Table 1_Supplementary Data:
A) Primers used for the amplification of CAPN3 cDNA in blood and muscle.
Forward Primer
(5’-3’)
Reverse Primer
(5’-3’)
1F:
ttgaactggatgtggacact
1R:
gcaggcagtcatctataacc
2R:
ccttctcatctttgtccaca
2R6+:
ggaatgattgtccggtc
2R:
ccttctcatctttgtccaca
3R:
gcgttgtacaggaagaagtc
4R:
tctcgtagctgttgatggtg
5R:
tgggacctttgaggtaaat
4R16+:
cttttgcttatcagggcttg
5R:
tgggacctttgaggtaaat
2F: gagccaacagaactgacatc
2F:
gagccaacagaactgacatc
2F6+:
ggctgctccattgatgat
3F
gtatgagacaagaatggcct
4F:
gtaccgtctgaagctcctg
5F:
aacaagattaaggcctggc
4F:
gtaccgtctgaagctcctg
4F16+:
atcatcttcgtttcggaca
Amplified
fragment (pb)
Position of amplified region on CAPN3 cDNA
(NM_000070)
680
174 (5’UTR)  853 (Exon 4)
810/666
650 (Exon 2)  1459 (Exon 9)
613
650 (Exon 2)  1262 (Exon 6-7 boundary)
367
1093 (Exon 5-6 boundary)  1459 (Exon 9)
624
1269 (Exon 7)  1892 (Exon 13)
863/845/731
1683 (Exon 11)  2545 (Exon 21)
393
2470 (Exon 20)  2862 (Exon 24)
508
1683 (Exon 11)  2208 (Exon 16)
753
2110 (Exon 16)  2862 (3’UTR)
- In bold, the combination of primers used to perform LGMD2A diagnosis.
- It should be noted that the design of these primers does not allow the identification of
those mutations which, if exist, would cause exon 6 or exon 16 skipping in
heterozygosis since we would be amplifying the wild-type allele. However, this
limitation could be resolved performing a RTQ-PCR designed in the exon of interest.
B) PCR programs:
1) 60-55 TouchDown program:
- Initial denaturation of 5 minutes at 94ºC
- 4 PCR cycles with an annealing temperature of 60ºC, 10 PCR cycles with a
decreasing annealing temperature of 0.5º/cycle (from 60ºC to 55ºC), 20 additional
PCR cycles with an annealing temperature of 55ºC
- Final extension of 5 minutes at 72º
2) 60-50 TouchDown program:
- Initial denaturation of 5 minutes at 94ºC
- 4 PCR cycles with an annealing temperature of 60ºC, 10 PCR cycles with a decreasing
annealing temperature of 1º/cycle (from 60ºC to 50ºC), 20 additional PCR cycles with
an annealing temperature of 50ºC
- Final extension of 5 minutes at 72º
3) Extaq Program:
- Initial denaturation of 5 minutes at 94ºC
- 30 cycles: 98ºC/10sec; 60ºC/30sec; 72ºC/1min30sec
- Final extension of 7 minutes at 72º