Development of a colorimetric test system for detection of point mutations via ligation of a tandem of short oligonucleotides on methacrylate beads |
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Authors: | L M Skobeltsyna D V Pyshnyi I G Shishkina D R Tabatadze G M Dymshits V F Zarytova E M Ivanova |
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Institution: | (1) Institute of Cytology and Genetics, Siberian Division, Russian Academy of Sciences, 630090 Novosibirsk, Russia;(2) Novosibirsk Institute of Bioorganic Chemistry, Siberian Division, Russian Academy of Sciences, 630090 Novosibirsk, Russia |
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Abstract: | 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. |
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Keywords: | point mutation DNA diagnostics oligonucleotides ligation T4 DNA ligase polymer support (beads) biotin streptavidin-alkaline phosphatase |
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