Chromosomes are entities containing genes, which are only visible under the microscope as a general structure. Genes are nucleotide sequences at the molecular level, whether they can be made visible under the microscope can also be made visible on the chromosome by special methods and determine the location of a gene on a chromosome, a method known as gene localization. Source: Tissue culture and molecular cytology techniques. (Beijing Publishing House)
Operation method
Radionuclide-labeled chromosomal in situ hybridization for gene localization
Principle
Genes are sequences consisting of an average of 1,000 to 3,000 nucleotides and are difficult to recognize under a light microscope. In order to display a specific gene on a chromosome, the following three important conditions must be present: 1. the need for a nucleotide sequence that can specifically join (complement) the target gene, i.e., a probe; 2. the need for markers that can bind to the probe (commonly used isotopes and fluorescein); and 3. the preparation of a good chromosome specimen. In localization, the first step is to make the marker and the probe to join (Lable: labeling), the formation of the probe / marker complex, and then make the complex and chromosomal DNA hybridization (Hybridization). Hybridization is the process by which two single-stranded DNAs of different origins complement each other to form double-stranded DNA. In general, the target gene and the probe that are localized on the chromosome are both double-stranded DNA, and hybridization between the two can only occur when they are unstranded, i.e., when they both become single-stranded. Temperature is the condition that makes double-stranded DNA unstranded, and DNA is unstranded (denatured) at a temperature of 80°C. Therefore, when the chromosome and the probe are placed on the chromosome, the DNA is unstranded. Therefore, when the chromosome and the probe placed in the same temperature conditions, the two will be unchained at the same time to become single-stranded DNA; when the temperature drops, the two DNA and back to the double-stranded state (denaturation); in the denaturation of the probe's single-stranded single-stranded with the target gene hybridization occurs. As the probe DNA Carrying markers, with the corresponding method can make the markers appear to become visible form. In the use of isotopes as markers, the use of radiation autoradiography to show the probe / isotope; fluorescein as a marker, the probe / fluorescein can be fluorescent under the fluorescence microscope, which can be observed and determine the probe site (that is, the presence of the target gene site). The method of hybridization between the labeled probe and the chromosomal gene (or cytoplasmic mRNA) is called In Situ Hybridization.
Materials and Instruments
Chromosome specimens Move I. Radionuclide Probe Admixture Procedures Common Problems I. Results In the successful case, the chromosomes were well banded, and scattered black round particles, 1-3 microns in diameter, were seen everywhere except on the chromosomes. Black round particles of 1 to 3 microns in diameter are scattered everywhere except the chromosomes, some of which are located on the chromosomes and some of which are located between the chromosomes. 50-100 split phases were observed and the presence of particles was counted. 50-100 cleavage phases were observed, and the presence of particles at regular sites on a particular chromosome was counted, as well as the presence of particles at specific sites on a particular chromosome. If the probability of the particles occurring on a specific chromosome accounts for 8% to 40% or more, it can be regarded as the locus of the gene. For more product details, please visit Aladdin Scientific website.
radionuclide
1. Probe preparation
2. Gap translation
(1) Isotope Decompression Concentration
Take 50 μl~100 μl of 3H-dTTP (1 μC/ml) and put it into Eppendorf tube. air depressurized and drained;
(2) Gap translation reaction
Take 1 μg of probe DNA and add it into Eppendorf tube, then add 10× nick translation buffer 5 μl. and add 5 μl of 10× nick translation buffer;
The composition of nick translation buffer was
Tris-Cl buffer 0.5 M
MgSO4 0.1 M
Dithiothreitol 1 mM
Bovine serum albumin 500 μg/ml
(3) Add 1 mM each of dATP, dGTP and dCTP; dilute to 50 μl with distilled water;
(4) Mixing
Transfer to a vacuum-dried Eppendorf tube containing 3H-dTTP and mix well;
(5) Add enzyme
Add 1 ml each of DNA polymerase I and DNA polymerase I and seal;
(6) Reaction
React at 16 ℃ for 10 minutes, add 0.5 M EDTA to the final reaction;
(7) Probe isolation
Add the above reaction solution to a Sephadex G50 column and elute with TE.
