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    Home > Biochemistry News > Biotechnology News > Sister chromosomal monosomal differentiation staining

    Sister chromosomal monosomal differentiation staining

    • Last Update: 2020-10-31
    • Source: Internet
    • Author: User
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    1.
    Experimental Principles Sister chromatid differentiation

    (
    SCD)
    is a chromosomal processing
    technique developed in
    2020s
    70s
    .
    1973 Latt
    added
    5-
    bromine deoxyridine nucleosides (
    5-Bromo de Oxyurdine
    ) to cells cultured

    ,
    brdU
    ), dyeing with
    Hoechst 33258
    fluorescent dyes, the chromatin reaction of the sister chromosomal monosome and the interchange between them were discovered.


    1974
    KO Renberg
    and
    Froeed-Lender
    improved this technique, dyeing
    Giemsa
    colors.
    SCD
    formation principle (Figure
    13-2
    ) is to add
    5-
    bromine deoxyuriaside to the
    cell culture
    process, when the cell's
    DNA
    is copied,
    Brdu
    can be incorporated into the newly synthesized
    DNA chain as a single replacement for thymus as a
    nucleotide

    presector. The second cycle is added to
    BrdU
    , the old template in each newly synthesized chromosome
    DNA
    double strand is not mixed, the new chain of semi-reserved replication is mixed with
    BUdR
    ; Two sister chromosomes, one consisting of a
    DNA
    chain containing
    BrdU
    in both strands and a
    DNA
    strand containing
    BrdU
    . The
    DNA
    helix of
    BrdU
    in both strands is reduced in structure, so the affinity for staining agents is low, and when dyed with
    Giemsa
    , only monoliths consisting of
    DNA
    strands containing
    BrdU
    are colored and colored differently. Using sister chromosomal monochromosomal differentiation to study the exchange of ion fragments between two monosomes from one chromosome at the same place, called sister chromotid exchange (
    sister chromotid exchange, SCE
    ).
    SCE
    is a phenomenon manifested by the interchange of two chromosomal mononucleotide sequences.Figure
    13-2 this technique is used to study cell cycles, chromosomal semi-retention replication, molecular structure and distortion of chromosomes, and the replication, damage and repair of
    DNA
    and a series of important theoretical issues. Because
    SCE
    can sensitively detect chromosomal changes, showing a dose
    -
    effect relationship, but also the frequency of sister chromosomal monoplate interchange as one of the usual indicators of detection of
    mutations
    and carcinogens.2.
    the cells examined by the subjects.3.

    reagents
    and equipment(
    1
    )
    BrdU
    storage fluid preparation:
    BrdU 5mg
    (first with
    0.5ml 1N NaOH
    dissolved) then add distilled water to
    5ml


    is
    1000
    μ
    g/ml
    storage liquid, because
    BrdU
    will be decomposed in light, storage liquid needs to be placed in brown bottles and black paper bottles to avoid freezing storage;(
    2
    )
    2
    ×
    SC
    , water bath,
    20W
    a UV lamp, Petri dish, lens paper.4.
    Experimental methods and steps(
    1
    )
    Brdu
    incorporated: cell culture
    24h
    after the addition of
    Brdu
    in the culture, the final concentration
    10
    μ
    g/ml
    , continue to culture under light-absorbing conditions.(
    2
    ) waiting for cells to undergo two cell cycles (
    48h
    ), before the end of culture
    4h
    plus autumn daffodils, autumn daffodil final concentration of
    0.02
    μ
    g/ml
    culture;(
    3
    ) conventional production, dyeing system film in
    37
    degrees C
    thermostat
    box aging
    24h
    ;(
    4
    ) UV light exposure: take the specimen slide placed in a large Petri dish, facing up, covered with lens paper, from the edge of the glass lens paper plus
    2
    ×
    SSC
    liquid caused the full lens paper immersion, moved into the
    60
    degrees C water bath pot, ultraviolet light distance
    5cm
    , Treatment
    30min
    ;(
    5
    ) staining: remove the post-treatment specimen, rinse with distilled water, and place
    pH6.8

    2% Giemsa
    staining
    10min
    .5.
    (
    1
    )
    BrdU
    is a strong mutant that should not be used at a high concentration, otherwise it will produce cytotoxic effects. (
    2
    ) factors that affect
    SCE
    : region and environment, concentration of
    Brdu
    , animal species, chromosome length, culture time and temperature.
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