Summary
A rapid, simple, nonradioactive method for detection of four common mutations causing cystic fibrosis (CF) has been developed combining multiplexing with allele-specific polymerase chain reaction amplification. This approach (MASPCR) provides an easy assay for direct genotyping of normal and mutant CF alleles in homozygotes and heterozygotes. 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 ΔF508, G551D, G542X, and N1303K mutations. Primers are chosen so that the size of the four PCR products differ, thereby facilitating detection on agarose gels following amplification in the same reaction. Patient samples are primed with either four normal or four mutant oligo mixtures, and PCR products run in parallel on gels to detect band presence or absence. This approach provides a simple and potentially automated method for cost-effective population screening.
Similar content being viewed by others
References
Ballabio A, Gibbs RA, Caskey CT (1990) PCR test for cystic fibrosis deletion. Nature 243:220
Beggs AH, Koenig M, Boyce FM, Kunkel LM (1990) Detection of 98% of DMD/BMD gene deletions by polymerase chain reaction. Hum Genet 86:45–48
Chamberlain JS, Gibbs RA, Ranier JE, Nguyen PN, Caskey CT (1988) Deletion screening of the Duchenne muscular dystrophy locus via multiplex DNA amplification. Nucleic Acids Res 16:11141–11156
Cremonesi L, Belloni E, Magnani C, Seia M, Ferrari M (1992) Multiplex PCR for rapid detection of three mutations in the cystic fibrosis gene. PCR Methods Applic 1:297–298
Cutting GR, Kasch LM, Rosenstein BJ, Zielenski J, Tsui L-C, Antonarakis SE, Kazazian HH Jr (1990) A cluster of cystic fibrosis mutations in the first nucleotide-binding fold of the cystic fibrosis conductance regulator protein. Nature 346:366–369
Cystic Fibrosis Genetic Analysis Consortium (1990) World-wide survey of the ΔF508 mutation: report from the Cystic Fibrosis Genetic Analysis Consortium. Am J Hum Genet 47:354–359
Dean M, White MB, Amos J, Gerrard B, Stewart C, Khaw K-T Leppert M (1990) Multiple mutations in highly conserved residues are found in mildly affected cystic fibrosis patients. Cell 61:863–870
Devoto M, Ronchetto P, Fanen P, Orriols JJT, Romeo G, Goossens M, Ferrari M, Magnani C, Seia M, Cremonesi L (1991) Screening for non-DeltaF508 mutations in five exons of the cystic fibrosis transmembrane conductance regulator (CFTR) gene in Italy. Am J Hum Genet 48:1127–1132
Fortina P, Monokian G, Conant R, Dotti G, Hitchcock W, Rappaport E, Schvvartz E, Surrey S (1991) Detection of 2 common β-thalassemia mutations in Mediterraneans using multiplex allele-specific PCR amplification. Blood 78 [Suppl 1]:194a
Fortina P, Conant R, Parrella T, Rappaport E, Scanlin T, Schwartz E, Robertson JM, Surrey S (1992) Fluorescence-based, multiplex allele-specific PCR (MASPCR) detection of the ΔF508 deletion in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Mol Cell Probes 6:353–356
Gibbs RA, Nguyen P-N, Edwards A, Civitello AB, Caskey CT (1990) Multiplex DNA deletion detection and exon sequencing of the hypoxanthine phosphoribosyltransferase gene in Lesch-Nyhan families. Genomics 7:235–244
Haliassos A, Chomel JC, Tesson L, Baudis M, Kruh J, Kaplan JC, Kitzis A (1989) Modification of enzymatically amplified DNA for the detection of point mutations. Nucleic Acids Res 17:3606
Kerem K, Rommens JM, Buchanan JA, Markiewicz D, Cox TK, Chakravarti A, Buchwald M, Tsui L-C (1989) Identification of the cystic fibrosis gene: genetic analysis. Science 245:1073–1080 (Erratum Science 245:1437)
Kerem B, Zielenski J, Markiewicz D, Bozon D Gazit E, Yahav J, Kennedy D, Riordan JR, Collins FS, Rommens JM, Tsui L-C (1990) Identification of mutations in regions corresponding to the two putative nucleotide (ATP)-binding folds of the cystic fibrosis gene. Proc Natl Acad Sci USA 87:8447–8451
Kwok S, Higuchi R (1989) Avoiding false positive with PCR. Nature 339:237
Lemna WK, Feldman GL, Kerem B, Fernbach SD, Zevkovich EP, O'Brien WE, Riordan JR, Collins FS, Tsui L-C, Beaudet AL (1990) Mutation analysis for heterozygote detection and prenatal diagnosis of cystic fibrosis. N Engl J Med 322:291–296
Ng ISL, Pace R, Richard MV, Kobayashi K, Kerem B, Tsui L-C, Beaudet AL (1991) Methods for analysis of multiple cystic fibrosis mutations. Hum Genet 87:613–617
Nozari G, Rahbar S, Wallace RB (1986) Discrimination among the transcripts of the allelic human beta-globin genes βA, βS and βC using oligodeoxynucleotide hybridization probes. Gene 43:23–28
Orita M, Iwahana H, Kanazawa H, Hayashi K, Sekiya T (1989) Detection of polymorphisms of human DNA by gel electrophoresis as single-strand conformation polymorphisms. Proc Natl Acad Sci USA 86:2766–2770
Poncz M, Solowiejzcyk D, Harpel B, Moroy Y Schwartz E, Surrey S (1982) Construction of human gene libraries from small amounts of peripheral blood; Analysis of β-like globin genes. Hemoglobin 6:27–33
Romeo G, Devoto M (1990) Population analysis of the major mutation in cystic fibrosis. Hum Genet 85:391–392
Rommens JM, Kerem B, Greer W, Chang P, Tsui L-C, Ray P (1990) Rapid nonradioactive detection of the major cystic fibrosis mutation. Am J Hum Genet 46:395–396
Saiki RK, Gelfand DH, Stoffel S, Scharf SJ, Higuchi R Horn GT, Mullis KB, Erlich HA (1988) Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239:487–489
Saiki RK, Walsh PS, Levenson CH, Erlich HA (1989) Genetic analysis of amplified DNA with immobilized sequence-specific oligonucleotide probes. Proc Natl Acad Sci USA 86:6230–6234
Wood WI, Gitschier J, Lasky LA, Lawn RM (1985) Base composition-independent hybridization in tetramethylammonium chloride: a method for oligonucleotide screening of highly complex gene libraries. Proc Natl Acad Sci USA 82:1585–1588
Zielenski J, Rozmahel R, Bozon D, Kerem B, Grzelczak Z, Riordan JR, Rommens JM, Tsui L-C (1991) Genomic DNA sequence of the cystic transmembrane conductance regulator (CFTR) gene. Genomics 10:214–228
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Fortina, P., Conant, R., Monokian, G. 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 90, 375–378 (1992). https://doi.org/10.1007/BF00220462
Received:
Revised:
Issue Date:
DOI: https://doi.org/10.1007/BF00220462