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MOLECULAR BIOLOGY: WORKING WITH DNA

MUTAGENESIS

The Incremental Truncation for the Creation of Hybrid EnzYmes (ITCHY) Protocol using α-Phosphothioate Nucleotide Analogs

The Incremental Truncation for the Creation of Hybrid EnzYmes (ITCHY) Protocol using α-Phosphothioate Nucleotide Analogs Title | Overview | Procedure | Solutions | BioChemicals | Hints | Printable Version

Overview Title | Overview | Procedure | Solutions | BioChemicals | Hints | Printable Version

Incremental truncation is a method to generate combinatorial libraries of single-basepair deletions between two DNA fragments of interest. Based on the original protocol by Ostermeier (see Protocol ID#2213), an alternative method was developed. The approach takes advantage of the nuclease-resistance of phosphothioate internucleotide linkages. PCR amplification of a DNA fragment of interest, using a mixture of standard dNTPs and α-phosphothioate nucleotide analogs results in the random, low-frequency incorporation of the latter. Upon treatment of the DNA with Exonuclease III, the digestion is terminated at the position of the phosphothioate linker, generating the incremental truncation library at once. In contrast to the original protocol, this approach enables the truncation of both DNA fragments in the same vector (Hint #1) and does not involve any gel purification steps of the library, simplifying the experimental protocol.

Procedure Title | Overview | Procedure | Solutions | BioChemicals | Hints | Printable Version

1. Add the following components to a 1.5 ml microcentrifuge tube:    1 μg plasmid DNA    5 μl of NEB Buffer #2    2 μl of Hind III    ddH2O to 50 μl    See Hint #1 2. Incubate the reaction for 2 hr at 37°C. 3. Add 10 μl of Gel Loading Buffer, mix by trituration, and load the reaction on 1% (w/v) Agarose gel in TAE buffer (see Protocol ID#1265) 4. Electrophorese the gel at 120 Volts until the linearized plasmid is well separated (see Hint #2). 5. Excise the linearized plasmid DNA from the Agarose gel with a clean razor blade and recover the DNA as instructed in the QIAquick™ manual (Qiagen) (see Hint #3). 6. Elute the DNA from the QIAquick™ column into a microcentrifuge tube with 50 μl of Buffer EB. 7. In a thin-wall PCR tube (200 μl volume), mix the following components on ice:    1 μl of linearized plasmid DNA (approximately 10 ng)    5 μl of Taq DNA Polymerase Buffer    2.5 μl of MgCl2    3.6 μl of dNTP Mix    2 μl of αS-dNTP Mix (see Hint #4)    1 μl of Primer A (see Hint #1)    1 μl of Primer B (see Hint #1)    1 μl of Taq DNA Polymerase (see Hint #5)    dd H2O to 50 μl 8. Place the tube in a PCR thermocycler and execute the following program:        1 cycle of 94°C for 5 min        94°C for 30 sec    53°C for 30 sec    72°C for 4 min and 30sec (see Hint #6)    Repeat for 30 cycles        72°C for 7 min 9. Purify the PCR product as instructed by the QIAquick™ manual (Qiagen). 10. Elute the DNA from the QIAquick™ column into a microcentrifuge tube with 50 μl Buffer EB. 11. Determine the DNA concentration of the PCR product by OD260 measurement (see Hint #7). 12. Add the following components to the remaining PCR product (approximately 45 μl):    5.5 μl of Exonuclease III Buffer    3 μl of Exonuclease III (see Hint #7) 13. Incubate the reaction at 37°C for 30 min. 14. Add 300 μl of Buffer PB to the reaction mixture and repeat Steps #9 through #10. 15. Add the following components to the DNA solution:    5.5 μl of Mung Bean Nuclease Buffer    1.5 μl of Mung Bean Nuclease (see Hint #8) 16. Incubate the reaction at 30°C for 30 min. 17. Add 300 μl of Buffer PB to the reaction mixture and repeat Steps #9 through #10. 18. Add the following components to the microcentrifuge tube:    6 μl of Klenow Buffer    2 μl of dNTP Mix 19. Pre-equilibrate the reaction at 37°C for 10 min. 20. Add 1 μl of Klenow Fragment DNA Polymerase to the tube (see Hint #9). 21. Continue the incubation of the tube at 37°C for 10 min. 22. Add 300 μl of Buffer PB to reaction mixture and repeat Steps #9 through #10. 23. Add the following components to the tube for the intramolecular ligation of the truncation library:    40 μl of Ligase Buffer    36 μl of PEG (50%)    24 Weiss Units of T4 DNA Ligase    dd H2O to 400 μl 24. Incubate the reaction at 4°C for 12 hr to overnight. 25. Add 1.2 ml of Buffer QG to the reaction and purify DNA according to the QIAquick™ manual (Qiagen). 26. Elute the DNA from the QIAquick™ column into a microcentrifuge tube with 30 μl of Buffer EB. 27. Transform bacterial host cells by electroporation (see Protocol ID#73)

Solutions Title | Overview | Procedure | Solutions | BioChemicals | Hints | Printable Version

