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Synthesis of Degenerate Oligonucleotide Inserts for Phage-Display Libraries
Contributor:
The Laboratory of George P. Smith at the University of Missouri
URL: G. P. Smith Lab Homepage
Overview
This procedure describes the preparation of double-stranded DNA fragments containing degenerate coding sequences for insertion into a phage-display vector (such as fUSE5 or f88-4). The library of phage produced by this method may then be screened for desired enzymatic activities and/or specific binding features.
The starting material is a pool of degenerate oligonucleotides whose sequence is flanked at either end by restriction sites to enable subsequent cloning in the appropriate reading frame in the phage vector of choice. A 20-nucleotide primer, complementary to the 3' end of the degenerate oligonucleotides, is used to generate a partial duplex that is then converted to a fully double-stranded fragment with the Klenow fragment of DNA Polymerase I. This product is then cleaved with appropriate restriction enzymes, purified, and ligated into the phage display vector.
Procedure
A. Klenow Fragment Extension
1. Combine the following in a 1.5 ml microcentrifuge tube (see Hint #2):
1.21 nmol Degenerate Oligonucleotide
1.21 nmol Primer
50 μl of 1 M Tris-HCl, pH 7.8
10μl of 1 M MgCl2
ddH2 to a final volume of 240 μl.
2. Vortex the sample briefly and microcentrifuge at medium speed to bring the solution to the bottom of the tube.
3. Incubate at 68°C for 5 min, at 37°C for 10 min, and then at room temperature for 10 min to anneal the oligonucleotides.
4. Place the microcentrifuge tubes on ice and add the following (see Hint #3):
10 μl of 5 mM dNTP
677 μl of ddH2O
375 Units of Klenow Fragment (New England BioLabs, approximately 75 μl)
5. Vortex gently and incubate at 30°C for 45 min.
6. Cool quickly by placing the tubes in an ice-water bath.
7. Electrophorese 3 μl of the sample as described in Section B.
8. Inspect the gel to verify that the DNA fragment has been extended to generate a DNA fragment of the expected size. If the DNA fragment is of the expected length, continue with Section C.
B. Polyacrylamide Gel Electrophoresis
Analysis of the DNA by gel electrophoresis is an important quality control for building a good library.
1. Remove 3 μl of the Klenow reaction to be analyzed. Combine this with 3 μl of TE Buffer and 1 μl 70/75/BPB Solution.
2. Immediately freeze the remaining reaction mixture at -80°C.
3. Electrophorese the sample on a 15% Polyacrylamide/1X TBE minigel with a suitable set of DNA markers (see Protocol ID#455).
C. DNA Isolation with a Centricon 30®
1. Remove the reaction mixture from the freezer and add 50 μl of 250 mM EDTA.
2. Thaw the liquid between the fingers and mix gently.
3. Incubate at 68°C for 10 min to inactivate the Klenow fragment.
4. Pipette half of the solution (approximately 525 μl) to a fresh 1.5 ml microcentrifuge tube.
5. Add an equal volume of Neutralized Phenol solution to each tube and vortex vigorously.
6. Microcentrifuge at maximum speed to separate the phases. Carefully draw off with a 200 μl pipette and discard the organic (lower) phase of the solution, leaving all of the interphase and upper phase in the microcentrifuge tube (see Hint #4).
7. Microcentrifuge at maximum speed to separate the phases once more and transfer the upper (aqueous) phase to a fresh microcentrifuge tube. Be very careful to avoid any of the interphase or lower (organic) phase (see Hint #5).
8. Add an equivalent volume of 100% Chloroform to the aqueous phase.
9. Extract the aqueous phase with the double-spin method (Step #C6).
10. Transfer the aqueous phase into a fresh microcentrifuge tube and add an equal volume of 100% Chloroform.
11. Extract the aqueous phase with the double-spin method (Step #C6).
12. Evaporate the extracted aqueous phase with a vacuum microcentrifuge (SpeedVac Concentrator, see Hint #6).
13. Dissolve each sample in TE Buffer and pool (see Hint #7).
14. Transfer the solution to a Centricon 10® filter (Amicon) and fill the Centricon® well to the 2 ml mark with TE Buffer (see Hint #8).
