Multiple fluorescence-based PCR-SSCP analysis using internal fluorescent labeling of PCR products.
The method to internally label PCR products with multiple colored fluorescent dyes was developed and applied to multiple fluorescence-based PCR single-stranded conformational polymorphism (MF-PCR-SSCP) analysis. PCR-amplified fluorescent DNA fragments, which were internally labeled by adding fluorescent dUTPs ([F]dUTPs) to the PCR mixture, were heat-denatured and applied to a nondenaturing polyacrylamide gel (SSCP gel) set on an automated DNA sequencer with a gel temperature-controlling system. The image data were analyzed by GENESCAN 672 software. In spite of differences in species and amount of integrated [F]dUTPs, the SSCP profiles were not significantly affected, even by the different labeling methods used-internal labeling or post-labeling at the 3' ends-in regard to the three different [F]dUTPs examined. However, the salt concentration of the solution containing the DNA samples affected the SSCP profiles. The internally labeled [F]dUTP-containing DNA fragments beyond 1000 bp in length were successfully digested with restriction endonucleases and subjected to SSCP analysis. MF-PCR-SSCP analysis with internal fluorescence labeling affords a simple and sensitive method to detect alterations in DNA sequences.
Iwahana
H
H
University of Tokushima, Japan.
Fujimura
M
M
Takahashi
Y
Y
Iwabuchi
T
T
Yoshimoto
K
K
Itakura
M
M
D004252:DNA Mutational Analysis / Q000379:methods*; D004262:DNA Restriction Enzymes; D004273:DNA, Neoplasm / Q000032:analysis / Q000235:genetics*; D004591:Electrophoresis, Polyacrylamide Gel; D005456:Fluorescent Dyes; D005470:Fluorometry; D011905:Genes, ras; D006801:Humans; D007091:Image Processing, Computer-Assisted; D016133:Polymerase Chain Reaction / Q000379:methods*; D018807:Polymorphism, Single-Stranded Conformational; D011516:Prothrombin / Q000235:genetics; D014407:Tumor Cells, Cultured
D004273:DNA, Neoplasm; D005456:Fluorescent Dyes; D011516:Prothrombin; D004262:DNA Restriction Enzymes