Probe and chromosome molecular in situ hybridization
1. Hybridization solution preparation
3H labeled probe 1.0 μg/ml
Formamide 50.0 %
Glucose sulfate 10.0 %
EDTA 5.0 mM
polyvinylpyrrolidone 0.04 %
Poly(sucrose) 0.04 %
Bovine serum albumin 0.04 %
Sodium chloride 300 mM
Sodium citrate 300 mM
Phosphate buffer 20 mM
Calf thymus DNA 50 μg/ml
The above components were mixed together at pH 7.0 ± 0.1.
2. Probe denaturation
The hybridization solution is mixed and placed in a water bath at 70 °C for 5 minutes, then the tube containing the hybridization solution is inserted into crushed ice.
3. Denaturation of chromosomal DNA
(1) To each chromosome specimen, add 50 μl of 100 μg/ml RNAase (for RNA removal), place a coverslip, and place in a moist dish at 37 ℃. Place a coverslip in a moist dish and incubate at 37 ℃ for 1 hour; remove the coverslip and immerse it in a 2×SSC tank and rinse it 4 times;
(2) Dehydration
Dehydrate in 70%, 80%, 90% and 100% alcohol;
(3) Denaturation
Place the chromosome specimen into 2×SSC formamide solution and process at 70 ℃ for 2 minutes (not too long);
(4) Rapidly put into 30% cold ethanol solution;
(5) Dehydration
Same as (2);
(6) Drying
Natural drying.
4. Probe and chromosome hybridization
(1) Add 50 μl of hybridization solution to each chromosome specimen and cover with a cover slip; hybridize for 16 hours at 42 ℃ in a humid dish;
(2) Elution
(2) Elution: At 30~40 ℃, wash 5 times in 50% formamide solution, 5 times in 2×SSC solution, and 3 times in 0.1×SSC solution (elution). solution for 5 times, 2×SSC solution for 5 times and 0.1×SSC solution for 3 times (excessive elution affects the hybridization rate);
(3) Dehydration
Same as before;
(4) Drying
Natural drying.
5. Radiographic autoradiography
(1) Smear
The film is coated with a diluted solution of domestic nuclear latex (1:1);
(2) Exposure
After 10 days of exposure in a refrigerator at 4 ℃ (specific activity of 107 cpm/μg or more), remove the film;
(3) Developing
Develop with D19B developer for 2-5 minutes;
(4) Fixing
Kodak F-5 fixer for 10 minutes;
(5) Washing
15 minutes.
6. Chromosome development
(1) Immerse the exposed film in the following solution
Na2SO4 50.0 mM
Na2B4O7 2.5 mM
37 ℃, pre-treatment for 10-30 seconds;
(2) Staining
(2) Rapidly diffuse into 10% Giemsa staining solution for 2~3 minutes;
(3) Wash, dry and observe under the microscope.
Discussion
Adequate denaturation of chromosomal DNA is good for hybridization with probes, but excessive denaturation is not good for banding. Adding 70% formamide in the denaturation is more effective. The effect of adding 70% formamide in the denaturation is better, which can ensure the denaturation and maintain the chromosome morphology at a lower temperature. The addition of 10% dextran sulfate to the hybridization solution is more effective. It is better to add 10% dextran sulfate to the hybridization solution, which can improve the hybridization rate, but it should not be too high, otherwise the background will also be high. Probe concentration should also be appropriate, too low need to extend the exposure time, the background increases; too high probe can be combined to affect the hybridization rate. The amount of probe added is related to the size of the DNA fragments. The amount of probe added is related to the size of DNA fragments, and the empirical value is that the DNA concentration of 10 kb fragment should be 0.5-0.05 mg/L. The amount of probe added is related to the size of DNA fragments.