Experiments to describe chromosomal structural abnormalities by comparative genomic hybridization
Experiments to describe chromosomal structural abnormalities by comparative genomic hybridization
Comparative Genomic Hybridization (CGH) is a molecular cytogenetic technique capable of characterizing, in a one-step genome-wide screening procedure, additions or subtractions of cytogenetic material that are not detectable in the G-band. CGH is superior to conventional Fluorescence In Situ Hybridization (FISH) and Multi-color FISH for Whole Chromosome Panel (wcp) in its ability not only to identify the chromosomal origin of the added, unknown segment, but to localize the segment to a specific chromosomal region. the fragment to a specific chromosomal region. HSH with a variety of probes to determine the origin of an added chromosomal segment is an expensive and laborious process because multiple wcp probes may be required before the origin of the chromosome can be identified. In addition, the number of locus-specific probes that can be obtained is limited, covering only a portion of the genome
Operation method
Experiments to describe chromosomal structural abnormalities by comparative genomic hybridization
Principle
Comparative genomic hybridization (CGH) is a molecular cytogenetic technique capable of characterizing, in a one-step genome-wide screening procedure, additions or subtractions of cytogenetic material that are not detectable by G-banding.CGH outperforms conventional fluorescence in situ hybridization (FISH) and multicolor FISH for whole chromosome painting (wcp) in its ability not only to identify the chromosomal origin of the added, unknown fragment but also to localize the fragment to a specific chromosomal region. the fragment to be localized to a specific chromosomal region. The process of employing various probes for HSH to determine the origin of the added chromosomal fragment is expensive and laborious because multiple wcp probes may be required before the origin of the chromosome can be identified. In addition, the number of locus-specific probes that can be obtained is limited, covering only a portion of the genome.
Materials and Instruments
Peripheral blood drawn from a karyotypically normal male Bovine serum albumin Move I. Culture of normal male peripheral blood cells 1. Add 500~650μL of fresh whole blood from a karyotypically normal male to 10mL of starter medium. 2. Cultivate at 37℃ for about 72h (3d). 3. Add 200mL of thymidine solution and mix gently. 4. Incubate at 37℃ for 14~18h (overnight). 5. Centrifuge at 155g for 10min. 6. Discard the supernatant, add 10mL of 1×PBS, and mix upside down. 7. Centrifuge at 155g for 10min. 8. Discard the supernatant, add 10mL of continuous medium, gently invert and mix. 9. Continue incubation at 37℃ for 4~5h. 10. Add 6 or 7 drops of colchicine amine solution and mix upside down. 11. Incubate at 37℃ in a water bath for 20min. . 12. Centrifuge at 170g for 10min. 13.Discard the supernatant and leave about 0.5mL of liquid. Flick the centrifuge tube several times to resuspend the cells thoroughly to avoid clumping. Quickly add 0.5mL 75mol/L KCl (preheated at 37°C), flick the centrifuge tube to mix thoroughly, and continue to add 4.5mL KC1. 14. Incubate at 37℃ in a water bath for 15min. 15. Using a Pasteur pipette, slowly add 1mL of freshly prepared fixative to each tube starting from the bottom of the tube, gently invert and mix. 16. Centrifuge at 170g for 10min. 17. Discard the supernatant, leaving about 0.5mL of liquid. 18. Add fresh fixative drop by drop to 5mL while mixing. 19. Incubate at room temperature for about 10min. 20. Centrifuge at 170g for 10min. 21. Discard the supernatant, leaving about 0.5mL of liquid, and break up the precipitate. 22. Add fresh fixative drop by drop to 5mL while mixing. 23. Repeat steps 20 to 22 2 or 3 times if necessary. 24. Discard the supernatant. 25. Dilute the precipitate to the appropriate concentration. The solution should be cloudy and lightly hazy (if more precipitate is obtained, add 1~1.5mL of fixative; if less precipitate is obtained, add <1mL of fixative). 26. Titrate the slides as described in the following two methods. Preparation of normal male intermediate chromosome specimens 1. Prepare a closed environment with a relative humidity of about 55% (can vary from 50% to 60%) and a temperature of 24~26.5°C. 2. Place a Pasteur pipette 2~4in above the glass slide and place a drop of cell suspension on the left side of the slide. Then quickly place a second drop of cell suspension on the right side of the slide (see Note 7) 3. Before all of the fixative evaporates, immerse the slide in a staining vat containing fresh fixative for 1 ~ 3 s. Remove the slide from the staining vat and dry the slides in a closed humidity/temperature environment as described in Step 1. 4. the quality of the chromosome specimen was examined under a light microscope T. 5. Chromosome specimens were left at room temperature overnight and then immersed in 1% formalin at 4°C for fixation for 5~10min. 6. Chromosome specimens were dehydrated in series of ethanol, 2min each in 70%, 85% and 100% ethanol. 7. The specimens were air-dried at room temperature or blown through the specimens in a laboratory fume hood to dry the specimens quickly. 