Development of a colorimetric test system for detection of point mutations via ligation of a tandem of short oligonucleotides on methacrylate beads

• Published in: Molecular biology (New York) 2000-05, Vol.34 (3), p.321-327
• Main Authors: Skobeltsyna, L M, Pyshnyi, D V, Shishkina, I G, Tabatadze, D R, Dymshits, G M, Zarytova, V F, Ivanova, E M
• Format: Article
• Language: English
• Subjects: Alkaline phosphatase; Bacteria; Biotin; Colorimetry; Complementary DNA; Deoxyribonucleic acid; DNA; Mutation; Oligonucleotides; Point mutation; Polymers; Streptavidin; Test systems
• ISSN: 0026-8933; 1608-3245
• DOI: 10.1007/BF02759660

A new approach for detection of point mutations has been developed. The nonradioactive test system proposed is based on enzymatic ligation of a tandem of three short oligonucleotides B∼pN8+pN4+pN′8 Bio in the presence of a complementary DNA template. The 5′-terminal octanucleotide B∼pN8 is immobilized on polymer methacrylate beads (B) and the 3′-terminal octanucleotide pN′8 Bio contains a biotin residue at the 3′-phosphate. Ligation of the tandem produces a 20-mer biotinylated oligonucleotide on a polymer bead, which is then visualized via subsequent treatments with streptavidin-alkaline phosphatase conjugate and chromogenic substrates. Intense staining of the polymer beads is observed when the amount of DNA template (20-mer oligonucleotide) is as low as ∼10−14 mol. It is shown that practically no polymer staining is observed when the complex formed by the tandem and the 20-mer DNA template contains a mismatch either in the tetranucleotide duplex or in the duplex of octanucleotide immobilized on the beads. This suggests a possibility of using the presented approach in test systems for detection of point mutations in PCR-amplified DNA fragments.