Molecular Techniques and Methods

Site-Specific In vitro Mutagenesis by Chemical Modifications

Copy Right © 2001/ Institute of Molecular Development LLC

INTRODUCTION

In this protocol, a large number of randomly distributed, nucleotide substitution mutations in cloned DNA fragments as large as 3 kb, is generated by chemical treatment.

(1) the DNA of interest is cloned into a vector that permits isolation of single-stranded circular DNA.
(2) the single-stranded DNA containing the region of interest is treated with a variety of chemicals each of which causes damage to specific bases.
(3) the mutated region is copied by extension of an appropriate oligonucleotide primer with reverse transcriptase (i.e. DNA-dependent DNA polymerase, as well as RNA-directed DNA polymerase). When the reverse transcriptase encounters damaged bases, incorrect nucleotides are incorporated at high frequency.
(4) The resulting double-stranded DNA is cleaved with restriction endonucleases and cloned into an appropriate vector. As many as 10-20% of the colonies obtained will contain DNA molecules with single-base substitutions.

Several different chemicals are used to obtain single-base substitutions at all positions of the target DNA. These reagents modify the bases without breaking the phosphodiester backbone.

Mutagen
Modification on DNA Strands
Nitrous acid
deaminates deoxycytosine to deoxyuridine, deoxyadenosine to deoxyhypoxanthine, and deoxyguanosine to deoxyxanthine.
Formic acid
depurinates DNA by breaking the N-glycosyl bonds of purine bases.
Hydrazine
breaks pyrimidine rings.

  • The concentrations of the chemicals are adjusted so that 10-20% of the target fragments are modified. This ensures that most of the mutants obtained contain only a single lesion.




    MATERIALS AND SOLUTIONS

    2 M Sodium nitrite (1 ml)
    Sodium nitrite ------------------------------------- 69 mg
    Add distilled H2O to make a final volume of ----- 1 ml


    Conc. Formic acid (18 M)


    Conc. Hydrazine (12 M)


    Carrier tRNA (10 mg/ml)
    Yeast tRNA ------------------------------------ 10 mg
    TE ---------------------------------------------- 1 ml
    Keep at -20oC.


    Oligonucleotide Primer (15-20 mer) (1 ug/ul)
    The primer is composed of a sequence that will allow it to anneal to the single-stranded vector immediately adjacent to the restriction sites that were used to clone the target fragment. For most applications, the target DNA fragment can be cloned into standard M13 vectors.


    2.5 mM dNTP Mix (1 ml)
    10 mM dATP -------------------------------- 100 ul of 100 mM dATP
    10 mM dCTP -------------------------------- 100 ul of 100 mM dCTP
    10 mM dGTP -------------------------------- 100 ul of 100 mM dGTP
    10 mM dTTP -------------------------------- 100 ul of 100 mM dTTP
    Distilled H2O --------------------------------- 600 ul




    PROCEDURES

    Preparation of Single-stranded Template

    1. Generate DNA fragment of interest by cleavage with two different restriction endonucleases.

    2. Clone the DNA fragment into an M13 phage vector (i.e. M13mpl8, M13mp19) or into a plasmid vector containing the M13 origin of replication.

    3. Prepare single-stranded DNA from a 100 ml culture of infected cells.



    Chemical Modification of DNA

    4. To each of three microcentrifuge tubes, add 40 ug of single-stranded DNA (1 mg/ml).

    5. Perform three separate chemical reactions as follow.

    Formic acid reaction:
    To one tube, add 60 ul conc. Formic acid.
    Incubate 10 min at room temperature.

    Hydrazine reaction:
    To another tube, add 60 ul conc. Hydrazine.
    Incubate 10 min at room temperature.

    Nitrous acid reaction:
    To the last aliquot of single-stranded DNA, add the following:
    2.5 M Sodium acetate (pH 4.3) ------- 10 ul
    2 M Sodium nitrite -------------------- 50 ul

  • Incubate 30-120 min at room temperature.
  • The nitrous acid reaction may turn yellow during the incubation; this color change does not affect subsequent steps.