8. Store specimens at room temperature for 2 to 4 weeks. 9. Perform CGH analysis on one of the chromosome specimen slices using a normal reference and a normal human sample or a sample with a known imbalance of chromosomal abnormalities. Extraction of high molecular mass DNA from patient and normal human samples DNA extraction is performed according to the instructions provided by the manufacturer. Optionally, DNA can be extracted by standard methods or any reliable DNA extraction kit. IV. Labeling of probes by notch panning method 1. Fluorescein-12-dUTP is used to label patient DNA and Texas Red-5-dUTP is used to label reference DNA. 2. prepare a 15°C water bath or add a small amount of ice to an ice box filled with tap water until the temperature drops to 15°C. 3. Set the other water bath to 72°C. 4. Add the following reagents to a 1.5 mL centrifuge tube (all reagents on ice): 1 μg of patient or reference DNA, 5 μL of 10× reaction buffer mixture, 5 μL of dNTP mixture, 1 μL of fluorescent moiety-dUTP (l mmol/L), 1 μL of DNAzyme (0.01 ~ 1 U/μL), and 2.5 μL of DNA polymerase I (10 U/VL). 5. Add water to a total volume of 50 μL. 6. Centrifuge the tube briefly so that all reagents sink to the bottom of the tube. 7. Incubate in a 15°C water bath for 60 min. 8. The reaction was terminated by incubation in a 72°C water bath for 10min. 9. Check the DNA fragment size by electrophoresis on a 1% agarose gel. The most suitable DNA fragment size for CGH is 300~2000bp (Figure 3). If the fragment is too long, add DNAzyme I and continue incubation in a 15°C water bath. 10. Notch pan labeling can be performed for a large number of samples. V. Preparation of CGH probe Add the following reagents to a 1.5mL centrifuge tube: 1. 200ng of normal (reference) DNA labeled by notch panning. 2. 200ng of patient DNA labeled by notch panning (10iLtL). 3. 20 to 30 μg Cot1-DNA (20 to 30 μL) (see Note 11). 4.1/10 volume of 3 mol/L acetate. 5. 150 μL (>2x volume) of 100% ice-cold ethanol. 6. 40~60min at -80°C. 7. Centrifuge at 29000g (or maximum speed of centrifuge) at 4℃ for 40min. 8. Gently pour off the ethanol. 9. Blot up the residual ethanol with a sterile cotton tipped applicator (do not touch the precipitate). 10. Place the centrifuge tube on a metal rack on a hot plate (42°C) and evaporate the residual ethanol for 2-3min. 11. Resuspend the precipitate with 101 μL of Hybrisol VII hybridization mixture, mix with up and down suction, and vortex for about 1 min. 12. The CGH probe can be used directly or stored at -20℃ to avoid light. Denaturation of mid-stage chromosome specimens 1. Add about 50mL of denaturing solution to the staining vat and place the vat in a 75~76℃ water bath. Preheat the denaturing solution to (73±2)℃. 2. Remove the specimens required for the experiment from the -20℃ refrigerator and dehydrate them in a series of ethanol at room temperature for 2min each. 3. Dry the slices at room temperature (5min), or dry the specimens by blowing the breeze through the specimens in the laboratory fume hood. 4. Preheat the slices on a 37~40℃ hot plate for 30s. 5. Immerse the specimen in denaturing solution at (73±1)℃ for 2.5~5min. 6. Remove the specimen quickly and put it into ice-cold 70% ethanol for 3min, then dehydrate it in 85% and 100% ethanol for 3min each. 7. Air-dry the specimen slide (5min), or blow the specimen through the breeze in the laboratory fume hood to dry the specimen. Probe denaturation In the middle chromosome specimen slides series of ethanol dehydration at the same time, the centrifugal tube containing the CGH probe in a (75 ± 1)℃ water bath denaturation for 10min. VIII. Hybridization 1. Rubbermaid was aspirated into the syringe barrel (which had the pillow removed). 2. Preheat the specimen slice on a 37 ~ 40 ℃ hot plate for about 1min. 3. With the specimen slice on the hot plate, add each CGH probe to the same hybridization area of the specimen, and cover each hybridization area with a 22mm×22mm glass coverslip. 4. Rubbermaid was squeezed with a syringe and sealed along the edge of the coverslip. 5. Incubate the specimens at 37°C in a wet box for 2~3d. IX. Elution after hybridization 1. Add about 50mL of 2×SSC solution to the staining vat, and preheat to bring the temperature to about 72°C (set the temperature of the water bath at about 75°C). 2. Add about 50mL of 4×SSC solution to the dyeing vat and preheat to a temperature of about 37°C (set the water bath temperature at about 39°C). 3. Add about 50mL of 4×SSC/0.1% Triton×-10O solution to the dyeing vat and preheat to a temperature of about 37°C (the water bath temperature is set at about 39°C). 4. Add about 50mL of 2×SSC solution to the staining vat and leave it at room temperature. 5. Add approximately 50mL of distilled water to the staining vat and leave at room temperature. 6. Remove the rubber cement from the specimen piece. 7. Gently slide off the cover piece (do not scratch the specimen). 8. Place the specimen slice in 2×SSC solution at (72±2)°C, shake the specimen slice for 2min, and then leave it in the solution for 5min. 9. The specimen slice was transferred to a 4×SSC solution at 37°C for 5min. 10. Transfer the specimen slice to a 4×SSC/0.1% TritonX-100 solution at 37°C for 5min. 11. Transfer the specimen slice to a 4×SSC solution at 37°C for 5min (the same solution as in step 9 can be used). 