Screening of fixed-point mutagenesis recombinant cloning experiments by hybridization with radiolabeled oligonucleotides
Screening of fixed-point mutagenesis recombinant cloning experiments by hybridization with radiolabeled oligonucleotides
This protocol mainly describes the screening of recombinant phage M13 clones, whereas one option is to screen bacterial clones containing phage particles. Finally, the detection of mutants by PCR is also described. This experiment comes from the next volume of the Laboratory Guide to Molecular Cloning (3rd edition) by [American] J. Sambrook D.W. Russell.
Operation method
Screening of fixed-point mutagenesis recombinant cloning experiments by hybridization with radiolabeled oligonucleotides
Materials and Instruments
Ammonium formate Oligonucleotide hybridization solution Oligonucleotide prehybridization solution TE Phage T4 polynucleotide kinase Restriction endonuclease Agarose gel Mutagenized oligonucleotide [γ-32P]ATP 2XYT Top agar and 2xYT agar flatware Move makings For more product details, please visit Aladdin Scientific website.
Blunt-end forceps Hypodermic needle (18-gauge) and India ink Incubator Cellulose nitrate membrane or nylon membrane Vacuum oven 68°C Water bath Whatman DEAE filter paper
Buffers and solutions
For various storage solutions, buffer components and related reagents, please refer to Appendix 1
Dilute the storage solution to the desired concentration.
Ammonium formate (0.2 mol/L)
Oligonucleotide hybridization solution
6XSSC
5X Denhardt's solution
106 to 107 dpm/ml radiolabeled oligonucleotides
Oligonucleotide prehybridization solution
6xSSC
5xDenhardt's solution
0.1% (m/V) SDS
6XSSC
6XSSC should be dawdled at the desired temperature, see steps 11, 12 and 13.
TE (pH 7.6)
Enzyme and buffer
Phage T4 polynucleotide kinase
Restriction endonucleases
See steps 18 and 19.
Gel
Agarose gel
See step 18.
Nucleic Acids and Oligosynucleotides
Mutagenized oligonucleotides (10 pmoles/ul)
Radioactive substances
[ γ-32P ]ATP (>5000Ci/mmole,10mCi/ml)
Culture medium
2XYT top agar and 2xYT agar petri dishes
Specialized equipment
Blunt-end tweezers (e.g., Millipore tweezers)
Needle for hypodermic injection (18-gauge) and India ink (optional)
Incubate at 65°C for prehybridization.
For incubator suitable for hybridization temperature, see note in step 7.
Nitrocellulose or nylon membrane.
Vacuum Oven
68°C water bath
Whatman DEAE Filter Paper (DE81)
Filter papers are stored at room temperature and handled with gloves.
Other Reagents
The reagents required for Step 4 of this protocol are listed in Chapter 10, Protocol 4.
Reagents required for Step 14 of this protocol are listed in Chapter 3, Option 4.
Reagents required for steps 15 and 16 of this protocol are listed in Chapter 2, Scheme 3, 4 or 5.
Step 17 of this protocol requires reagents listed in Chapter 3, Scheme 3.
Reagents needed for step 20 of this protocol are listed in Chapter 6, Areas 8 and 10.
Vectors and Bacterial Strains
Mutagenized phage M13 recombinant
Please use the petri dish containing phage M13 phage plaque generated in Scheme 2.
Unmutated phage M13 recombinant
Use as a negative control; see Step 5.
E. coli TG1 or corresponding strain
Methods
Radiolabeling of oligonucleotides by phosphorylation reaction
1. Mix the following reagents in a sterilized microcentrifuge tube:
Mutagenized oligonucleotide (10 pmoles/ul) 1ul
10x Phage T4 Polynucleotide Kinase Buffer 1ul
10mCi/ml [γ-32P]ATP (10~50pmoles) 1ul
Water 6ul
5~10 units/ul phage polysorbate kinase 1ul
Incubate the mixture at 37°C for 30 min.
2. Add 90ul of TE (pH 7.6), dilute the mixture to 100ul and heat at 68°C for 10 min to inactivate polysporinic acid kinase.
3. Measure the efficiency of transfer of 32P to the oligonucleotide as follows and estimate the specific activity by chromatography on DE81 filter paper.
a. Cut a piece of DE81 filter paper about 1 cm wide and 7-10 cm long, and use a soft-core pencil to draw a thin, parallel line 1.5 cm from one end. Mark the origin of the start of chromatography.
b. Spot 1.0ul of diluted phosphorylated reactant at the origin. Add about 25-50 ml of 0.2 ml/L ammonium formate to a depth of about 0.5 cm in a 250 ml beaker. Place a strip of DE81 filter paper vertically in the beaker so that the radioactive sample at the origin is just above the buffer. Cover the beaker with a piece of glass or aluminum foil and allow the chromatography to progress until the front of the solvent migration is near the top of the beaker.
c. Wrap a strip of DE81 filter paper in Sharon wrapping film and radiographically self-develop it in a very short time. Using the developed X-ray film as a reference, cut off the area of the solvent front that is radioactive, the region of origin, and any other areas that are radioactive. The radioisotope intensity of each section is determined using a liquid flash counter.
