A New Approach to Revealing Point Mutations in DNA Analyzed by Colorimetric Detection

Abstract

A new approach to detection of point mutations in an amplified DNA was developed. The approach is based on highly selective ligation (T4 DNA ligase) of a tandem of short oligonucleotides one of which contains the biotin group. The ligation product is formed only when the hybridization complex DNA/tandem is formed and the tandem is perfect. The hybridization complex DNA/(biotinylated ligation product) was separated from the biotinylated component of the tandem by UV‐immobilization of the reaction mixture on a nylon membrane. The immobilized hybridization complex was detected colorimetrically by a streptavidin‐alkaline phosphatase cojugate with a chromogenic substrate.

Keywords:

• T4 DNA ligase
• Ligation
• Hybridization
• High‐specific probe
• Colorimetric detection
• Point mutation
• HIV
• ΔF508

Acknowledgments

This work was supported by the MCB Program, Russian Academy of Sciences, Lavrentyev Competition of Young Scientists, Siberian Division of the Russian Academy of Sciences (2002), RFBR‐INNO (grant No. 02‐04‐08077), and integration projects of Siberian Division of the Russian Academy of Sciences (No. 126) and CRDF NO‐008‐X1.