15. Centrifuge the Centricon 10® at 5,000 rpm with a Sorvall® SS-34 rotor (3,000 X g) 4°C for approximately 45 min.
16. Discard the solution in the reservoir and add 2 ml of TE Buffer to the Centricon 10® well.
17. Centrifuge the Centricon 10® at 5,000 rpm with a Sorvall® SS-34 rotor (3,000 X g) 4°C for approximately 45 min (or until the volume remaining in the Centricon 10® well is approximately 100 μl).
18. Wash the Centricon 10® with TE Buffer two more times (Steps #C12 to #C14).
19. Back-centrifuge the Centricon 10® to collect the retentate (see Hint #9).
20. Transfer the retentate into a fresh 1.5 ml microcentrifuge tube.
21. Add approximately 100 μl of TE Buffer to the Centricon 10® and vortex the filter apparatus.
22. Back-centrifuge the Centricon 10® once again and pool the wash with the original retentate (see Hint #10).
23. Electrophorese 1 μl of the pooled retentate as in Section B above with suitable DNA size markers and estimate the amount of DNA from the band intensity.
D. Restriction Fragment Digest
1. Thaw the reaction mixture by rubbing the tubes between your fingers.
2. Digest the double-stranded product with the appropriate Restriction Enzyme according to the reaction conditions suggested in the accompanying product information. (see Hint #11).
3. Electrophoresis approximately 100 to 300 ng DNA on a polyacrylamide gel (Section B) to confirm that cleavage has occurred. The gel should resolve uncleaved, singly-cleaved, and doubly-cleaved fragments---the latter being the target product. Cleavage is satisfactory as long as there is a substantial amount of doubly-cleaved product.
E. Degenerate Oligonucleotide Collection
1. Add sufficient 250 mM EDTA to the frozen bulk digest in order to bring the final EDTA concentration to 1.25 times the Mn2+ concentration.
2. Thaw the reaction mixture by rubbing the tubes between your fingers.
3. Add 1X TE Buffer to bring the total volume to 1 ml.
4. Extract, concentrate, and wash as described in Steps #C4 to #C21.
5. Prepare a 15% Polyacrylamide/1X TBE gel with 1 mm spacers and a 1 mm comb with a 7.2 cm wide preparative well and a small marker well (see Protocol ID#455, also see Hint #12).
6. Add one-sixth volume of 70/75/BPB Solution to the washed DNA sample and mix lightly.
7. Load up to 50 μg of DNA into a single well on the preparative gel (see Hint #13).
8. Load suitable DNA markers into the marker well (see Hint #14).
9. Electrophorese the DNA sample (conditions will vary depending on oligonucleotide size; use a time and voltage that is appropriate).
10. Place the gel in a shallow pan, completely cover the gel with 1X TBE, and agitate gently for a couple of minutes.
11. Remove the 1X TBE solution and completely cover the gel with Ethidium Bromide Solution.
12. Incubate at room temperature for 20 min with constant but gentle shaking.
13. Remove the Ethidium Bromide Solution (and discard appropriately as hazardous waste).
14. Completely cover the gel with 1X TBE, incubate for a couple of minutes with gentle shaking and then discard the 1X TBE solution appropriately as hazardous waste.
15. Use a scalpel blade (or single edged razor blade) and an old piece of X-Ray film to aid in excising the DNA band of interest. Use a small spatula and a clean weigh boat to aid in the manipulation of the gel fragment. Caution: Always wear gloves when handling gels stained with Ethidium Bromide.
16. Place the stained gel onto the piece of X-ray film and illuminate it with a long-wave UV Transilluminator. Working as quickly as possible to minimize the exposure of the DNA to the UV light, excise the double-cleaved band (which is roughly 72 x 3 mm) with the scalpel and transfer it with the spatula to the clean weigh boat.
17. Cut the excised band evenly into eight 9 x 2 mm strips and place two strips into each of four Centrilutor® capped inserts (Amicon, see Hint #15).
18. Prepare the Centrilutor® electroeluter apparatus (Amicon) to accommodate the four Centricon 10®s as described in the manufacturer's instructions.
19. Fill the top and bottom reservoirs with 1X TBE Buffer and dip each Centrilutor® insert (with gel fragments inside) in the upper buffer a few times to ensure that air bubbles are removed.
