Non-banded chromosomes are difficult to identify except for some chromosomes based on their morphology, whereas the banding technique allows the appearance of different bands along the longitudinal length of the chromosome to identify all the chromosomes. gTG is short for G-banding, Trypsin, and Giemsa. giemsa dye is a thiazide-eosin dye, and the chromosome coloration depends on the formation of a thiazide-eosin (2:1) precipitate in situ. Giemsa dye is a thiazine-eosin dye, and the coloration of chromosomes depends on the formation of a thiazine-eosin (2:1) precipitate in situ.

Operation method

basic program

Principle

Non-banded chromosomes are difficult to identify except for some chromosomes based on their morphology, whereas the banding technique allows the appearance of different bands along the longitudinal length of the chromosome to identify all the chromosomes.GTG is short for G-banding, Trypsin, and Giemsa.The Giemsa dye is a thiazine-eosin dye, and the coloration of chromosomes depends on the formation of a thiazine-eosin (2:1) precipitate in situ. Giemsa dye is a thiazine-eosin dye, and chromosome coloration depends on the formation of a thiazine-eosin (2:1) precipitate in situ. Coloration depends firstly on the binding of two thiazine molecules to the DNA, on top of which an eosin molecule is bound, and secondly on a hydrophobic environment that facilitates the precipitation of the dye for coloration. The hydrophobic proteins in the negative G-band region can be removed, or their structure made more hydrophobic, by tryptic banding pretreatment. It follows that the proteins extracted in the G-banding tend to be hydrophobic and come mainly from the negative G-banding region.

Materials and Instruments

Chromosome specimens
Trypsin HCl Ba(OH)2 Giemsa stain SSC
Microscope Oven Thermostat Staining vat

Move

I. Supplies and reagents
0.25% trypsin (0.85% NaCl formulated at pH 7.2-7.4), HCl, Ba(OH) ₂, Giemsa staining solution, SSC.
II. Operational steps
1. Aging of specimens
Prepare human peripheral blood lymphocyte chromosome specimens (unstained) air-dried as per routine and oven-dried at 78°C for 2-3 hours. Remove and place in 37℃ constant temperature box for 3 days after pretreatment.
2. Pancreatic enzyme pretreatment
Chromosome specimens were immersed in trypsin solution (which had been stabilized in a 4°C refrigerator for more than 1 hour) for 40-50 seconds, removed and immediately washed with water to remove excess trypsin.
3. Giemsa stain
1:10 Giemsa staining solution for 5-10 minutes, wash off excess dye and air dry.
4. Microscopy

Oil microscopy shows well-dispersed chromosomes with different shades of banding on the longitudinal axis, i.e., readable specimens.

Caveat

1. routinely prepared chromosome specimens with a high number of split phases, well dispersed, and moderate chromosome length.

2. The key to GTG banding is the time of trypsin treatment. If the chromosomes are still darkly colored and the banding is unknown, the pretreatment time is insufficient and the pretreatment time should be extended; if the chromosomes become thicker and show rough edges, or even mushy, the pretreatment is excessive and the treatment time should be shortened appropriately.

3. Giemsa staining time should be moderate, short time, coloring is not enough, the contrast between the dark and light bands is small; time is too long, coloring is deep, also affects the banding contrast, not easy to identify.

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