Multiple Fluorescence-Based PCR-SSCP Analysis Using Internal Fluorescent Labeling of PCR Products. (September 1996)

• Record Type: Journal Article
• Authors: Iwahana, H.; Fujimura, M.; Takahashi, Y.; Iwabuchi, T.; Yoshimoto, K.; Itakura, M.
• Is Part Of: Biotechniques. Volume 21:Number 3(1996)
• Page Start: 510
• Page End: 519
• Publication Date: 1996-09
• Subjects: Biology, Experimental -- Periodicals; Molecular biology -- Periodicals; Medical technology -- Periodicals; Biology, Experimental; Medical technology; Molecular biology; Clinical Laboratory Techniques -- Periodicals; Research -- Periodicals; Medical Laboratory Science -- Periodicals; Periodicals; Electronic journals; 570
• DOI: 10.2144/96213rr05
• Languages: English
• ISSNs: 0736-6205

Abstract

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-PCRSSCP) analysis. PCR-amplified fluorescent DNA fragments, which were internally labeled by adding fluorescent dUTPs ([F]dUTPs) to the PCR mixture, were heatdenatured 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 TM 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.