Analysis of radioisotope-labeled DNA sequence determination

Summary

Radioisotope-labeled DNA sequencing analysis can be applied to analyze gene structure-function relationships.

Operation method

Analysis of radioisotope-labeled DNA sequencing

Principle

Using a single-stranded DNA template or a denatured double-stranded DNA template and an appropriate DNA synthesis primer, DNA polymerase synthesizes the exact complementary strand using the single-stranded DNA template, and during synthesis, some sort of dNTP is exchanged for ddNTP, which is then used as a substrate by DNA polymerase to terminate the growth of the DNA strand by incorporating it into the 3' end of the oligonucleotide chain without 3'-OH at the 3' end. The DNA polymerase uses 2',3'-dideoxyribonucleoside triphosphate as a substrate and dopes it into the 3' end of the oligonucleotide chain, resulting in the absence of 3'-OH at the 3' end, thus terminating the growth of the DNA strand. The different types of dideoxyribonucleotides and the different doping positions result in the termination of complementary strands of different lengths at different specialized positions. The sequence of the complementary strand of the template DNA can be read out by doping radioactive nucleotides and polyacrylamide gel electrophoresis.

Materials and Instruments

DNA Samples
Silicone Solution PAGE Gel Plasmid DNA Alkaline Denaturation Solution T7 Sequencing Kit Anhydrous Ethanol
Syringes Sequencing plates Electrophoresis apparatus Centrifuge

Move

1. Siliconize the sequencing plate, wash with running water and ddH2O, wash with anhydrous ethanol and air dry.

2. gel filling: mount the sequencing plate, place the glass plate at an angle of 15-30° tilted, fill the gel between the two pieces of glass with a 50ml syringe, insert the flat end of the shark-tooth comb into the upper edge of the gel, about 0.5-1cm deep. clamp it well and place the sequencing plate horizontally. Pre-electrophoresis after polymerization for about 3hr.

3. Pre-electrophoresis: install the electrophoresis device according to the instructions, and inject 1×TBE into the upper and lower tanks. set the temperature at 50°C, power at 100W, and time at about 30min.

(Prepare sequencing reaction at the same time)

4. Double-stranded base denaturation of plasmid DNA: dissolve 3-5μg of DNA in 32μl of ddH2O, add 8μl of 2N NaOH, mix well, and let it stand at room temperature for 15min. add 7μl of 3M NaAc, 120μl of anhydrous ethanol, mix well and let it stand at -20℃ for 30min. centrifugation is 12000g×15min, discard the supernatant and wash with 500μl of 70% ethanol once, centrifugation is 12000g×7min. Centrifuge at 12000g×7min, discard the supernatant, dry the precipitate, and dissolve it in 10μl of sterilized ddH2O. 5.

5. Template and primer recovery: add 2μl of sequencing primer and 2μl of Annealing buffer, mix well, centrifuge slightly, incubate at 65℃ for 5min, and then immediately place at 37℃ for 10min, room temperature for 5min or more.

6. Labeling reaction: add 3 μl labeling mixture, 1 μl of the corresponding radioisotope (10 μCi), 2 μl of T7 sequencing enzyme (diluted 1:5 with diluent before use), mix, centrifuge, and place at 37 ℃ for 5 min.

7. Prepare four 0.5ml microcentrifuge tubes, labeled A, C, G, T, in which 2.5μl of ddATP, ddCTP, ddGTP, ddTTP were added.

8. Chain termination reaction: add 4.5μl of reaction solution to each of the four microcentrifuge tubes of A, C, G and T, 37℃ for 5min. add 5μl of stop solution and centrifuge briefly.

9. Sampling: Turn off the power of pre-electrophoresis, rinse the gel plane, and insert the shark's teeth into the gel plane about 1-2mm to form a sampling hole. Place four reaction tubes at 80℃ for 2min, and immediately take 1.5-2μl and add it to the sampling hole.

10. Electrophoresis: 50℃, 100W, electrophoresis 2.5hr, pause the electrophoresis, in the reserved holes after the second sample, continue electrophoresis 2.5hr.

11. Lower gel: stop electrophoresis, remove the sequencing plate, pour off the electrophoresis buffer. Disassemble the sequencing plate, the gel should be stuck on the unsiliconized glass plate. Cut a piece of filter paper slightly larger than the gel, lay it flat on the gel, lift up the filter paper and the gel will be lifted up together.

12. Pressing: Cover the gel with plastic wrap and use a monitor to read the intensity of the radiation to estimate the exposure time. In the darkroom covered with X-ray film, placed in the dark folder, -70 ℃ radiation self-development.

13. Wash and read the film: remove the dark folder and return to room temperature. After D72 development, acidic fixing solution fixation, washing, drying. Read the sequence on the X-ray film lamp.

Caveat

1. The cleaning and siliconization of sequencing plate directly affects the filling and gluing. Poor handling will easily lead to the generation of bubbles when filling the gel, and the electrophoresis gel will be easily damaged when placing the gel.

2. Avoid the leakage of the gel when filling the gel; when filling the gel between two pieces of glass with syringe, pay attention to the speed control to prevent the generation of air bubbles.

3. When sequencing reaction and electrophoresis sampling, pay attention to the careful operation to prevent radioactive isotope contamination, and at the same time, strengthen the protection of oneself in the whole process of subsequent experiments.

4. standardize and properly dispose of radioisotope-contacted articles and wastes.

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