20. Load the Centrilutor® into the Centricon 10®.
21. Using a 9-inch Pasteur pipette whose end has been bent into the shape of a hook, squirt 1X TBE buffer vigorously under each Centricon® to expel trapped air bubbles (see Hint #16).
22. Electroelute the sample at 100 V for 2 hr.
23. Disassemble the Centrilutor® apparatus while being very careful not to lose any of the solution in the Centricon 10® well (see Hint #17).
24. Concentrate the electroelutes by centrifuging the Centricon 10® at 5,000 rpm with a Sorvall® SS-34 rotor (3,000 X g) at 4°C for approximately 45 min.
25. Back-centrifuge from three of the Centricon 10®s to collect the retentate (see Hint #9).
26. Transfer the retentate from the three Centricon 10®s to the fourth Centricon 10® well.
27. Add 200 μl of TE Buffer to the three Centricon 10®s, vortex the filter apparatus, and back-centrifuge the three Centricon 10®s.
28. Pool the TE Buffer wash from the three Centricon 10®s into the fourth Centricon 10®s well (see Hint #18).
29. Concentrate the electroelute by centrifuging the Centricon 10® at 5,000 rpm using a Sorvall® SS-34 rotor (3,000 X g) at 4°C for approximately 45 min.
30. Discard the solution in the reservoir and add 2 ml of TE Buffer to the Centricon 10® well.
31. Centrifuge the Centricon 10® at 5,000 rpm with a Sorvall® SS-34 rotor (3,000 X g) at 4°C for approximately 45 min (or until the volume remaining in the Centricon 10® well is approximately 100 μl).
32. Repeat washing the Centricon 10® with TE Buffer two more times (Steps #E29 to #E31).
33. Back-centrifuge the Centricon 10® to collect the retentate (see Hint #9).
34. Collect the retentate into a 1.5 ml microcentrifuge tube.
35. Add approximately 100 μl of TE Buffer to the Centricon 10® and vortex the filter apparatus.
36. Back-centrifuge the Centricon 10® again and pool the wash with the original retentate (see Hint #9).
37. Electrophorese approximately 0.5 μg of DNA, as in Section B above, with a suitable set of DNA markers. Estimate the amount of DNA from the band intensity.
Solutions
1 M MgCl2
1 M Magnesium Chloride
Ethidium Bromide Solution
0.5 μg/ml Ethidium Bromide (CAUTION! See Hint #19)
Prepare in 1X TBE
250 mM EDTA
Neutralized Phenol
Allow phases to separate and remove the aqueous (upper) phase
Equilibrate with Tris once more
Use water-saturated Phenol (CAUTION! See Hint #19)
Shake or vortex vigorously to equilibrate phases
Add 0.1 volume of 1 M Tris-HCl, pH 8.0
Use the lower phase as Neutralized Phenol
TBE (5X)
Store at room temperature
31 g Boric Acid (H3BO3, CAUTION! See Hint #19)
60.5 g Tris-HCl
3.7 g Disodium EDTA (Na2EDTA-2H2O)
Do not adjust the pH
Add 1 liter ddH2O
70/75/BPB Solution
70% (v/v) Glycerol
75 mM EDTA
0.3% (w/v) Bromophenol Blue
TE Buffer
Autoclave and store at room temperature
10 mM Tris-HCl, pH 8.0
1 mM EDTA
5 mM dNTP
5 mM dGTP
5 mM dCTP
5 mM dATP
5 mM dTTP
Also available from commercial sources
1 M Tris-HCl, pH 8.0
Autoclave
1 M Tris-HCl, pH 8.0
1 M Tris-HCl, pH 7.8
1 M Tris-HCl, pH 7.8
Autoclave
BioReagents and Chemicals
Phenol
Tris-HCl
EDTA
Klenow Fragment
Oligonucleotide
Disodium EDTA
Glycerol
Bromophenol Blue
Boric Acid
dCTP
dTTP
dGTP
dATP
Ethidium Bromide
Magnesium Chloride
Chloroform
Protocol Hints
1. An example of a pair of oligonucleotides for making a degenerate insert for vector fUSE5 is shown in Image #1. The degenerate oligonucleotide strand is on the top, the primer strand in the middle, and the intended amino acid sequence at the N-terminus of mature pIII is on the bottom. The two oligonucleotides are complementary over a 10-base-pair overhanging region, enough for the Klenow fragment to extend both 3' ends.
