Montse Bernat, Esther Titos and Joan Clària

DNA Unit, Hospital Clínic, Institut d´Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Barcelona 08036, Spain

Correspondence author: J. Clària

E-mail: jclaria@clinic.ub.es

Genet. Mol. Res. 1 (1): 72-78 (2002)

Received March 7, 2002

Published March 28, 2002

ABSTRACT. We describe the application of two different fluorescence-based techniques (ddNTP primer extension and single-strand conformation polymorphism (SSCP)) to the detection of single nucleotide polymorphisms (SNPs) by capillary electrophoresis. The ddNTP primer extension technique is based on the extension, in the presence of fluorescence-labeled dideoxy nucleotides (ddNTP, terminators), of an unlabeled oligonucleotide primer that binds to the complementary template immediately adjacent to the mutant nucleotide position. Given that there are no unlabeled dNTPs, a single ddNTP is added to its 3’ end, resulting in a fluorescence-labeled primer extension product which is readily separated by capillary electrophoresis. On the other hand, the non-radioisotopic version of SSCP established in this study uses fluorescent dye to label the PCR products, which are also analyzed by capillary electrophoresis. These procedures were used to identify a well-defined SNP in exon 7 of the human p53 gene in DNA samples isolated from two human cell lines (CEM and THP-1 cells). The results revealed a heterozygous single-base transition (G to A) at nucleotide position 14071 in CEM cells, proving that both fluorescence-based ddNTP primer extension and SSCP are rapid, simple, robust, specific and with no ambiguity in interpretation for the detection of well-defined SNPs.

Key words: Single nucleotide polymorphisms, Fluorescence-based techniques, THP-1 cells, CEM cells, p53 gene