Oligonucleotides were retained at the origin, while ATP and inorganic phosphorus migrated in the same direction as the solvent. The inorganic iodine migrates slightly behind the solvent front, and the ATP is located at an equal distance between the origin and the inorganic phosphorus The transfer of phosphoric acid from [ γ-32P ]ATP to the oligonucleotide results in the appearance of radioactivity at the origin, and the specific activity of the radiolabeled probe, as well as the specific activity of the [ γ-32P ]ATP in the reaction and the efficiency of the transfer of radioactivity to the oligonucleotide, is measured by an oligonucleotide molar count. More than 50% of the radioactivity of the reaction mixture in Step 1 should be transferred to the oligonucleotide.
4. (Optional) Precipitate unbound radioactivity from the oligonucleotide with cetylpyridinium bromide according to the procedure described in Scheme 4 in Chapter 10.
Under normal circumstances, the reaction mixture can be used directly as a probe. This step is not normally required unless the hybridization background continues to cause problems.
Screening for phage M13 phage spots
5. Prepare replicates of phage M13 phage spots for screening with radiolabeled oligonucleotide probes according to the following procedure:
a. Place a petri dish containing 100-500 phage spots at 4°C for at least 30 min. at least one petri dish containing the original wild-type recombinant M13 phage spot should be included. This dish serves as a negative control for the hybridization and membrane washing steps.
b. When the dishes are completely chilled, remove the dishes from the cold chamber and immediately cover the agar surface of each dish with dry nitrocellulose or nylon membrane as a marker. Use an 18-gauge needle to create a series of holes through the membrane and the underlying agar. These holes will be used in a later step as markers for matching the filter membrane to the underlying agar.
A small amount of India ink dabbed on the tip of the needle prior to needling the filter membrane creates an indelible marking spot.
c. After 30 s to 4 min, carefully remove each filter membrane from the petri dish with blunt-ended forceps. Lay the phage-stained filter membrane face up on a stack of blotting paper. Wrap the Petri dish with Sharon wrapping film and store at 4°C until needed.
d. When the membranes are dry (about 30 min at room temperature), bake in a vacuum kiln at 80°C for 1h.
Since the single-stranded DNA can be released from the phage during baking, there is no need to denature the DNA with a base when screening for phage M13 phage spots.
6. Transfer all membranes to a heat-sealable plastic bag (e.g., Sear seal-A-Meal), a suitable diameter evaporating dish, or a roller for hybridization. Add the oligonucleotide prehybridization solution (approximately 10 ml of prehybridization solution for 82 mmol/L membranes when working in a plastic bag or evaporating dish, or approximately 5 ml of prehybridization solution for 82 mmol/L membranes when working in a hybridization roller). Seal the bag, cover the mouth of the evaporation dish or the lid of the hybridization drum with Sharon packaging film, and incubate at 65°C for 1-2 h. The incubation time should not exceed 2 hours.
7. Discard the prehybridization solution and add the hybridization solution containing the oligonucleotide probe (about 5 ml/82 mmol/L membrane). Reseal the pouch, or cover the evaporating dish, or cover the hybridization roller. Carry out the hybridization reaction at the appropriate temperature for 4~6 h.
The hybridization reaction should be performed at a temperature 5-10°C below the Tm value of the radiolabeled oligonucleotide. The formula for calculating the Tm value of the oligonucleotide is as follows:
Tm=4(G+C)+(A+T)
G+C is the total number of G and C residues in the oligonucleotide and A+T is the total number of A and T residues in the oligonucleotide.
8. At the end of hybridization, quickly transfer the filter membrane to a dish containing 200-300 ml of 6XSSC solution. Cover the dish with Sharon wrapping film and shake in a rotary shaker at room temperature for 15 min, replacing the wash solution every 5 min. Simultaneously transfer the remaining radioactive hybridization solution from the bag, evaporating dish or roller to a disposable plastic tube. Close the bottle tightly and store the radioactive solution at -20°C until it is reused for positive phage spot screening (step 13).
Properly label and store the hybridization solution.
9. At the end of the membrane wash, quickly transfer the hybridization filter membrane to a sheet of Sharon Wrap Membrane laid flat on the bench. Cover the membrane with another sheet of Sharon packaging film. Fold the edges of the two sheets of Sharon Wrapping Film membrane to seal. Attach the dot with the radioactive material or fluorescent ink to the outside of the wrap. With the use of a sensitizing screen, the process of radiographic self-development of hybridization filter burst X-ray film usually takes 1 to 2 h. The process of radiographic self-development of hybridization filter burst X-ray film takes about 1 to 2 hours.
