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

A new approach for detection of point mutations has been developed. The non radioactive test system proposed is based on enzymatic ligation of a tandem of three short oligonucleotides B similar to pN(8) + pN(4) + pN(8)' Bio in the presence of a complementary DNA template. The 5'-terminal octanucleoti de B similar to pN(8) 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 oligonu cleotide on a polymer bead, which is then visualized via subsequent treatme nts with streptavidin-alkaline phosphatase conjugate and chromogenic substr ates. Intense staining of the polymer beads is observed when the amount of DNA template (20-mer oligonucleotide) is as low as similar to 10(-14) mel. It is shown that practically no polymer staining is observed when the compl ex formed by the tandem and the 20-mer DNA template contains a mismatch eit her in the tetranucleotide duplex or in the duplex of octanucleotide immobi lized on the beads. This suggests a possibility of using the presented appr oach in test systems for detection of point mutations in PCR-amplified DNA fragments.