In-situ notch panning technique

Summary

The in situ nick translation technique is also known as in situ nick translation. It was first used to study DNA replication in vitro, and has since been extended to study the effectiveness of DNA transcription in chromosomes, cultured cells and tissue sections.

On the one hand, it is sensible and easy to observe and record; on the other hand, it can also be used for semi-quantitative (counting silver particles) or quantitative (collecting cells for liquid-flash assay) measurements, and its advantages are obvious.

This technique reflects changes in genetic material at the molecular level and is therefore much more sensitive than, for example, chromosomal aberrations, breaks, changes in sister chromatid exchange frequency, micronucleus formation, and alkaline elution and fluorescence analysis.

In practice, there are two kinds of DNase I and no DNase I. The former is mainly used to distinguish the presence or absence of transcripts.

The former is mainly used to distinguish between chromatin or genes with or without transcriptional activity or potential transcriptional activity, while the latter is mainly used to detect breakage damage to cellular DNA by factors such as carcinogens, mutagens or radiation.

Principle

The basic principle of the in situ nick panning technique is that Escherichia coli DNA polymerase I (E. coli DNA polymerase I) excises nucleotides from the 5' end of the nick (nick) of a DNA strand while ligating nucleotides from the 3' end of the nick, and proceeds sequentially in that order, with the resultant cuts panning along the DNA.

Thus, the amount of new synthesis reflects the extent of the break (damage) to the DNA strand. The in situ nick translation technique is a combination of nick displacement and isotope radioautography, which allows the extent of DNA damage to be reflected in the nucleus or chromosomes.

Appliance

The in situ incision panning technique is commonly used to differentiate between chromatin or genes with or without transcriptional activity or potential transcriptional activity, and to detect the effects of fracture damage to cellular DNA by factors such as carcinogens, mutagens, or radiation.

Operation method

In-situ notch panning technique

Principle

The basic principle of the in situ nick panning technique is that Escherichia coli DNA polymerase I (E. coli DNA polymerase I) excises nucleotides from the 5' end of the nick (nick) of a DNA strand while ligating nucleotides from the 3' end of the nick, and proceeds sequentially in that order, with the resultant cuts panning along the DNA. Thus, the amount of new synthesis reflects the extent of the break (damage) to the DNA strand. The in situ nick translation technique is a combination of nick displacement and isotope radioautography, which allows the extent of DNA damage to be reflected in the nucleus or chromosomes.

Materials and Instruments

Equipment:
①Microscope
②PCR instrument
③ ultraviolet detector
④ high-speed clutch, constant temperature water bath, high-speed vacuum dryer
Reagents:
①Materials: cultured cells
②Carnoy fixative
③500 mmol/L Tris-HCl (pH 7.4), 50 mmol/L MgCl2, 100 mmol/L La-mercaptoethanol
④10 mmol／L dATP, dGTP, dCTP, dTTP, 3H-dTTP
⑤ BSA
⑥ E. coli Polymerase I

Move

The basic process of the in situ incision panning technique can be divided into the following steps:

(1) Cell culture and processing The experiment can be performed with either normal or malignant cells, depending on the purpose of the experiment, and the cells are inoculated in small-sized plastic petri dishes (3.4 cm in diameter) with pre-positioned cover slips (2.2 cmx2.2 cm).

2 ml of medium (e.g. DMEM) containing appropriate amount of serum with antibiotics was added to each dish. The total number of cells per dish was ^3x105. The dishes were placed in a 37 °C _CO2 incubator.

After 24 h, the cells were well adhered to the cover slip, the medium was aspirated and washed twice with serum-free DMEM, and serum-free DMEM or DMEM containing a certain concentration of the test drug (e.g., MNNG, Cotton Phenol Acetate, for example, was used in this experiment) was added.

(2) Fixation of cells After a certain period of time by the action of the above test drugs, the cover slip was removed and washed with PBS (pH 7.4) to remove cell debris and so on.

Then, the cover slip was fixed with 3:1 glacial acetic acid-methanol Carnoy solution for 15 min. After drying, the cover slip with cells attached was fixed to one end of the carrier plate with gum.

After 1 d, the gum dried up and the next step of the in situ notch shift reaction was carried out.

(3) In situ notch panning reaction On the above cover sheet, add 30 μl of notch panning reaction solution dropwise to submerge the cells and cover with another clean cover sheet, being careful not to have air bubbles to ensure that all cells are impregnated with the reaction solution.

To do this, prepare Mix A first and then the reaction solution. This experiment uses 10 specimens as an example to show how much solution needs to be prepared.

①Mix A 500 mmol/L Tris-HCl (pH 7.4),188 μl; 50 mmol/L MgCl₂,188 μl; 100 mmol/L α-mercaptoethanol, 188 μl; 10 mmol/L dATP, 5.64 μl; 10 mmol/L dGTP, 5.64 μl; 10 mmol/L dCTP 5.64 μl; 10 mmol/L dTTP, 5.64 μl.

(ii) Reaction solution Mix A, 96.3 μl; 3H-dTTP, 16 μl; E. coli polymerase I (200 U/ml), 30 μl; BSA (2 mg/ml), 15 μl; double-distilled water, 145.4 μl.

(4) The reaction was terminated 15 min later by removing the overlying cover slip and washing with 50 mmol/L Tris-HCI (pH 7.4) several times to remove the reaction solution.

Then, the coverslips were macerated in 95% ethanol for 30 min, dried (about 30 min), and subjected to the isotope autoradiography procedure.

(5) Radiographic autoradiography The coverslips that have undergone the in situ notch shift reaction described above are impregnated with Kodak Radiographic Autoradiography Latex (Nuclear IN Latex can also be used) in a dark room, but the latex should be pre-warmed at room temperature, and it is usually possible to dilute the latex by a factor of one.

After drying (about 30 min), it was placed in an airtight container with desiccant and exposed for 3 d at 4 ℃.

(6) Developing and fixing agents for radiographic autoradiography. Here we recommend D19 solution, developing for 5 min, fixing for 10 min, and then rinsing in tap water for 15 min.

(7) Staining of the specimen The specimen can be stained with Giemsa stain. The author recommends lichen red (orcein) staining, the process is as follows: 2% lichen red staining for 10 min;

The procedure is as follows: 2% orcein staining for 10 min; washing with water for a few seconds; washing with glacial acetic acid-water mixture (1:1) for a few seconds; treating with 95% ethanol for 1 min; treating with n-butanol, n-butanol-xylene mixture (1:1), xylene I, and xylene II for a few seconds; and sealing with neutral gum.

(8) Observation and recording Under the oil microscope, the number of silver particles in each nucleus was counted, and the number of background particles of equal area was subtracted, which was the number of true silver particles in each nucleus.

Generally, the field of view should be taken randomly, and a total of 100 cells or more should be observed, and micrographic records should be made at the same time. If quantitative analysis is done, the cells can be collected, liquid-flash assay can be performed, and the groups can be compared with each other.

Caveat

(1) In situ notch shift reaction solution kits are available for sale, so it is not necessary to prepare your own.(2) If 3H-dTTP is not available, 3H-TdR can be used instead.(3) This technique can also be used in tissue and animal experiments.

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