in
50-μl volume and consisted of 5 μl of 10× PCR buffer, 200uM dNTPs (Qiagen Mississauga, ON,
Canada), 0.56 μM final concentration of each primer (Invitrogen Life Technologies), and 1
unit of HotStart Taq (Qiagen, Mississauga, ON, Canada), 1mM MgCl2 (Qiagen), and 10 μl of Q
solution (Qiagen) were added to exon 1 amplification. The protocol used is a Touchdown
protocol from 65°C to 60°C for all primer pairs: 95°C for 15 minutes, 10 cycles of 95°C for 45
seconds, 65°C for 45seconds ( -0.5°C increment at every cycle) and 72°C for 45 seconds.
Followed by 32 cycles of 94°C for 45 seconds, 60°C for 45 seconds and 72°C for 45 seconds.
Final extension was at 72°C during 5 minutes. The PCR products were purified and then
sequenced. Sequence data were analyzed by using the Lasergene software from DNASTAR
(Madison, Wisconsin).
1A: Primers used in Ghent
Exon Forward 5’ to 3’
Reverse 5’ to 3’
1
2a
2b
3
4
5
6
7
8
9
AGGCGAGAGAACGAAGACTG
CCATAATTGTGTTTTTCCAACACCTG
GAATTGCATACATTTATCAAGAAGGGA
GGTCTCAGATGGGCACAAATGC
GGTTATTTTTTCATGCTTATCAAACAC
CGCTATTTTGACATTTCTAGAC
GAGAATCAAATGAAAGAGATAAGA
CCTGTAAGTGTTCATTCAGTATAA
CCGCCCATCAATATCAAAATCCC
CACAGAATCATATTTAGGTATTTT
TCCGCTTTACGTCTGACGTC
CATGTTACACTTTTAAATCTCTAA
CCTTCTGTTCAGCACTAGATG
CCTTAGATCATCATCATGATTTGG
GCCAATACATCCAAACAGGTAAAA
CAAATCTAATATTATCTCTTCTG
CTTGGCCAGACTGGTCTACTTG
GTATAACCAAGTCAGTAAGGCCA
CAGTCATTGGACTTTTAGGTTGCTT
GTTACTTAAAAATATTTCTAAGAT
1B: primers used in Montréal
Exon Forward 5’ to 3’
Reverse 5’ to 3’
1
2
3
4
5
6
7
8
9
GTAAACATGGACGTGGGAGG
TGGTTTCCTGACGATAGTACAAAAT
GCTGTGGCATTTCTCATTTTG
TTGTAGGTCAAGGAAGGAAGAGA
TGGAAACCAACCAAACGTAAC
ATCTAACGGTACTGTGCTTAGTGCT
CAGACAAGGCAACAAAAGTGTC
TTTGGGGACAATGTTCTAAGC
GGCCACATGAGATCAGCTTT
AAATGGGATTTTGGGGAATC
CCTAGCATCACTGTTGTCTACAAAT
GACATTTCTGTTGCCTTGGG
TTTTGCTATAATTTGTCATCTTTCAG
TTACTGTTCCAGGCATTGGG
ATGCCACCATGTCTGGTTAAT
GAATAATGATTTGCAGTATTTCCC
CATACGGGTAATTTGAAGGGTG
CGCCTGGCCCTAGAATAAA
Size
(bp)
340
317
281
324
295
281
242
216
373
377
Size
(bp)
471
461
472
413
430
345
400
384
491