Molecular Techniques and Methods

Site-directed In vitro Mutagenesis with Oligonucleotides

Copy Right © 2001/ Institute of Molecular Development LLC

INTRODUCTION

In this protocol, an oligonucleotide with the desired sequence change is used to alter a single-stranded DNA sequence. This altered DNA sequence is then converted into a biologically active circular DNA strand by using the oligonucleotide to prime in vitro synthesis on a single-stranded circular template. This protocol uses a DNA template containing a small number of uracil residues in place of thymine. Use of the uracil-containing template allows rapid and efficient recovery of mutants.




E.coli mutant
Phenotype
dut-
lack the enzyme dUTPase and contain elevated concentrations of dUTP which effectively competes with TTP for incorporation into DNA.
ung-
lack the enzyme uracil N-glycosylase which normally removes uracil from DNA.
dut- ung-
deoxyuridine is incorporated into DNA in place of thymidine and is not removed. Thus, standard vectors containing the sequence to be changed can be grown in a dut- ung- host to prepare uracil-containing DNA templates for site-directed mutagenesis.




MATERIALS AND SOLUTIONS

Single-Stranded M13 Bacteriophage Vector with Insert


E. coli CJ236 (dut- ung- F' strain)


5 x Phage Precipitation Solution (100 ml)
15% (w/v) Polyethylene glycol 8000 ------------------ 15 g
2.5 M NaCl ------------------------------------------ 50 ml of 5 M NaCl
Add deionized H2O to make a final vol. of ----------- 100 ml


5 x Polymerization Mix (1 ml)
100 mM Tris-HCl (pH 8.8) --------------------------- 100 ul of 1 M Tris-HCl
10 mM DTT ------------------------------------------ 10 ul of 1 M DTT
50 mM MgCl2 ---------------------------------------- 50 ul of 1 M MgCl2
2.5 mM dATP ---------------------------------------- 25 ul of 100 mM dATP
2.5 mM dCTP ---------------------------------------- 25 ul of 100 mM dCTP
2.5 mM dGTP ---------------------------------------- 25 ul of 100 mM dGTP
2.5 mM dTTP ---------------------------------------- 25 ul of 100 mM dTTP
5 mM ATP ------------------------------------------- 50 ul of 100 mM ATP
Distilled H2O ----------------------------------------- 690 ul
  • Use HPLC-purified dNTPs.
  • The use of high concentration of dNTPs (500 uM each) serves to optimize DNA synthesis and to reduce the 3'-exonuclease activity of the T4 DNA polymerase.



    PROCEDURES

    Preparation of Uracil-Containing Single-Stranded M13 DNA Template

    1. Pick a M13 plaque containing the DNA of interest with a Pasteur pipet.

    2. Place in 1 ml sterile 1 x YT medium in a microfuge tube.

    3. Incubate 5 min at 65oC to kill cells.

    4. Vortex vigorously for 1 min to release phage from the agar.

    5. Centrifuge at 13,000 rpm for 1 min to pellet E.coli cells and agar.

    6. Place 100 ul of M13 phage supernatant into 1 liter flask containing 100 ml 1 x YT medium supplemented with uridine to 0.25 ug/ ml.

    7. Add 5 ml of a mid-log culture of E. coli CJ236 (dut- ung- F').
  • These proportions result in a multiplicity of infection of considerably less than one per cell. Thus, all of the input phage infect cells and are passaged through the dut- ung- strain.

    8. Incubate with vigorous shaking at 37oC for 6-18 hr.
  • The good aeration provided by vigorous shaking is important for high phage titers.

    9. Centrifuge at 5,000g for 30 min to pellet E.coli.
  • The clear supernatant should contain phage at a titer of about 1011 pfu/ml.

    10. Titer the phage on any E. coli ung- (e.g., CJ236) versus ung+ strain (e.g., JM105, JM107, or JM109).
  • Phage containing uracil in the DNA have normal biological activity in the ung- host, but > 100,000-fold lower survival in the ung+ host.

    11. Add 1 volume of 5 x Phage Precipitation Solution to 4 volume of phage supernatant.

    12. Mix well and incubate 1 hour at 0oC.

    13. Centrifuge at 5,000g for 15 min to pellet phage.

    14. Resuspend the phage pellet in 5 ml TE.
    Vortex vigorously.

    15. Place the resuspended phage solution on ice for 1 hr.
    Centrifuge at 5,000g for 15 min.
  • This step removes residual cell debris and endogenous low-molecular-weight DNA, which can nonspecifically prime in subsequent in vitro DNA polymerase reactions.

