Rapid capillary zone electrophoresis in isoelectric histidine buffer: high resolution of the poly-T tract allelic variants in intron 8 of the CFTR gene.
The poly-T tract in intron 8 of the cystic fibrosis conductance transmembrane regulator (CFTR) gene exists in three variants, 5T, 7T, and 9T. The 7T and 9T variants generate a predominantly normal transcript, whereas the 5T variant engenders an anomalous product. The analysis of the poly-T tract is assuming increasing relevance, both to assess the implication of the CFTR gene in congenital bilateral absence of the vas deferens and to evaluate genotype-phenotype correlation in cystic fibrosis. Mapping of the poly-T tract has been performed by cumbersome and time-consuming methodologies. Capillary zone electrophoresis, combined with laser-induced fluorescence detection, was introduced for a rapid separation of the poly-T tract amplified products. As separation buffer, we adopted 200 mmol/L histidine (pH = pI = 7.6), and the capillary was filled with 10% polyacrylamide, allowing separations in less than 10 min. Capillary zone electrophoresis results were in perfect agreement with dot-blot analysis.
Gelfi
C
C
Instituto Di Technologie Biomedicrye, Consiglio Nazionale delle Ricerche, Milan, Italy.
Perego
M
M
Righetti
P G
PG
Cainarca
S
S
Firpo
S
S
Ferrari
M
M
Cremonesi
L
L
D000483:Alleles; D002021:Buffers; D002648:Child; D003550:Cystic Fibrosis / Q000235:genetics / Q000473:pathology; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000235:genetics*; D019075:Electrophoresis, Capillary; D005453:Fluorescence; D006639:Histidine; D006801:Humans; D007438:Introns / Q000235:genetics*; D007834:Lasers; D008297:Male; D011071:Poly T / Q000235:genetics*; D016133:Polymerase Chain Reaction; D014649:Vas Deferens / Q000002:abnormalities
D002021:Buffers; C505032:CFTR protein, human; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D011071:Poly T; D006639:Histidine