Rapid identification of single nucleotide polymorphisms by fluorescence-based capillary electrophoresis
Genet. mol. res. (Online)
; 1(1): 72-78, Mar. 2002.
Article
in English
| LILACS
| ID: lil-417650
Responsible library:
BR1.1
RESUMO
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
Full text:
Available
Collection:
International databases
Health context:
SDG3 - Target 3.4 Reduce premature mortality due to noncommunicable diseases
Health problem:
Leukemia
Database:
LILACS
Main subject:
Leukemia, Lymphoid
/
DNA Primers
/
Electrophoresis, Capillary
/
Polymorphism, Single Nucleotide
Type of study:
Diagnostic study
/
Observational study
Limits:
Humans
Language:
English
Journal:
Genet. mol. res. (Online)
Journal subject:
Molecular Biology
/
Genetics
Year:
2002
Document type:
Article
Affiliation country:
Spain
Institution/Affiliation country:
Institut d' Investigacions Biomèdiques August Pi i Sunyer/ES