Synthetic double-stranded oligonucleotides can be used to screen cDNA expression libraries constructed in λ phage to identify clones corresponding to specific DNA-binding proteins (Singhetal.1988,Vinsonetal.1988). This experiment was derived from the next volume of the Laboratory Guide to Molecular Cloning (3rd edition) by [American] J. Sambrook D.W. Russell.
Operation method
Screening of DNA binding proteins using λ phage libraries
Materials and Instruments
ATP binding buffer Denaturing solution Dithiothreitol EDTA Ethanol kinase Ligase buffer Phenol Chloroform Screening buffer SDS Sodium acetate TE T4 Phage DNA ligase T4 Phage polynucleotide kinase Synthesized oligonucleotide [a-32P]ATP Non-denaturing polyacrylamide gel Move makings For more product details, please visit Aladdin Scientific website.
Baking dish Crystallization dish Flat-tip tweezers Filled with waterproof black ink Syringe Cellulose nitrate filter membrane Sephadex G-75 centrifugal column Water bath Whatman 3 MM filter paper
Buffers and solutions
See Appendix 1 for components of storage solutions, buffers and reagents.
Dilute the storage solution to the appropriate concentration.
ATP (100 mmol/L)
Dilute 100 mml/L storage solution with 25 mmol/L Tris-Cl (pH 8.0) to prepare ATP solutions at 10 mmol/L and 50 mmol/L concentrations, respectively.
10X binding buffer
250 mmol/L HEPES (pH 7.9)
30 mmol/LMgCl2
40 mmol/LKCl
lx binding buffer with 1 mmol/L dithiothreitol
This solution is used to dilute the fully denatured solution to prepare the series of washes in this section. See steps 11-13 to calculate the volume of solution required.
1X Binding Buffer with 1 mmol/L Dithiothreitol and 0.25% Skimmed Milk Powder
Approximately 75 ml of 1X Binding Buffer is required for each 82 mm membrane used for screening, and 180 ml for 138 mm filtration. do not reduce the volume of this buffer used during the process.
1X Binding Buffer with 5% (m/V) Skimmed Milk Powder
Requires 10 ml per 82 mm membrane or 25 ml per 138 mm membrane for screening.
1X Binding Buffer with 0.25% (m/V) Skimmed Milk Powder
Requires 10 ml per 82 mm membrane or 25 ml per 138 mm membrane for screening.
1X Binding Buffer
Approximately 10 ml of this sealer is required per 82 mm membrane or 25 ml per 138 mm membrane.
Denaturing Solution (freshly prepared)
Dilute 10X Binding Buffer with 5 times the volume of distilled water (see above) and add the appropriate amount of solid guanidine hydrochloride to the resulting 2X Binding Buffer to make a 6mol/L solution. When the guanidine hydrochloride is completely dissolved, adjust the 1X binding buffer with distilled water and add dithiothreitol to a final concentration of 1 mmol/L. Approximately 15 ml of complete denaturing solution (6 mol/L) per 82 mm membrane or 25 ml per 138 mm membrane is required for screening.
Dithiothreitol (1 mol/L)
EDTA (0.5 ml/L)
Ethanol
10x Kinase/Ligase Buffer
50 mmol/L Tris-Cl (pH 7.6)
100 mmol/LMgCl2
Phenol/chloroform (1:1, v/v)
Screening buffer
1x Binding Buffer
0.25% (m/V) skimmed milk powder
1 mmol/L dithiothreitol
10ug/ml denatured salmon sperm DNA or herring sperm DNA
Approximately 10 ml of screening buffer per 82 mm membrane or 25 ml per 138 ml membrane is required for preparation of denatured salmon or herring sperm DNA as described in Chapter 6, Protocol 10.
SDS (20% m/V)
Sodium acetate (3 mol/L, pH 5.2)
TE (pH 8.0)
Enzyme and buffer
T4 Phage DNA Ligase
T4 Phage polynucleotide kinase
Nucleic acids and oligonucleotides
Synthesized oligonucleotides
After purification by gel electrophoresis and Sep-Pak affinity chromatography as described in Scheme 1 of Chapter 10, single-stranded oligonucleotides of complementary sequences of 20 to 25 nucleotides in length are dissolved in TE solution at pH 7.6 to a final concentration of 0.2 mg/ml. upon reannealing, the central region of the oligonucleotide forms the optimal form for binding to target proteins at the site of gel retention or Southwestem blotting. double-stranded monomeric form. At least one complementary base pair of the "mutant" oligonucleotide is required for the second screen. When annealed, the central region of the "mutant" oligonucleotide duplex forms a defective form of the optimal binding site and fails to bind the target protein. Sequential screening with a positive probe (the best double-stranded binding sequence) and a negative probe (the closely related but mutated double-stranded sequence) eliminates most false positives.