2. Start with large amounts of the degenerate oligonucleotide and the primer (enough theoretically to make 70 μg of the 88 base pair double-stranded product in the case of the oligonucleotides described in Hint #1). This amount is more than that which can be loaded on the "check-gel" described in Section B. However, working on a large scale helps to compensate for losses.
3. Final reaction concentrations are as follows:
50 μM dNTPs
1.2 μM Degenrate Oligonucleotide
1.2 μM Primer
50 mM Tris-HCl
10 mM MgCl2
375 Units/ml Klenow Fragment.
4. The purpose of removing the organic phase is to lower the interphase into the narrow tip of the microcentrifuge tube so that the aqueous phase can be drawn off with a high yield. Avoid removing the aqueous phase.
5. The contributor recommends the double-centrifugation method to increase the yield of the aqueous phase from each organic extraction.
6. Evaporate the DNA sample to remove any excess Chloroform. The Centricon 10® used in the following steps is not resistant to Chloroform.
7. Any volume of TE Buffer can be used to dissolve the DNA so that the pooled samples will have a final volume of less than 2 ml. Make sure the DNA is completely dissolved before loading the sample onto the Centricon 10®.
8. The Centricon 10® has a molecular weight cut-off of 10 KDa.
9. Follow the manufacturer's instructions to collect the retentate.
10. The final volume recovered from the Centricon 10® filter should be approximately 200 μl, and the theoretical concentration is approximately 100 to 350 μg/ml (depending on the size of the Oligonucleotide).
11. Use HindIII and PstI for the f88-4 inserts. Use SfiI or BglI for the fUSE5 inserts. Typically, the reaction is done in REact 2 Buffer (Gibco-BRL) for 2 hr at 37°C. The density of the restriction sites on these small synthetic duplexes is much higher than in natural DNA; therefore, use approximately 10 Units Restriction Enzyme per μg of DNA to ensure a good digestion. In order to keep the volume of enzyme at a reasonable level, it is advantageous to use high-concentration forms of the enzymes available from many suppliers.
12. These dimensions are for the BioRad Mini Protean II minigel system; other systems will have similar dimensions. The key is to prepare a gel with a large preparative well, allowing one to load a sizeable amount of DNA into a single lane.
13. The maximum amount of DNA (50 μg) is for a well that is 7.2 cm wide; appropriately increase or decrease the maximum amount of DNA loaded onto the gel depending on the size of the preparative well in use.
14. Load enough DNA marker sample so that the marker bands can be seen even with light staining by ethidium bromide.
15. The Centrilutor® capped inserts have holes in the bottom and top to allow the current to pass through and to allow eletroeluted DNA molecules to pass through the bottom.
16. Briefly expose the middle segment of the thin section of a 9-inch Pasteur pipette to a Bunsen burner flame, then quickly bend the tip of the thin section to form a U-shape (hook shaped). The shape of the Pasteur pipette will allow water to be forced into the underside of a Centricon® loaded into the Centrilutor®. Use the mirror on the bottom of the lower tank buffer reservoir to aid in sighting the bubbles. The Centrilutor® instructions advise sucking the bubbles out; however, the contributor of this protocol has found that to be much more difficult and time consuming than squirting the trapped air out with vigorous positive upward pressure.
17. Carefully lift the upper buffer reservoir (with Centricons) off the lower reservoir, pour out the buffer from the lower reservoir, re-install the upper buffer reservoir, and by pulling one of the stoppers from an unused hole, drain the upper buffer reservoir into the lower one. Slowly and carefully lift the inserts out of the Centricons®, allowing any buffer therein to drain back into the Centricons®. Gently pull the Centricons® out of their holes in the upper buffer reservoir while being very careful not to allow the buffer in the Centricons to spill out.
18. The fourth Centricon 10® should now contain all of the electroeluted DNA. This includes the original retentates from three of the Centricon 10®s, as well as the TE Buffer washes from the three Centricon 10®s.
19. CAUTION! This substance is a biohazard. Consult this agent's MSDS for proper handling instructions.
