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    Home > Biochemistry News > Biotechnology News > Oligonucleotide probe synthesis.

    Oligonucleotide probe synthesis.

    • Last Update: 2020-10-27
    • Source: Internet
    • Author: User
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    Using oligopolytic
    nucleotides
    automatic synthesizer, oligonucleotide probes (e.g. 15 to 50bp) can be easily synthesized, and probes have the following advantages:
    (1) short probes are faster and more specific than long probes.
    (2) can be prepared in large quantities in a short period of time.
    (3) is marked in the composition to make a probe.
    (4) can be synthesized single-stranded probe, avoiding the use of double-
    . DNA
    probes are self-complex in hybridization, improving the efficiency of hybridization.
    (5) oligonucleotide probe can detect small DNA fragments and, under strict hybridization conditions, can be used to detect misaligning of single base pairs in a sequence.
    , the study of oligonucleotide probes is of great significance for improving the specificity and sensitivity of
    nucleic acid
    hybridization technology and expanding the application range.there are three commonly used oligonucleotide probes in
    : a single oligonucleotide probe with a specific sequence of

    (
    1);
    are more than 32P labeled oligonucleotide probes, e.g.
    1) by T4
    phages
    polynucleotides
    kinases
    catalyzed
    phosphate
    reaction mark synthetic oligonucleotides Probe, in the synthetic oligonucleotide period 5' end is missing a phosphate base, so easy to use T4 phage polynucleotide kinase phosphatization reaction, and α-32P from the .γ-32P ATP to its 5' end. This phosphate reaction can add up to one 32P atom to each oligonucleotide molecule.
    2) Synthesized oligonucleotide probes are labeled with E. coli DNA polymerase i Klenow fragments, which are more active than each oligonucleotide molecule with several radioactive atoms and can be as high as 2×1010 count/(min× mg).
    non-radioactive marking of oligonucleotide probes, the following methods can be used:
    1. Enzyme extension synthesis and probe purpose
    gene
    3' - end of a complementary oligonucleotide nucleotide sequence, this sequence of 5'-end plus an A, with the purpose gene fragments to aside, and then extended with Klenow enzyme, so that bio-dUTP mixed into the 3' end of the probe.
    2. 5' Phosphoric acid end marker method with 5' - oligonucleotide probe of phosphoric acid, treated with water-soluble carbon dimiamine (EDC) in the methylene buffer, can produce active phosphate methylene intermediates, with excessive ethyldiamine action, you can introduce a arm with amino. A 5'-phosphate-labeled oligonucleotide probe can be obtained by biotin markers.
    3. Enzyme standard probe method uses dual-functional joint agents such as nicotine double hydroxysamine to connect oligonucleotides and alkali phosphorusases, can produce 1:1 enzyme label oligonucleotide probe. This method omits the intermediate step of biotin-pro-pyridine and reduces nonse specific reactions.
    4. Biotin azimate cytosine modification method under the sulphate catalysis, biotin azithyl can replace the oligonucleotide probe on the cytosine amino and get biotinized oligonucleotide probe.
    5. Oligonucleotide's enzyme-promoted tail marking method, under the effect of the end
    transsiase
    , uses non-radioactive material modified nucleotides (biotin dATP; biotin-dUTP; geocosin-dUTP) can be added to the 3' end of dna, each probe DNA can be added 10 to 20 modified base.
    (1) take -0.5 ml siliconized plastic centrifugal tube, insert it into an ice bath, add oligonucleotides (3pmol) × sl, 5× add tail buffer 20 sl, 5.0mmol/l dUTP 4 sl (Final concentration of 200 smol/L), modified dNTP (biotin-dUTP; biotin-dATP; geosin-dUTP) × sl (final concentration of 100 smol/L), added to 100 sl, mixed and then added to the end transfer enzyme 5u. Reaction 1h at 37 degrees C.
    (2) probe purification: ethanol precipitation method: add 50 sg tRNA, 15ul 4mol/L sodium acetate and 375 sl waterless ethanol, mix well, -20 degrees C1h, high-speed centrifugation 10min, discarded clean, with 70% ethanol and waterless ethanol and then repeatedly washed precipitation, drying, and then dissolved in water, concentration of 500ng/ml.
    .
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