    16. Do phenol: chloroform: IAA extraction.
    Do chloroform: IAA extraction.

    17. Add 1/10th vol of 3 M Sodium acetate and 2.5 volume of ethanol.

    18. Precipitate phage DNA in ice for 5 min.

    19. Centrifuge at 13,000 rpm for 5 min to pellet phage DNA.

    20. Determine the single-stranded M13 phage DNA concentration at 260 nm.
  • (1 OD260 = 36 ug/ ml for single-stranded DNA).



    Phosphorylation of Oligonucleotides

    21. To a 1.5 ml microcentrifuge tube add the following:

    10 x PNK Buffer
    2 ul
    10 mM ATP
    2 ul
    Oligonucleotides (15-50 nucleolides long) with the desired sequence change
    Add distilled H2O to make a final vol. of
    20 ul

  • The amount of oligonucleotide used depends upon the desired molar ratio of oligonucleotide to single-stranded template.
  • Ratios from 2:1 to 10:1 are routine. For primers that are 15-20 bases in length, this corresponds to 4-30 ng/ ug single-stranded M13 phage DNA template.

    22. Add 2 U of T4 Polynucleotide kinase and incubate 60 min at 37oC.

    23. Add 1 ul of 0.5 M EDTA to terminate the reaction.

    24. Heat to 70oC to denature the enzyme.



    Second Strand Synthesis by Primer Extension

    25. Add the following in a microfuge tube:

    Phosphorylated oligonucleotides from step 24
    20 ul
    Single-stranded circular uracil-containing DNA template from step 20
    1 ug/ul
    20 x SSC
    1.25 ul

  • Mix thoroughly.

    26. Place tube in 500 ml beaker of water at 70oC.

    27. Allow to cool to room temperature to anneal the oligonucleotides to DNA template.

    28. Spin to collect condensation and place tube on ice.

    29. Add the following in a microfuge tube for second strand synthesis:

    Oligonucleotides/ Single-stranded DNA Hybridization mixture from step 28
    5 x Polymerization Mix
    20 ul
    T4 DNA polymerase
    2.5 U
    T4 DNA ligase
    2 U
    Add distilled H2O to make a final vol. of
    100 ul


    30. Mix thoroughly, then incubate as follow:

    Step
    Incubation time
    Temperature
    1
    5 min
    0oC
    2
    5 min
    room temperature
    3
    2 hours
    37oC

  • The reaction is begun at lower temperatures to polymerize a small number of bases onto the 3' end of the oligonucleotide, thus stabilizing the initial duplex between the template DNA and the mutagenic oligonticleotide primer.

    31. Add 3 ul of 0.5 M EDTA to terminate the reaction.

    32. Analyze 20 ul of the reaction mixture (200 ng of DNA) by Agarose gel electrophoresis in a 0.8% agarose gel.
  • For comparison, adjacent lanes should contain the following standards:
  • Single-stranded circular viral DNA.
  • Double-stranded, closed circular DNA (RFI).
  • Nicked double-stranded circular DNA (RFII).
  • A successful reaction should convert essentially all the single-stranded template DNA into RFI and RFII DNA.



    Transformation and DNA Sequence Analysis

    33. Based upon an estimate from the gel analysis, use 1-100 ng of double-stranded DNA product to transform any desired ung+ strain of E. coli (e.g., JM105, JM107, or JM109).

    34. Resulting clones (as phage plaques or colonies) can be selected or, if no phenotype is known, chosen randomly for isolation of pure genetic stocks. These can be analyzed by sequencing the DNA.




    NOTES

  • Highly efficient mutagenesis depends upon a good template and a successful DNA polymerase reactions. The template should exhibit a strong difference in survival in ung- versus ung+ hosts.

  • T4 DNA polymerase is preferred over the Klenow fragment because it will not strand displace the mutagenic oligonucleotide after synthesis is completed; this permits efficient ligation and expression of the mutation. With some oligonucleotide template combination, this enzyme does not work well, in which case 1 U Klenow fragment can be substituted.

  • The oligonucleotide should be of high quality; i.e., purified away from lower molecular weight contaminants that arise from incomplete DNA synthesis. In some cases, especially for oligonucleotides larger than 40 nucleotides, purification by polyacrylamide gel electrophoresis may be necessary.




    KIT INFORMATION




    REFERENCES

  • Kunkel TA (1985) Rapid and efficient site-specific mutagenesis without phenotypic selection. PNAS. 82: 488-492.

  • Smith M (1985) In vitro mutagenesis. Ann. Rev. Genet. 19: 423-463.


  • Please send your comment on this protocol to "editor@MolecularInfo.com".

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