ABCMdb

PMID: 1282898

Fortina P, Conant R, Monokian G, Dotti G, Parrella T, Hitchcock W, Kant J, Scanlin T, Rappaport E, Schwartz E, et al.
Non-radioactive detection of the most common mutations in the cystic fibrosis transmembrane conductance regulator gene by multiplex allele-specific polymerase chain reaction.
Hum Genet. 1992 Dec;90(4):375-8., [PubMed]

Sentences

No. Mutations Sentence Comment

3 ABCC7 p.Gly551Asp ABCC7 p.Asn1303Lys ABCC7 p.Gly542* The strategy involves multiplex PCR of exons 10, 11, and 21 within the cystic fibrosis transmembrane conductance regulator (CFTR) gene in a single reaction containing three common oligoprimers and either the four normal or four mutant oligos corresponding to the AF508, G551D, G542X, and N1303K mutations. Login to comment 11 ABCC7 p.Gly551Asp ABCC7 p.Arg553* ABCC7 p.Asn1303Lys ABCC7 p.Gly542* Five mutations have been identified (AF508, G551D, R553X, G542X, and N1303K), which account for 84% of all Caucasian CF chromosomes, and a rapid, reliable and cost-effective screening method is required that includes detection of these prevalent mutations. Login to comment 18 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp ABCC7 p.Arg553* ABCC7 p.Arg553* ABCC7 p.Asn1303Lys ABCC7 p.Asn1303Lys ABCC7 p.Gly542* ABCC7 p.Gly542* Oligonucleotide primers used for mutation detection by multiplex allele-specific polymerase chain reaction (MASPCR) Primers ~ Sequence b pMoV CF-W1468-N CF-508RP AF508 5' IVS-11 R553X-N R553X-M G551D-N G551D-M G542X-N G542X-M 5' IVS-2I N1303K-N N1303K-M 5'-GGCACCATTAAAGAAAATATCATCTT-3' l5 5'-TAGTGTGAAGGGTTCATATGCATAAT-3' 15 5'-GGCACCATTAAAGAAAATATCATTGG-3' 15 5'-CAACTGTGGTTAAAGCAATAGTGT-3' 20 5'-CTAAAGAAATTCTTGCTCG-3' 10 5'-CTAAAGAAATTCTTGCTCA-3' 10 5'-GAAATTCTTGCTCGTTGA C-3' 5 5'-GAAATTCTTGCTCGTTGAT-3 ' 5 5'-GTGTGATTCCACCTTCTCC-3' 2(1 5'-GTGTGATTCCACCTTCTCA-3' 20 5'-AAGAATGATACAAAGCAGACATG-3' 40 5'-CACTGTTCATAGGGATCCAAG-3' 40 5'-CACTGTTCATAGGGATCCAAC-3' 4(1 a Normal (N) and mutant (M) primers b Bold letters indicate single-base mutations c Amount (pMol) of each primer used in multiplex reactions single lane of a gel. Login to comment 22 ABCC7 p.Gly551Asp ABCC7 p.Arg553* ABCC7 p.Gly542* DNA samples were obtained from CF patients with the AF508, G551D, R553X, and G542X mutations. Login to comment 30 ABCC7 p.Gly551Asp ABCC7 p.Gly542* Each reaction contained the three common primers (CF-508RP, 5' IVS-11, and 5' IVS-21) and either four normal or four mutant primer sets for the AF508, G551D, G542X, and NI303K sequences (Table 1). Login to comment 31 ABCC7 p.Gly551Asp ABCC7 p.Arg553* ABCC7 p.Arg553* Screening for the R553X mutation was done in two additional PCR reactions (with normal or mutant primer) since detection of the G551D and R553X mutations was not possible in the same lane on electrophoresis. Login to comment 33 ABCC7 p.Gly551Asp ABCC7 p.Gly542* 5' IVS-11 5' IVS-21 f,f A AA CF-W1468-N.--1~(139 bp) d'---- CF-508RP AFS08 .~1P.(136bp) <1~ CF-508RP ._~ (174 bp) ~ G542X (N or M) 5' IVS-11 (202 bp) ~ G551D (N or M) 5' IVS-21 ~ (240 bp) ~I~N1303K (N or M) Fig. 1. Login to comment 36 ABCC7 p.Gly551Asp ABCC7 p.Gly542* ABCC7 p.Gly542* Multiplex amplification is accomplished using common primers (CF-508RP, 5' IVS-I1, and 5' IVS-21) and a mixture of either the four normal (CF-W1468-N, G551D-N, G542X-N, and N 1303K-N) or four mutant primer (AF508, G55 l DM, G542X-M, and NI303K-M) sets (Table 1) in each PeR reaction. Login to comment 47 ABCC7 p.Gly551Asp ABCC7 p.Asn1303Lys ABCC7 p.Gly542* All amplifications were done with either normal (N) or mutant (M) primers corresponding to the AF508, G551D, G542X, and N1303K regions of the human CFTR gene. Login to comment 48 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp ABCC7 p.Asn1303Lys ABCC7 p.Asn1303Lys ABCC7 p.Gly542* ABCC7 p.Gly542* Amplifications were done with DNA from normal controls using normal primers (lane 2 N1303K-N and 5' IVS-21; lane 4 G551D-N and 5' IVS-11; lane 6 G542X-N and 5' IVS-11; and lane 8 CF-W1468-Nand CF-508RP) or mutant primers for the same regions (lane 3 N1303K-Mand 5' IVS-21; lane 5 G551D-M and 5' IVS-11;lane 7 G542X-M and 5' IVS-11; and lane 9 AF508and CF-508RP). Login to comment 52 ABCC7 p.Gly551Asp ABCC7 p.Asn1303Lys ABCC7 p.Gly542* Multiplex amplification of four regions in normal DNA (lanes 2-5) encompassing AF508, G551D, G542X, and N1303K mutations with normal primer (N; lanes 2 and 4) or mutant (M) primer sets (lanes 3 and 5). Login to comment 53 ABCC7 p.Gly542* ABCC7 p.Gly542* Multiplex amplification was done using separate reactions containing a mixture of either the normal or corresponding mutant primer sets with DNA from a heterozygote for the AF508mutation (lanes 6 and 7), DNA from a heterozygote for the G542X mutation (lanes 8 and 9), and DNA from a compound heterozygote for the AF508and G542X mutations (lanes 10 and 11), respectively. Login to comment 61 ABCC7 p.Gly551Asp ABCC7 p.Gly542* We have successfully detected the G542X, G551D, and AF508 mutations using this multiplex approach. Login to comment 62 ABCC7 p.Asn1303Lys Patient samples with the N1303K mutation were not, however, available for testing. Login to comment 63 ABCC7 p.Gly542* ABCC7 p.Gly542* Data are shown confirming the following genotypes: a AF508 heterozygote (lanes 6 and 7), a G542X heterozygote (lanes 8 and 9), and a AF508/G542X compound heterozygote (lanes 10 and 11). Login to comment 73 ABCC7 p.Gly542* For example, in one sample, the four normal primers produce normal amplification products, while the mutant primer set yields only a 174-bp fragment, consistent with heterozygosity for the G542X mu- tation. Login to comment