12. transfer the specimen slice to stay in 2×SSC solution at room temperature for 5min. 13. the specimen piece is transferred to stay in water at room temperature for 5min. 14. Remove the specimen from the water and place it in a dark place to dry at room temperature; or blow the specimen in a laboratory fume hood to dry it quickly. 15. Add 10 μL of DAPI re-staining agent to the target area of the specimen. 16. Cover the coverslip. 17. Place the specimen slice in the dark for 5~10min, and then perform CGH image capture and analysis. 18.Photograph at least 10 of each autosomal chromosome and 7 of each sex chromosome. Caveat 1. A normal male control is usually chosen so that both the X and Y chromosomes are present in a single interim chromosome specimen slice. 2. If the initial cytogenetic analysis has been done in another laboratory, the patient's blood sample should be placed in a tube with sodium heparin, which can be subsequently used for DNA extraction or, if needed, peripheral blood culture for FISH. In this case, the volume of blood collected should ideally be more than 3 mL. If the patient is an infant, the volume of blood collected is usually less than 3 mL, as long as the volume is sufficient for DNA extraction. 3. Large quantities of male and female reference DNA can be extracted and stored at -20°C for backup. 4. Each new batch of DNAzyme should be subjected to a concentration gradient of 0.0~0.1 U/mL, and agarose gel electrophoresis should be performed to determine which concentration will yield the best-sized DNA fragments. 5.-The labeling of more than 1μg of DNA can get a large amount of labeled reference DNA, which can be stored at -20℃. 6. A specimen slide should be prepared for pre-testing to determine the mitotic index and to assess the need to dilute the cell suspension with a fixative or to resuspend the cells with a smaller volume of fixative after centrifugation. 7.- Drop two regions on one carrier sheet; two different CGH experiments can be performed simultaneously on one sheet, but it should be ensured that chromosomes are well dispersed in both regions. 8. Carry out the preparation of mid-stage chromosome specimen slices with the following objectives: (i) to obtain mid-stage chromosomes that are evenly dispersed, with fairly straight chromosome arms and less overlap between chromosomes; (ii) to have as little cytoplasmic residue as possible on the slide and chromosomes; and (iii) to observe mid-stage chromosomes as gray [not too dark and/or too bright, and not too light or hazy] under a phase contrast microscope. 9. The quality of the mid-stage chromosome specimen slices is the most critical factor in the CGH process. Each batch of specimen slices should be pre-tested, and the better quality specimen slices should be stored at -20°C. Pre-experimentation is performed approximately two weeks after drop preparation, and once the results are satisfactory, the batch of specimen slices is stored at -20°C. Generally the quality of the whole batch of specimen slices is similar, and if a batch of specimen slices does not give satisfactory results, the batch of specimen slices can be used for other experiments such as FISH, or thrown away. In laboratories that are proficient in mid-stage chromosome specimen preparation, the mid-stage chromosome preparation method they commonly use should be preferred. In many cases, midblock chromosomes that meet the criteria in Note 8 are also suitable for CGH analysis. 10. When preparing CGH probes, consideration should be given to preparing two different probes, a reference probe of the same sex as the patient and a reference probe of a different sex. The gender-identical probe is important in revealing unbalanced chromosomal abnormalities of chromosomes, including sex chromosomes. If two probes are used for two hybridized regions of a slice (see Note 7), the sex-differentiated reference probe can be used as an internal control because different color ratios are expected throughout the sex chromosomes. 11. approximately 20 μg of Cot1-DNA should be used in marker cases and approximately 30 μg of Cot1-DNA should be used in all other cases. the vast majority of marker chromosomes contain mitotic grains, and in some cases less Cot1-DNA is used to improve the detection efficacy. 12. The denaturing solution should be pre-warmed for at least 30 min. the solution can be pre-warmed in a microwave oven on the highest setting for 20 s to accelerate the pre-warming process. 12. Determine the optimal denaturation time for each batch of specimen slices. Suitable specimens can be denatured for a wide range of times to obtain good hybridization results and DAH reversal band patterns. An initial denaturation time of 3.5~4min can be tried. For more product details, please visit Aladdin Scientific website.
Freshly prepared fixative Colchicine Amine PRMI Fetal bovine serum Penicillin Streptomycin L-glutamine Phytohemagglutinin HEPES buffer KCl Dulbecco's Phosphate buffer Thymidine solution Starter medium Continuous medium Formalin fixative Fluorescein-2-dUTP Texas Red-5-dUTP Complete set dNTP 2-phosphoethanol dNTP Mixture TAE Ethidium bromide Agarose gel
Pasteur pipette Staining vat PureGene DNA purification kit Water bath