Compositions and methods for random nucleic acid mutagenesis

Details Compositions and methods for random nucleic acid mutagenesis Compositions and methods for random nucleic acid mutagenesis

2002-05-23

2004-10-12

Horlick, Kenneth R. (Department: 1637)

Chemistry: molecular biology and microbiology

Micro-organism, tissue cell culture or enzyme using process...

Preparing compound containing saccharide radical

Reexamination Certificate

active

06803216

ABSTRACT:

TECHNICAL FIELD
The invention relates to random nucleic acid mutagenesis using exo− DNA polymerases.
BACKGROUND
PCR-based random mutagenesis is widely used for elucidating structure-function relationships of proteins, and for improving protein function (e.g., directed protein evolution) (Cadwell, R. C. and Joyce, G. F. 1992. Randomization of genes by PCR mutagenesis. PCR Methods Appl. 2:28-33; Leung, D. W., Chen, E., and Goeddel, D. V. 1989. A method for random mutagenesis of a defined DNA segment using a modified polymerase chain reaction. Technique 1:11-15). The procedure involves amplifying a gene or portion of a gene under mutagenic conditions, cloning the PCR fragments, and then screening the resulting library for novel mutations that affect protein activity (Melnikov, A. and Youngman, P. J. 1999. Random mutagenesis by recombinational capture of PCR products in
Bacillus subtilis
and
Acinetobacter calcoaceticus
. Nucleic Acids Res. 27:1056-1062; Wan, L., Twitchett, M. B., Eltis, L. D., Mauk. A. G., and Smith, M. 1998. In vitro evolution of horse heart myoglobin to increase peroxidase activity. Proc. Natl. Acad. Sci. U.S.A. 95:12825-12831; You, L. and Arnold, F. H. 1996. Directed evolution of subtilisin E in
Bacillus subtilis
to enhance total activity in aqueous dimethylformamide. Protein Eng. 9:77-83). Mutations are deliberately introduced during PCR through the use of error-prone DNA polymerases and reaction conditions. To analyze structure-function relationships, mutation rates of 1 mutation per gene are desired to assess the contribution of individual amino acids to protein function (Vartanian, J. P., Henry, M., and Wain-Hobson, S. 1996. Hypermutagenic PCR involving all four transitions and a sizeable proportion of transversions. Nucleic Acids Res. 24:2627-2631). For directed evolution, mutagenesis rates of 2 to 7 mutations per gene are considered the most effective for creating mutant libraries and isolating proteins with enhanced activities (Cherry, J. R. Lamsa, M. H., Schneider, P., Vind, J., Svendsen, A., Jones, A., and Pedersen, A.H. 1999. Directed evolution of a fungal peroxidase. Nat. Biotechnol. 17:379-384; Shafikhani, S., Siegel, R. A., Ferrari, E., and Schellenberger, V. 1997. Generation of large libraries of random mutants in
Bacillus subtilis
by PCR-based plasmid multimerization. BioTechniques 23:304-310; Wan, L., Twitchett, M. B., Eltis, L. D., Mauk. A. G., and Smith, M. 1998. In vitro evolution of horse heart myoglobin to increase peroxidase activity. Proc. Natl. Acad. Sci. U.S.A. 95:12825-12831; You, L. and Arnold, F. H. 1996. Directed evolution of subtilisin E in
Bacillus subtilis
to enhance total activity in aqueous dimethylformamide. Protein Eng. 9:77-83). Mutation rates greater than 7 mutations per gene typically result in loss of protein activity, although proteins with improved activities have been successfully isolated from highly mutagenized libraries exhibiting up to 20 mutations per gene (Daugherty, P. S., Chen, G., Iverson, B. L., and Georgiou, G. 2000. Quantitative analysis of the effect of the mutation frequency on the affinity maturation of single chain Fv antibodies. Proc. Natl. Acad. Sci. U.S.A. 97:2029-2034).
Conventional methods employ Taq DNA polymerase, as it lacks proofreading activity and is inherently error prone. To achieve useful mutation frequencies, the error rate of Taq (1 mutation per ~125,000 bases (Cline, J., Braman, J. C. and Hogrefe, H. H. 1996. PCR fidelity of Pfu DNA polymerase and other thermostable DNA polymerases. Nucleic Acids Res. 24:3546-3551) is further increased by employing PCR reaction buffers that contain Mn
2+