The two pairs of oligonucleotides should be designed to have protruding, sticky ends that can be attached to each other.
Radioactive compounds
[ a-32P ]ATP (10mCi/ml,5000Ci/mmole)
Gel
Non-denaturing polyacrylamide gel
See step 7.
Specialized equipment
Reusable chemiluminescent markers in radiolucent ink or chemiluminescent labeled adhesive labels are available from Strawgene (Glogos). The markers can be used multiple times, except that they need to be exposed to fluorescent light before each new round of self-development.
Baking dish (12 inches X 8 inches)
Crystallization dish
Flat-tip tweezers
Syringe with waterproof black ink (India ink) and 18-gauge needle
Nitrocellulose filter membrane
Sephadex G-75 column equilibrated with TE (pH 7.6) solution
Water baths pre-adjusted to 16°C, 65°C and 85°C
Whatman 3 MM Filter Paper
Additional Reagents
The reagents required for steps 8 and 10 of this protocol are listed in Protocol 1 of this chapter.
Methods
Preparation of radioactive multiplex probes
1. Prepare phosphorylation reactions of two synthetic oligonucleotides separately and then anneal both:
Oligonucleotide 200ng
10X kinase-ligation buffer (DDT) 2.5ul
100 mmol/L dithiothreitol (DDT) 2.5ul
32P ATP 100uCi
Water to 23ul
T4 phage polynucleotide kinase (8~10 units/ul) 2.0ul
Incubate at 37°C for 1 h.
2. Mix the two phosphorylation reactions together and anneal the oligonucleotides by incubating the mixture as follows, most conveniently on a thermal cycler
85°C for 2 min
65°C 15 min
37°C 15 min
22°C 15 min
4°C 15 min
3. Add 4ul of T4 phage DAN ligase (lWeiss units/ul) and 1ul of 50 mml/L ATP and incubate the mixture for 12 h at 16°C.
4. Add 0.5 mol/L EDTA (pH 8.0) to a final concentration of 5 mol/L. 5 mol/L EDTA (pH 8.0) was added to the mixture.
5. Separate labeled oligonucleotides from unconsumed γ-32P, single-stranded oligonucleotides and unlinked double-stranded oligonucleotides by column chromatography on a Sephadex G-75 column (see Appendix 8).
6. Estimate the specific activity of the final probe.
The specific activity of the labeled probe should be greater than 2x106cpm/pmole.
After annealing the 3' ends of unlabeled polycomb oligosiderophores by leveling, which may also yield the specific activities required in this section, set up end leveling reactions as described in Scheme 7 of Chapter 10, using 2-fold [ a-32P ]dNTF in each reaction whenever possible to increase the specific activity of the radiolabeled product. At the end of the reaction, the labeled oligosiderophores were separated from the unconsumed dNTP using an affinity chromatography column (please see Appendix 8).
7. Analyze the size of the radiolabeled DNA using non-denaturing polyacrylamide gel electrophoresis and radioautography (see Appendix 9). If everything goes well with the annealing and radiolabeling tests described above, the radiolabeled polycomb DNA should be seen as a ladder.
Preparation of Filter Membranes
8. To prepare plates containing the λ phage expression library, number the nitrocellulose filter membranes exactly as described in Steps 1 to 8 of Scheme 1.
If possible, use unamplified expression libraries. This is because if the desired protein is toxic to bacteria or inhibits the growth of the recombinant phage, the clone may rarely be expressed in the amplified library.
9. Remove the numbered nitrocellulose filter membrane from the phage spot with flat-tipped tweezers (e.g., Millipore valves) and place it, phage-contact side up, on a Whatman 3 MM filter paper. Allow to dry at room temperature for 15 min.
