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L. M. Skobeltsyna D. V. Pyshnyi I. G. Shishkina D. R. Tabatadze G. M. Dymshits V. F. Zarytova E. M. Ivanova 《Molecular Biology》2000,34(3):321-327
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. 相似文献
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Pyshnyi D. V. Skobeltsyna L. M. Gushchina E. N. Pyshnaya I. A. Shishkina I. G. Dymshits G. M. Zarytova V. F. Ivanova E. M. 《Molecular Biology》2000,34(6):840-851
Ligation of a tandem of short oligonucleotides was proposed for detecting single-base substitutions in amplified DNA fragments. An octamer–tetramer–octamer tandem was ligated on a 20-mer template with T4 DNA ligase. As shown with radiolabeled oligonucleotides, the efficiency and selectivity of ligation did not change with an octamer linked to a water-soluble carrier based on polyethylene glycol (MPEG), while ligation was somewhat lower with the octamer immobilized on methacrylate beads (DMEG). In both cases, polymer attachment improved the discrimination of 20-mer templates with single-base substitutions in the binding site for the tetramer or for the immobilized octamer. Tandems with a radiolabeled or biotinylated component were also efficiently ligated on amplified DNA fragments. The data obtained with DNA fragments of HIV-1 strains bru and rf demonstrate the possibility of reliable detection of single-base substitutions via ligation of a tandem and colorimetric detection of the immobilized ligation product with the streptavidin–alkaline phosphatase technique. 相似文献
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Larisa M. Skobeltsyna Dmitrii V. Pyshnyi Eugenia M. Ivanova Vadim A. Stepanov Valery P. Puzyrev Grigory M. Dymshits Vladimir N. Kharkov Valentina F. Zarytova 《Molecular biotechnology》2010,45(1):1-8
We propose a novel universal methodology, Short Oligonucleotide Tandem Ligation Assay (SOTLA), for SNP genotyping. SOTLA is
based on using a tandem of short oligonucleotide (TSO) probes consisting of three fragments: the core oligonucleotide and
two flanking oligomers, one of which is immobilized onto a solid support and another one contains the biotin label. TSO is
self-associated on a complementary DNA template, forms the complex containing two nicks, which are efficiently ligated with
DNA ligase giving biotinylated oligonucleotide covalently bound to polymer beads. No ligation of TSO on an imperfect DNA template
bearing the base substitution in the core binding site is occurred. We used SOTLA for the highly selective SNP analysis in
different DNA fragments of human Y chromosome. Comparison of SOTLA results with those of PCR-RFLP and allele-specific PCR
techniques demonstrates that SOTLA ensures the univocal reliable SNP analysis in different PCR fragments varying in length
and base composition. The fundamental difference between SOTLA and well known OLA approaches while using T4 DNA ligase is
that the accuracy of SNP analysis in OLA is ensured only by the specificity of ligase while that in SOTLA is provided by the
specificity of both ligation and hybridization of TSO probes. 相似文献
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