Mung Bean buffer (10X)    500 mM Sodium Acetate pH 5.0 300 mM NaCl 10 mM ZnSO4 Exonuclease III    200 Units/μl Exonuclease III Exo III buffer (10X)    660 mM Tris-HCl, pH 8.0 6.6 mM MgCl2 Klenow fragment    5 Units/μl Klenow Fragment DNA Polymerase Taq DNA polymerase    2.5 Units/μl Taq DNA Polymerase Klenow buffer (10X)    50 mM MgCl2 100 mM Tris-HCl, pH 7.5 NEB Buffer #2 (10X)    500 mM NaCl 10 mM Dithiothreitol (DTT) 100 mM MgCl2 100 mM Tris-HCl, pH 7.9 Hind III    20 Units/μl Hind III Restriction Enzyme MgCl2    25 mM MgCl2 Taq DNA polymerase buffer (10X) Taq DNA polymerase buffer (10X) Taq DNA polymerase buffer (10X)    500 mM KCl 100 mM Tris-HCl, pH 9.0 1% (v/v)Triton X-100 Primer B    See Hint #1 1 μM 3' Oligonucleotide αS-dNTP Mix Primer A    See Hint #1 1 μM 5' Oligonucleotide αS-dNTP Mix T4 DNA Ligase    3 Weiss Units/μl T4 DNA Ligase αS-dNTP Mix PEG (50%)    50% (v/v) Polyethylene Glycol 8000, aqueous solution αS-dNTP Mix    0.5 mM αS-dTTP 0.5 mM αS-dCTP 0.5 mM αS-dATP 0.5 mM αS-dGTP Ligase Buffer (10X)    10 mM ATP 300 mM Tris-HCl, pH 7.5 100 mM Dithiothreitol (DTT) 100 mM MgCl2 Mung Bean Nuclease    10 Units/μl Mung Bean Nuclease dNTP Mix    2.5 mM dGTP 2.5 mM dCTP 2.5 mM dTTP 2.5 mM dATP Buffer PE (QIAquick™ kit, Qiagen) Buffer PB (QIAquick™ kit, Qiagen) Buffer QG (QIAquick™ kit, Qiagen) Buffer EB    10 mM Tris-HCl, pH 8.5 Gel Loading Buffer (6X)    300 μl of ddH2O 200 μl Blue/Orange Loading Dye (Promega) 500 μl 50% (v/v) Glycerol

BioReagents and Chemicals Title | Overview | Procedure | Solutions | BioChemicals | Hints | Printable Version

Restriction enzyme, Hind III Ligase, T4 DNA Alpha-phosphothioate dTTP Alpha-phosphothioate dCTP Alpha-phosphothioate dGTP Alpha-phosphothioate dATP Magnesium Chloride Tris-HCl Polyethylene Glycol, 8000 Exonuclease III Potassium Chloride DNA Polymerase, Klenow Sodium Chloride Nuclease, Mung Bean Glycerol DNA Polymerase, Taq dCTP dTTP Sodium Acetate dGTP Dithiothreitol dATP Zinc Sulfate ATP Triton X-100

Protocol Hints Title | Overview | Procedure | Solutions | BioChemicals | Hints | Printable Version

1. For details on the vector and primer design for the ITCHY method, see Citation #1. 2. Following digestion with Hind III restriction enzyme, the contributor of the protocol recommends the purification of the DNA by Agarose gel electrophoresis. Otherwise, even the smallest amount of undigested vector will be carried through the protocol and upon transformation, bias the library. 3. The contributor of this protocol recommends the use of this product because of superior results for DNA recovery/purification from gels and solutions compared to Ethanol precipitation or other DNA purification procedures. 4. Racemic mixtures of α-phosphothioate nucleoside triphosphates are commercially available from Promega and Amersham/Pharmacia. Adjust the concentration of the nucleotide analogues with ddH2O accordingly. Depending on the length of the truncation sequence desired, the ratio between standard dNTPs and α-phosphothioate nucleotides requires empirical determination to yield optimal results. The described 10:1 mixture was successfully applied for truncations over a range of 200 to 1000 bases. 5. The DNA polymerase may be added after the initial denaturing step ("HotStart" conditions) to improve the quality and quantity of the PCR reaction. The contributor of this protocol recommends using Taq DNA Polymerase for the reaction to maintain high fidelity. Polymerases with exonuclease activity are not suitable for this reaction (for details, see Citation #1). 6. Primer extension time is determined by the size of the linearized DNA fragment. As a general rule, the contributor of this protocol recommends the rate of 1 min per kilobase plus 30 seconds (e.g., the primer extension time for a 4 kb plasmid would be 4 min plus 30 sec). 7. The total amount of DNA determines the amount of Exonuclease III to add in the next step. The contributor of this protocol recommends the use of 120 Units of Exonuclease III per microgram of DNA (e.g., a standard 50 μl PCR reaction yields 5 μg of DNA. This requires 600 Units of Exonuclease III, which corresponds to 3 μl at 200 Units/μl) 8. The contributor of this protocol recommend the use of 3 Units of Mung Bean Nuclease per microgram of DNA (e.g., based on the original 5 μg of DNA, the sample requires 15 Units of Mung Bean Nuclease, which corresponds to 1.5 μl at 10 Units/μl) 9. The contributor of this protocol recommends the use of 1 Unit of Klenow Fragment DNA Polymerase per microgram of DNA.

Citation and/or Web Resources Title | Overview | Procedure | Solutions | BioChemicals | Hints | Printable Version

1.Lutz, S., Ostermeier, M., Benkovic, S.J. (2001) Nucleic Acids Res. 29(4):e16.

   

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