In situ hybridization (ISH) refers to the use of specifically labeled nucleic acid probes with known base sequences to hybridize with homologous DNA sequences in mid-stage chromosome slide specimens based on the principle of base complementary pairing. Chromosomal in situ hybridization is highly sensitive and specific, capable of precise localization, qualitative and relative quantitative analysis of specific nucleic acid sequences.
Principle
The basic principle of chromosomal in situ hybridization experiments is to use specifically labeled nucleic acid probes with known base sequences to hybridize with homologous DNA sequences in mid-stage chromosome slide specimens based on the principle of base complementary pairing.
Labeling can be done with radioactive isotopes (3H, 35S, 32P), followed by autoradiography; or non-radioactive fluorescein biotin, digoxin, and then observed with a fluorescence microscope, i.e., fluorescent in situ hybridization (FISH) technique.
Operation method
in situ hybridization of chromosomes
Principle
The basic principle of chromosomal in situ hybridization experiments is to use specifically labeled nucleic acid probes with known base sequences to hybridize with homologous DNA sequences in mid-stage chromosome slide specimens based on the principle of base complementary pairing. Labeling can be done with radioactive isotopes (3H, 35S, 32P), followed by autoradiography; or non-radioactive fluorescein biotin, digoxin, and then observed with a fluorescence microscope, i.e., fluorescent in situ hybridization (FISH) technique.
Materials and Instruments
Equipment: Move The basic process of a chromosomal in situ hybridization experiment can be divided into the following steps: (1) Notch panning method A The total volume of the labeling reaction is 50 μl, containing 1 μg of DNA probe to be labeled, 5 μl of 10x Incision Panning Buffer (composition depends on the kit), 50 μmol of each of dATP, dGTP, and dTTP, 50 μmol of [α-32P]dCTP (50 μCi), 5 ng of DNase I, and 5 U of DNA polymerase I. B Add the above reagents into a 0.2 mL Eppendorf tube. B Add the above reagents into a 0.2 mL Eppendorf tube, mix well, and place in a constant temperature water bath at 16 ℃ for 1 h. Detect the labeled products by 0.8% agarose gel electrophoresis. The length of the DNA fragments should be about 300~500 bp. If the fragment is large, appropriate amount of DNaseI should be added to continue the digestion until the length of the DNA fragment is appropriate, then add 2 μl of termination buffer (0.5 mol/L EDTA), and heat at 75 ℃ for 10 min to terminate the reaction. (2) Random primer method A Dissolve the DNA probe to be labeled (25 ng) in 20 μl of double-distilled water, denature in boiling water bath for 5 min, and then quickly put it into ice-water mixture for use. B The total volume of the labeling reaction is 50 μl, containing 25 ng of DNA probe to be labeled, 5 μl of 10x random primer buffer (composition depends on the kit), 50 μmol of each of dATP, dGTP, and dTTP, 50 μmol of [α-32P]dCTP (50 μCi), 10 μg of hexa-base random primer, and 3 U of Klenow fragment. C Add the above reagents into a 0.2 mL Eppendorf tube, mix well, and then place it in a 25 ℃ constant temperature water bath for 1 h. Add 2 μl of termination buffer (0.5 mol/L EDTA) to terminate the reaction. The random primer method is simple and produces a more uniform length of the radiolabeled product, which is more reproducible in the hybridization reaction. (3) Probe purification A Add 5 μl of 3 mol/L NaAc (pH 5.2) to the labeled DNA probe, mix well, and leave it at room temperature for 5-10 min. B Add 2.5 times the volume of pre-cooled anhydrous ethanol and mix well. C Place at 4 ℃ or -20 ℃ for overnight precipitation. D Centrifuge at 12000 g for 30 min at 4 ℃, discard the supernatant and air dry (do not over dry). (1) Pretreatment of chromosome slide specimen A Put the chromosome slide specimen into 2x SSC prepared RNAase (10 μg/mL) solution, and put it in a water bath at 37 ℃ for 1 h. B Rinse the specimen with 2x SSC. B Rinse the slide with 2x SSC three times for 5 min each time. C The slides were dehydrated in 70%/85%/100% gradient ethanol for 5 min each and dried in air. D Immerse the slide in 70% formamide prepared by 2x SSC and denature at 70 ℃ for 2 min. E Dehydrate the slides in 70%/85%/100% gradient ethanol for 5 min each and dry in air. (2) Hybridization A Add 20-50 μl of hybridization solution to each chromosome slide specimen, cover the slides with coverslips (previously treated with silanization) and seal with rubber cement. B Place the slides in a wet box and hybridize at 37 ℃ overnight. C Remove the hybridization slide, remove the sealer and coverslip, and rinse with 50% deionized formamide (2x SSC preparation) preheated at 39 ℃ for 10 min. D Rinse the slide with 2x SSC preheated at 39 ℃ for 10 min. E The slides were dehydrated in 70%/85%/100% gradient ethanol for 5 min each and air dried. Caveat 1 Do not use a vortex shaker to mix reagents during probe purification. For more product details, please visit Aladdin Scientific website.
① Staining vat
② Micro-sampler
③ Fluorescence microscope
③ Fluorescence microscope ④ Constant temperature water bath
⑤ Wet box
⑥ Human mid-stage chromosome slide specimen (unstained white slides)
⑦ Human G-banded chromosome slide specimen.
Reagents:
① 10x Incision panning buffer
② 10x random primer buffer
③ 4x dNTP (0.5 mmol/L each) ④ 0.2 mmol/L dNTP (0.5 mmol/L each)
③ 4x dNTP (0.5 mmol/L each) ④ 0.2 mmol/L [a-32P] dCTP (10 μCi/μl)
⑤ DNase I (1 mg/mL, diluted 1000 times before use)
⑥ DNA polymerase I (5 U/μl)
⑦ Klenow fragment (3 U/μL)
⑧ Six-base random primer (1 mg/mL)
⑨ 3 mol/L NaAc (pH 5.2)
⑩ 70% deionized formamide (DF, 2x SSC preparation)
⑪ 50% Deionized formamide (DF, 2x SSC prepared)
⑫ 0.5 mol/L EDTA
⑪ RNAase (1 mg/mL)
⑭ 70 mmol/L NaOH
⑮ 1% formaldehyde (PBS preparation)
⑯ 70%/85%/100% gradient ethanol