Table 1. Screening for FLG mutations: PCR, Restriction digest and/or sequencing conditions.
| Mutation | Primers | Cycling
conditions |
Restriction digest or sequencing strategy |
|---|---|---|---|
| 501X | F 5’ CAC GGA AAG GCT GGG CTG A 3’
R 5’ ACC TGA GTG TCC AGA CCT ATT 3’ |
94°C 5min (x1)
94°C 30sec 57°C 30sec 72°C 1min (x35) 72°C 5min (x1) |
R501X was screened by restriction digest of a
312bp product R501X introduces a NlaIII restriction site. To determine presence of the R501X mutation digest PCR product with NlaIII: Run digests on a 3% (w/v) agarose gel. Wildtype allele cuts to give fragments of 182bp, 109bp and 21bp. Mutant allele cuts to give fragments of 127bp, 109bp, 55bp and 21bp. |
| 2282del4 | F 5’ AAT AGG TCT GGA CAC TCA GGT 3’
R 5’ GGG AGG ACT CAG ACT GTT T 3’ |
94°C 5min (x1)
94°C 30sec 57°C 30sec 72°C 1min (x35) 72°C 5min (x1 |
2282del4was screened by restriction digest of a
811bp product 2282del4 introduces a DraIII restriction site. To determine presence of the 2282del4 mutation digest PCR product with DraIII: Run digests on a 2% (w/v) agarose gel. Wildtype allele 811bp. Mutant allele cuts to give fragments of 671bp and 140bp |
| R2447X | F 5’ CCA CAC GTG GCC GGT CAG CA 3’
R 5’ GTC CTG ACC CTC TTG GGA CGT 3’ |
94°C 5min (x1)
94°C 30sec 64°C 30sec 72°C 1min (x35) 72°C 5min (x1 |
R2447X was screened by restriction digest of a
185bp product. R2447X introduces a NlaIII restriction site. To determine presence of the R2447X mutation digest PCR product with NlaIII: Run digests on a 10% TBE polyacrylamide gel. Wildtype allele cuts to give fragments of 95bp, 69bp and 21bp. Mutant allele cuts to give fragments of 69bp, 55bp, 40bp and 21bp. |
| S1040X | F 5’ CCAGACAATCAGGAACTCC 3’
R 5’ ATGAGTGCTCACCTGGTAGAT 3’ |
94°C 3 min (x1)
94°C 30sec 62°C 30sec 72°C 1 min (x34) 72°C for 5 min |
S1040X was screened by restriction digest of a
375bp PCR product. S1040X creates a BtsI site, digestion with this enzyme yields fragments of 251bp and 124bp (mutant allele) whereas the wildtype allele is uncut (375bp). |
| G1139X | F 5’ CCAGACAATCAGGAACTCC 3
R 5’ ATGAGTGCTCACCTGGTAGAT 3 |
94°C 3 min
94°C 30sec 62°C 30sec 72°C 1 min (x34) 72°C 5 min |
G1139X was screened by restriction digest of a
653bp PCR product. G1139X abolishes a TspRI site, digestion with this enzyme yields fragments of 393bp, 157bp, 51bp, 31bp, 15bp and 6bp (wildtype allele) whereas the mutant allele generates fragments of 550bp, 51bp, 31bp, 15bp and 6bp |
| R3419X | F 5’GCCCATGGGCGGACCAGGA 3’
R 5’GCTTCATGGTGATGCGACCA 3’ |
94°C 3 min
94°C 30sec 61°C 30sec 72°C 1 min(x34) 72°C 5 min |
R3419X was screened by restriction digest of a
332bp PCR product R3419X creates a NlaIII site and digestion with this enzyme yields fragments of 307bp, 14bp, 7bp and 4bp (wildtype allele) whereas the mutant allele produces fragments of 252bp, 55bp, 14bp, 7bp and 4bp |
| 3702delG | F 5’ GCA AGC AGA CAA ACT CGT AAG 3’
R 5’CAG ACA ACC TCT CGG AGT CG 3’’ |
94°C 3 min
94°C 30sec 62°C 30sec 72°C 1 min (x34) 72°C 5 min |
3702delG was screened by direct sequencing of
a ~217bp PCR product. Use reverse primer as the sequencing primer. Sequenced on Applied Biosystems (Waltham, Massachusetts, USA) 3100 DNA sequencer |
| Y2092X | F 5’ CA CAG TCA GTG TCA GCA CAG 3’
R 5’ GGC TAA CAC TGG ATC CCC GGG 3’ |
94°C 3 min
94°C 30sec 62°C 30sec 72°C 1 min (x34) 72°C 5 min |
Y2092X was screened by direct sequencing
of a 574bp PCR product. Use forward primer a sequencing primer Sequenced on Applied Biosystems (Waltham, Massachusetts, USA) 3100 DNA sequencer |
| S3247X | F 5’ GTA ATG AGG AAC AAT CAG GAG ACA 3’
R 5’ CTG GGG TGT CTG GAG CCG TGC 3 |
94°C 5min
94°C 30sec 64°C 30sec 72°C 45sec (x34) 72°C 5min |
S3247X was screened by direct amplification of a
268bp product. Use forward primer a sequencing primer Sequenced on Applied Biosystems (Waltham, Massachusetts, USA) 3100 DNA sequencer |