and/or unbalanced nucleotide concentrations (Cadwell, R. C. and Joyce, G. F. 1992. Randomization of genes by PCR mutagenesis. PCR Methods Appl. 2:28-33; Leung, D. W., Chen, E., and Goeddel, D. V. 1989. A method for random mutagenesis of a defined DNA segment using a modified polymerase chain reaction. Technique 1:11-15). In the presence of 7 mM MgCl
2
, 0.5 mM MnCl
2
, 1 mM dCTP and TTP, and 0.2 mM dGTP and dATP, Taq incorporates 4.9 to 6.6 mutations per kb per PCR (Cadwell, R. C. and Joyce, G. F. 1992. Randomization of genes by PCR mutagenesis. PCR Methods Appl. 2:28-33; Shafikhani, S., Siegel, R. A., Ferrari, E., and Schellenberger, V. 1997. Generation of large libraries of random mutants in
Bacillus subtilis
by PCR-based plasmid multimerization. BioTechniques 23:304-310). Under these conditions, mutational bias is regarded as minimal or skewed to favor mutations at AT base pairs. Lower mutation frequencies can be obtained by reducing MnCl
2
concentration (1-2 mutations per kb), while higher mutation frequencies (>6 mutations per kb) are achieved by performing consecutive PCRs or by selectively increasing dGTP concentration (Melnikov, A. and Youngman, P. J. 1999. Random mutagenesis by recombinational capture of PCR products in
Bacillus subtilis
and
Acinetobacter calcoaceticus
. Nucleic Acids Res. 27:1056-1062; Nishiya, Y. and Imanaka, T. 1994. Alteration of substrate specificity and optimum pH of sarcosine oxidase by random and site-directed mutagenesis. Appl. Env. Microbiol. 60:4213-4215; You, L. and Arnold, F. H. 1996. Directed evolution of subtilisin E in
Bacillus subtilis
to enhance total activity in aqueous dimethylformamide. Protein Eng. 9:77-83).
Although widely used, Taq-based methods exhibit significant drawbacks that limit the utility of PCR random mutagenesis methods. First, amplification under mutagenic conditions (Mn
2+
, unbalanced nucleotide pools) reduces the activity of Taq and limits random mutagenesis to DNA sequences less than 1-kb in length (Leung, D. W., Chen, E., and Goeddel, D. V. 1989. A method for random mutagenesis of a defined DNA segment using a modified polymerase chain reaction. Technique 1:11-15; Stemmer, W. P. 1994. DNA shuffling by random fragmentation and reassembly: in vitro recombination for molecular evolution. Proc. Natl. Acad. Sci. USA 91:10747-10751). Second, PCR products are amplified in lower yield using mutagenic reaction conditions (Vartanian, J. P., Henry, M., and Wain-Hobson, S. 1996. Hypermutagenic PCR involving all four transitions and a sizeable proportion of transversions. Nucleic Acids Res. 24:2627-2631), which can reduce cloning efficiency and library size. Third, preparing and using multiple buffers (varying MnCl
2
and dNTP concentrations) to construct a series of libraries with different mutation frequencies is time-consuming and can produce variable results. Finally, altering nucleotide ratios to achieve high mutation frequencies (>6 mutations per kb) can lead to strong bias in the types of mutations produced. For example, selectively increasing dGTP concentration favors AT→GC transitions, which accounted for 70% of all mutations in one study (You, L. and Arnold, F. H. 1996. Directed evolution of subtilisin E in
Bacillus subtilis
to enhance total activity in aqueous dimethylformamide. Protein Eng. 9:77-83).
There is a need in the art for random mutagenesis of nucleic acid longer than 1 kb. There is also a need to improve the yield of the final mutated product to facilitate subsequent cloning of the product. There is further a need for a novel error prone DNA polymerase which minimizes mutation bias or produces a different mutational bias than a given polymerase produces. Finally, there is a need for a simplified PCR mutagenesis conditions to achieve various mutation frequencies.
SUMMARY OF THE INVENTION
The invention is related to novel compositions and methods for nucleic acid mutagenesis.
The invention provides a composition for PCR mutagenesis comprising an archaeal exo− DNA polymerase which substantially lacks 3′ to 5′ exonuclease activity, and PCR enhancing factor.
In a preferred embodiment, the archaeal exo− DNA polymerase is selected from the group consisting of: exo−Tli DNA polymerase, exo− Pfu DNA polymerase, exo− KOD DNA polymerase, exo− JDF-3 DNA polymerase, and exo−PGB-D DNA polymerase.
The invention also provides a composition compr

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