ONLINE METHOD SUPPLEMENT
Genotyping methods
For the detection of the F508del mutation and the TGmTn variants, exon 10 and exon 9 were
respectively amplified in a 50µl volume polymerase chain reaction (PCR), containing 1xGold Buffer
(Applied Biosystems, California, USA), 0.2 mM dNTPs, 2mM MgCl2, 2.5 U AmpliTaq Gold (Applied
Biosystems), 5 pmol of each primer and 150ng of DNA. The primers used for amplification can be
found in supplementary table 1. Reactions were performed using the following amplification
profile in a PCR System 9700 thermal cycler (Applied Biosystems): denaturation at 94˚C for 5
minutes, followed by 30 cycles of 94˚C for 30 seconds, 57˚C for 30 seconds and 72˚C for 1 minute,
and a final extension step for 5 minutes at 72˚C. Subsequently, individuals carrying the F508del
variant were detected by heteroduplex formation analysis of the exon 10 fragments in nondenaturing 6% polyacrylamide gel electrophoresis (PAGE), as previously described (1).
For the identification of the TGmTn variants, the PCR reaction of exon 9 was followed by a
sequencing reaction using the sense primer 5'-TAA TGG ATC ATG GGC CAT GT-3', and antisense primer 5'-AAA AAA GAG ACA TGG ACA CCA-3', run on an ABI PRISM® 3100 genetic
analyser (Applied Biosystems). Sequences were then analysed using Sequencing Analysis
Software and SeqScape Software (Applied Biosystems).
For the genotyping of the 4 additional CFTR polymorphisms (rs1800503, rs213950, rs1042077 and
rs1800136), we used a multiplex PCR and multiplex single nucleotide primer extension assay. The
primers used for amplification of the genomic regions and the single nucleotide primer extension
oligonucleotides for the CFTR SNPs in these regions are given in supplementary table 1 and 2.
Initial PCR reactions were performed in a volume of 50µl containing 1xGold Buffer (Applied
Biosystems), 0.2 mM dNTPs, 2 mM MgCl2, 2.5 U AmpliTaq Gold (Applied Biosystems) and 150ng
of DNA; primer concentrations ranged from 2.5 to 10 pmol. The mixture was then incubated in a
thermal cycler (PCR System 9700, Applied Biosystems) using the following amplification profile:
denaturation at 95˚C for 5 minutes, 45 cycles of 95˚C for 40 seconds, 54˚C for 40 seconds and
72˚C for 2 minutes, followed by a final extension step for 10 minutes at 72˚C. PCR products were
verified by electrophoresis on 2% ethidium bromide stained agarose gels.
For the single nucleotide primer extension assays, the SNaPshot® Multiplex System SNP
detection kit (Applied Biosystems) was used. This assay is based on a dideoxynucleotide
triphosphate (ddNTP) single base extension of an unlabelled oligonucleotide primer, which ends
just 5’ to the SNP that will be typed. By using varying lengths of primers, different SNPs can be
typed in a multiplex reaction. Free primers and dNTPs of the PCR reaction were then removed by
incubating 7.5 µl of the PCR reaction with 2.5 U shrimp alkaline phosphatase (SAP, Amersham
Biosciences) and 2 U of Exonuclease I (ExoI, Amersham Biosciences, Buckinghamshire, United
Kingdom) at 37˚C for 1 hour. Subsequently, the enzymes were inactivated at 75˚C for 15 min. The
extension reaction was prepared with 5 µl SNaPshot Multiplex Ready Reaction Mix (Applied
Biosystems), 2.8 µl of SAP/ExoI treated PCR products, and 0.2–1.25 mM of each unlabelled
primer. The extension was performed in a thermal cycler (PCR System 9700, Applied Biosystems),
using the following temperature profile: 25 cycles of denaturation at 96˚C for 10 seconds,
annealing at 50˚C for 5 seconds, and extension at 60˚C for 30 seconds. Unincorporated ddNTPs
were then removed with 1 U of SAP for 1 hour at 37˚C, followed by inactivation of the enzyme at
75˚C for 15 min. Then, 0.8 µl of the SNaPshot® reaction was mixed with 9 µl of Hi-Di™ formamide
(Applied Biosystems) and 0.5 µl GeneScan™-120 Liz™ size standard (Applied Biosystems). After
denaturation at 95˚C for 5 minutes, the mixture was resolved on an ABI PRISM® 3100 genetic
analyser (Applied Biosystems) and analysed with GeneScan® v2.5 analysis software (Applied
Biosystems).
References
(1) Cuppens H, Loumi O, Marynen P, Cassiman JJ. Identification of a new frameshift mutation
and a duplication polymorphism in the CFTR gene in the Algerian population. Hum Mol
Genet 1992 Jul;1(4):283-284.
Target SNP
rs number
Amplified
fragment
1001+11C/T
rs1800503
Exon 6b
TGmTn
rs4148705
variants
rs1805177
1540 A/G
M470V
Primer 1
5'-CAT TTG ATT GTC ACA AAC ATC-3'
Primer 2
5'-CAG TGA AAG ACT TGT GAT TAC C-3'
Primer 1
5'-ACA GTG TTG AAT GTG GTG CA-3'
Primer 2
5'-TAA TGG ATC ATG GGC CAT GT-3'
Primer 1
5'-GCA GAG TAC CTG AAA CAG GA-3'
Primer 2
5'-CAT TCA CAG TAG CTT ACC CA-3'
Primer 1
5'-GTA TAC ATC CCC AAA CTA TC-3'
Primer 2
5'-CAC TTA GAT TCA AGT AAT ACT ATT C-3'
Primer 1
5'-ACT ATT GCC AGG AAG CCA TT-3'
Primer 2
5'-GGA CAC AGC AGT TAA ATG TG-3'
Exon 9
rs213950
Exon 10
2694 T/G
rs1042077
Exon 14a
T854T
4521 G/A
rs1800136
Q1463Q
Nucleotide sequence
Exon 24
Supplementary table 1. Primers for PCR amplification of CFTR fragments.
Target SNP
rs number
Nucleotide sequence
Product
length
1001+11C/T
rs1800503
5'-AAA AAA GAA AAC TTA AGA CAG TAA GTT GTT-3'
31
rs213950
5'-AAA AAA AAA AAA AAT TTC CAG ACT TCA CTT CTA ATG-3'
37
1540 A/G
M470V
2694 T/G
rs1042077
T854T
4521 G/A
rs1800136
5'-AAA AAA AAA AAA AAA AAA AAA TGA CTA CAT GGA ACA CAT ACC TTC
GAT ATA TTA C-3'
5'-CGG AAC TCA AGC AAG TGC AAG TCT AAG CCC CA-3'
Q1463Q
Supplementary table 2. Single nucleotide extension primers for CFTR polymorphisms.
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