Point: Do not allow the filter film on the Sharon wrap to dry.
10. Compare the hybridization filter membrane pattern with the phage spot distribution of the petri dish. In this step, it is normal to find that all phage spots hybridize to the probe. However, certain phage spots showed stronger hybridization signals than others, and these phage spots usually carried mutants.
11. Transfer the membranes to a plastic box containing 100-200 ml of 6XSSC preheated to 10°C below Tm.
Vibrate for 2 min (see section on Shake for 2 min (see note below) and then transfer the membrane to a Sharon wrap for re-radiation autoradiography as described in step 9. This step often identifies two types of phage, one with a reduced intensity of the radioactive signal and the other without change.
If using short (<20bP) oligonucleotide probes, do not wash the membrane for more than two minutes at temperatures below 10°C below the Tm value. Otherwise, the intact hybrid formed between the radioactive oligonucleotide and the mutagenized target sequence will dissociate. Longer oligonucleotide probes require longer wash times that have been developed empirically. A handheld microradiometer can be used to monitor the intensity of the radioactive signal during the wash and estimate the appropriate wash conditions based on the decrease in the intensity of the radioactive signal.
12. Repeat the membrane wash and autoradiography, raising the temperature of the wash solution by 2-10°C per cycle, in order to find the appropriate temperature that will cause dissociation of the mismatched hybrid molecules (e.g., mutant oligonucleotides and wild-type sequences) without affecting the perfectly matched hybrid molecules.
With continued practice, it may be possible to omit some of the rinsing and autoradiography by using a handheld micro-radiation monitor to examine the filter membrane after each rinse.
Purification of Positive Phage
13. Hybridization-positive phage spots are usually a mixture of mutant and wild-type sequences. Therefore, it is necessary to purify phage from positive hybridized phage spots as follows:
a. Touch the surface of the positive hybridized phage plaque with the blunt end of a sterilized wooden disposable toothpick.
b. Place the toothpick into a sterile test tube containing lml of sterile TE (pH 7.5). Place the test tube at room temperature for 10-15 min, and shake the test tube continuously to dislodge the phage particles in the solution.
c. Perform a l0x series of dilutions of the phage suspension with TE (pH 7.6). Mix 10ul of 10-3, 10-4 and 10-5 times diluted phage with 100ul of overnight culture of E. coli (e.g. TG1), respectively.
d. Add 2.5 ml of 2XYT Top Agar (melted and cooled to 45°C) to each culture and spread the above mixture on individual YT spheroplast dishes. Incubate the dishes at 37°C for 16 h to allow phage plaque formation.
Incubate the dishes at 37°C for 16 h to allow phage plaque formation. e. Rescreen the phage plaques with radiolabeled oligonucleotide probes as described in Steps 5 to 12. It is not necessary to gradually increase the temperature of the membrane wash solution during the second round of screening. The membrane can be transferred directly from room temperature to a preheated (see step 12) empirically determined temperature 6XSSC.
14. Pick two phage spots from each of the three hybridization-positive mutants. Prepare single-stranded DNA from phage-infected small volume cultures derived from phage spots as described in Chapter 3, Scheme 4.
15. Sequence the DNA containing the target sequence by double deoxy chain termination (see Scheme 3 or 4 in Chapter 12). Sequencing universal primers can be used, or synthetic primers that bind 50-100 nucleotides upstream of the mutation site can be used.
16. When the mutant has been identified, sequence analysis is used to confirm the full sequence of the target DNA cloned into phage M13 to ensure that no unintended mutations have occurred during propagation of the phage M13 recombinant. This process usually involves designing primers that are complementary to the target DNA at 200-400bp intervals.
17. Purify the recombinant phage plaque with the desired mutation and no unexpected mutation in the target DNA region (Step 14), infect the bacteria, and isolate the replicative phage M13DNA from the infected culture medium. For the method to isolate and purify the replicative phage M13DNA, please refer to Scheme 3 in Chapter 3.
18. Digest phage M13 replicative DNA with appropriate restriction enzymes and prepare a gel electrophoresis to recover the mutated target DNA sequence. Clone the target DNA into the desired vector.
19. Digest the recombinant unmutated target sequence with several different restriction endonucleases or digest the recombinant target sequence with mutations.
20. Separate the restriction enzyme-digested DNA fragments by gel electrophoresis and transfer the DNA onto a nitrocellulose or nylon membrane as described in Scheme 8 in Chapter 6. Southern hybridization is performed at 10°C below the Tm value using 32P-labeled mutagenic oligonucleotides as probes. The membranes were washed under different conditions and subjected to radioautoradiography.
The last autoradiography should only show a signal that hybridizes to the relevant mutant target DNA fragment. 