10. Place the second (numbered) IPTG-impregnated filter membrane onto the moss (see Step 4 of this protocol). Using an 18-gauge needle, mark each membrane with a hole corresponding to the position on the first membrane. Continue to incubate at 37°C for 2 h, then remove the membranes and allow to dry at room temperature as described above.
Important: The following steps were performed at 4°C. The first set of membranes was not subjected to hydrochloric acid. The first set of membranes is labeled without guanidine hydrochloride denaturation (i.e., steps 11-13 are omitted), while the second set of membranes is labeled according to steps 11-14.
11. Place the membranes in a 12" X 8" dry baking dish containing denaturing solution at 4°C. Slowly shake the membranes on a shaker platform for 5 minutes at 4°C. Discard the denaturing solution and replace with fresh denaturing solution. Shake again for 5 min.
12. Pour the second addition of denaturing solution into a measuring cylinder and dilute with an equal volume of 1X binding buffer containing dithiothreitol. Then pour it into a clean glass dish, sheet by sheet will be put into the solution of the filter membrane, pay attention to be sure to make each filter membrane can contain dithiothreitol denaturing solution in full contact.
13. Repeat step 12 four times or more, each time diluting the denaturing solution two-fold so that the concentration of guanidine hydrochloride in the denaturing solution is 3 mol/L (step 11), 1.5 mol/L, 0.75 mol/L, 0.375 mol/L, 0.187 mol/L, 0.094 mol/L. Finally, wash the membranes two times with dithiothreitol-containing 1x buffer.
14. Place both sets of membranes (i.e. denatured and non-denatured) in 1X binding buffer containing 5% skimmed milk powder. Shake gently at 4°C for 30 min.
15. Wash the membranes with 1X binding buffer containing 0.25% skimmed milk powder.
Detection of cured proteins with a radioactive probe
16. In a crystallization dish, add the 32P-labeled multiplex probe obtained in step 5 to the hybridization solution prepared in screening buffer (10 ml per 82 mm membrane or 25 ml per 138 mm membrane).
The best results in the screening reaction are obtained when the final concentration of the labeled probe (holding activity 2xl08~5x108cpm/ug) net is 25ng/ml. However. If the amount of probe is limited, then a minimum concentration of 2.5 ng/ml can be used. In the earliest application of this method (Singh et al. 1988), poly(dI:dC) was used as a nonspecific competitor. When sonicated denatured salmon or calf thymus DNA is used, the background can generally be reduced. Some DNA-bound fusion proteins may be obscured by any DNA, including DNA used as a competitive template or other complexes. Therefore, sensitivity tests to various sequestering agents such as salmonid DNA, poly(dl:dC), poly(A), and other reagents should be done prior to screening during gel lag experiments or Southwestern blotting (see Chapter 17).
17. Transfer the membrane to the radiolabeled probe solution in a crystallization dish. Shake slowly on a rotating platform at 4°C for 2~12 h.
The ionic composition of the binding/screening buffers selected in this section is used for a wide range of DNA-protein interactions; however, in some experiments it is also necessary to adjust the salt and magnesium ion concentrations to optimize binding conditions in gel lag experiments or other methods. An alternative binding buffer recommended in this section is one containing 25 mmol/L HEPES (pH 7.9), 25 mmol/L NaCl, 5 mmol/L MgCl2, and 0.5 mmol/L DTT (Singh 1993).
18. Rinse the membranes for 5 min at 4°C with large volumes (25 ml per 82 mm membrane, 60 ml per 138 mm membrane) of binding buffer containing lmmol/L dithiothreitol and 0.25% skimmed milk powder.
19 Repeat step 18 twice.
20. Discard the buffer from the last wash. Place the wet membrane on a sheet of Saran wrap and cover with a sheet of Saran wrap. Apply adhesive label paper with radioactive ink or chemiluminescent markings to several asymmetrical locations on the Saran wrap.
Cover the radioactive label with Scotch tape to prevent radioactive ink from contaminating the x-ray film holders or intensifier screens.
21. Perform radiographic self-development in accordance with Appendix 9.
22. Pick positive spots and rescreen with specific and nonspecific probes as described in the Introduction to this protocol.
The probes can be removed from the membrane by repeating the denaturation and denaturation process so that the membrane can be rehybridized with other DNA-binding protein cDNAs.