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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.