    6. Add the following to each tube to precipitate DNA and to remove chemicals.
    2.5 M Sodium acetate (pH 5.5) ------------ 100 ul
    Disitlled H2O ------------------------------- 200 ul
    Carrier tRNA (10 mg/ml) ------------------- 30 ug/ 3 ul
    Absolute Ethanol --------------------------- 1 ml

    7. Keep in dry ice (-80oC) for 10 min

    8. Centrifuge at 13,000 rpm for 10 min.

    9. Repeat ethanol precipitation as in step 6 to remove the chemical agents. Repeat two times.

    10. Resuspend the chemically treated DNAs in 70 ul TE.



    Second-strand Synthesis by Primer Extension and Cloning

    11. Synthesis a second-strand as the following.
    Chemically treated DNAs from step 10 ---------------- 70 ul
    Oligonucleotides (15-20 mer) -------------------------- 2-5 ug/ 10 ul
    2.5 mM dNTP Mix ------------------------------------ 10 ul
    10 x Reverse Transcriptase Buffer --------------------- 10 ul

    12. Incubate 5 min at 65oC.

    13. Slowly cool down at room temperature.

    14. Add 1 ul (30-40 U) of AMV (or, M-MuLV) Reverse Transcriptase.

    15. Incubate 1 hour at 37oC.

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

    17. Precipitate the DNA by adding as follow.

    2.5 M Sodium acetate (pH 5.5) ------------------- 100 ul
    Disitlled H2O -------------------------------------- 200 ul
    Carrier tRNA (10 mg/ml) -------------------------- 30 ug/ 3 ul
    Absolute Ethanol ---------------------------------- 1 ml

    18. Keep at -80oC dry ice for 10 min

    19. Centrifuge at 13,000 rpm for 10 min.

    20. Resuspend the DNA pellets in 100 ul of 1 x Restriction Enzyme Buffer and cleave with the appropriate Restriction Enzymes to excise the target fragment from the vector.

    21. Do phenol: chloroform extraction.
    Do chloroform: IAA extraction.

    22. Precipitate the DNA by adding as follow.

    2.5 M Sodium acetate (pH 5.5) -------------------- 100 ul
    Disitlled H2O -------------------------------------- 200 ul
    Carrier tRNA (10 mg/ml) -------------------------- 30 ug/ 3 ul
    Absolute Ethanol ----------------------------------- 1 ml

    23. Keep in dry ice (-80oC) for 10 min

    24. Centrifuge at 13,000 rpm for 10 min.

    25. Resuspend the DNA in 10-15 ul TE.

    26. Add 1 ul of RNase A (1 mg/ml).

    27. Incubate 15 min at 37oC to degrade the carrier tRNA.
  • The tRNA is removed so that it will not interfere with the visualization of the excised target fragment on the preparative gel.
  • The RNase treatment can be omitted if the target fragment is larger than 150 bp.

    28. Load the DNA samples (mixed with Glycerol Dye Mix or Ficoll Dye Mix) onto a nondenaturing polyacrylamide gel (5-7%) and electrophoresis for a length of time appropriate to bring the excised target fragment about midway down the gel.

    29. Stain the gel with ethidium bromide (final concentrafion 0.5 ug/ml) and visualize the DNA fragments under UV light.
  • Generally, DNAs corresponding to single- and double-stranded vector sequences run as a smear near the origin of electrophoresis.

    30. Using a razor blade, excise the regions of the gel containing the target fragments, transfer the gel pieces to microcentrifuge tubes, and purify the target fragments away from the gel material.

    31. Ligate the purified insert DNA into a plasmid or bacteriophage vector that contains ends compatible with those of the target fragments.

    Plasmid vector -------------------------------------- 5-50 ng
    Insert DNA ---------------------------------------- 1-10 ng
    5 x Ligation Buffer --------------------------------- 2 ul
    T4 DNA Ligase ----------------------------------- 1 U
    Add distilled H2O to make a final volume of ------ 10 ul

    32. Incubate at room temperature for 1 hour.



    Transformation and Isolation of Mutant Clones

    33. Transform the ligation mixture into competent bacterial cell.
  • 10 to 20% of the colonies will carry plasmids with one or more mutations in the target sequence.

    34. Screen for mutants on the basis of a biological property.

  • In cases where no biological selection or screen is available, screen mutants by direct DNA sequencing.




    NOTES

  • For many purposes, it is appropriate to use conditions in which 10 to 20% of the target sequences are modified by the chemical. This efficiency of chemical modification ensures that most of the mutants obtained (85 to 95%) contain single-base substitutions rather than multiple substitutions.

  • The times for chemical treatment necessary to achieve 10 to 20% mutagenesis of a target fragment of 150 bp are as follows:

    2 M Sodium nitrate
    60 min
    Conc. Formic acid (18 M)
    10 min
    Conc. Hydrazine (12 M)
    10 min




    KIT INFORMATION




    REFERENCES

  • Myers, RM, Lerman, LS, Maniatis, T (1985) A general method for saturation mutagenesis of cloned DNA fragments. Science 229:242-246.

  • Myers, RM, Fischer, SG, Lerman, LS, Maniatis, T (1985) Nearly all single-base substitutions in DNA fragments joined to a GC clamp can be detected by denaturing gradient gel electrophoresis. Nucl. Acids Res. 13:3131- 3145.


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

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