Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2000-10-26
2003-06-24
Horlick, Kenneth R. (Department: 1656)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091100, C435S091200
Reexamination Certificate
active
06582914
ABSTRACT:
BACKGROUND OF THE INVENTION
A. Field of the Invention
The present invention is related to the generation of libraries of mutant nucleic acid molecules from a precursor nucleic acid template or templates. The mutant library is then useful for selecting or screening purposes to obtain improved nucleic acid, protein or peptide product. More particularly, the present invention provides a novel method for the generation of combinatorial mutations.
B. Description of the State of the Art
Developing libraries of nucleic acids that comprise various combinations of several or many mutant or derivative sequences has recently been recognized as a powerful method of discovering novel products having improved or more desirable characteristics. A number of powerful methods for mutagenesis have been developed that when used iteratively with focused screening to enrich the useful mutants is know by the general term directed evolution.
For example, a variety of in vitro DNA recombination methods have been recently developed for the purpose of recombining more or less homologous nucleic acid sequences to obtain novel nucleic acids. For example, recombination methods have been developed comprising mixing a plurality of homologous, but different, nucleic acids, fragmenting the nucleic acids and recombining them using PCR to form chimeric molecules. For example, U.S. Pat. No. 5,605,793 generally comprises fragmentation of double stranded DNA molecules by DNase I. U.S. Pat. No. 5,965,408 generally relies on the annealing of relatively short random primers to target genes and extending them with DNA polymerase. Each of these disclosures relies on polymerase chain reaction (PCR)-like thermocycling of fragments in the presence of DNA polymerase to recombine the fragments. Other methods have taken advantage of the phenomenon known as template switching, described in, e.g., Meyerhans, A., J.-P. Vartaanian and S. Wain-Hobson (1990)
Nucleic Acids Res.
18, 1687-1891. One shortcoming of these PCR based recombination methods however is that the recombination points tend to be limited to those areas of relatively significant homology. Accordingly, in recombining more diverse nucleic acids, the frequency of recombination is dramatically reduced and limited.
In many contexts, it is desirable to be able to develop libraries of mutant molecules that mix and match mutations which are known to be important or interesting due to functional or structural data. Several strategies toward combinatorial mutagenesis have been developed. In Stemmer et al., Biotechniques, vol. 18, no. 2 pp. 194-196 (1995), the authors use their “gene shuffling” methods in combination with a mixture of specifically designed oligonucleotide primers to incorporate desired mutations into the shuffling scheme. In another example, Osuna et al., Gene, vol. 106, pp. 7-12 (1991) designed an experiment in which synthetic DNA fragments comprising 50% wild type codon and 50% of an equimolar mixture of codons for each of the 20 amino acids at positions 144, 145 and 200 of EcoRI endonuclease. The mutagenic primers were added to a solution of ssDNA template and the primers for the 144 and 145 mutations used separately from the primers for the 200 site. The separate mixtures from each experiment were hybridized to the template ssDNA and extended for one hour with Polik polymerase. The fragments were isolated and ligated to produce a full length fragment with mutations at all three sites. The fragment was amplified with PCR and purified and cloned into a vector. While Osuna predicted that a balanced distribution of each of the 20 mutants would be obtained at each position, the authors were unable to verify whether the predicted distribution was attained. Tu et al., Biotechniques, vol. 20, no. 3, pp 352-353 (1996) describes a method for generation of combination of mutations by using multiple mutagenic oligonucleotides which are incorporated into a mutagenic polynucleotide by a single round of primer extension followed by ligation. Merino et al., Biotechniques, vol. 12, no. 4, pp. 508-509 (1992) describes a method for single or combinatorial directed mutagenesis which utilizes a universal set of primers complementary to the areas that flank the cloning region of the pUC/M13 vectors used in the mutagenesis scheme for the purpose of optimizing yield of mutants. In PCT Publication No. WO 98/42728 (California Institute of Technology) several variations on the theme of recombination of related families of nucleic acids are described. In particular, the authors describe the use of defined primers in combination with recombination based generation of diversity, the defined primers being used to encourage cross-over recombination at sites not otherwise likely to be cross-over points.
While it is apparent that a number of methods exist, it is desirable to develop further and more efficient methods of producing libraries of mutant nucleic acids and particularly for combinatorial mutagenesis. For example, significant advantages accrue from the ability to develop customized mutant nucleic acid libraries which have designed specific biases towards certain mutations. In addition, it is desirable to introduce contiguous and discontiguous mutations in a simple straightforward manner, as opposed to many current processes for discontiguous combinatorial mutation which are particularly cumbersome.
In the present invention, the inventors herein have determined a method for the combinatorial mutagenesis of nucleic acids which allows for optimization of the mutational scheme based on knowledge of the function and/or structure of the protein, while still developing a significant number of mutants with the potential for dramatically improved performance.
SUMMARY OF THE INVENTION
According to the present invention, a method of producing a library of mutant nucleic acid molecules is provided comprising the steps of: (a) obtaining a template nucleic acid; (b) preparing an oligonucleotide primer pair corresponding to the ends of said said template nucleic acid; (c) preparing two mutagenic oligonucleotide primers corresponding to a first and a second desired mutation within said template nucleic acid; (d) mixing the oligonucleotide primers prepared in said steps (b) and (c); (e) combining said mixture in said step (d) with the template nucleic acid under conditions to facilitate the polymerase chain reaction, wherein said mutagenic oligonucleotides are present in a concentration that is less than saturation concentration.
In a preferred embodiment, the template nucleic acid is a single nucleic acid. In another preferred embodiment of the invention, the mixture of oligonucleotide primers further includes non-mutagenic oligonucleotide primers corresponding to either or both of said first and second oligonucleotides. In a further preferred embodiment of the invention, the primers are added in a pre-defined ratio.
In a another embodiment of the invention, the invention comprises a method of producing a library of mutant nucleic acid molecules comprising the steps of obtaining a template nucleic acid; preparing an oligonucleotide primer corresponding to a first desired mutation within said template nucleic acid; preparing an oligonucleotide primer corresponding to a second desired mutation within said template nucleic acid; mixing the oligonucleotide primers prepared in the previous two steps; combining said mixture in said step (d) with the template nucleic acid under conditions to allow hybridization of said oligonucleotides with said template nucleic acid, wherein said oligonucleotides are present in a concentration that is less than saturation level; extending said primers to produce a library of mutant template nucleic acids using the polymerase chain; transforming said mutant template nucleic acid from said library into a competent host cell; expressing protein corresponding to said mutant nucleic acid in said host cell; and screening said expressed proteins for desired characteristics.
REFERENCES:
patent: 5093257 (1992-03-01), Gray
patent: 5270170 (1993-12-01), Schatz et al.
paten
Caldwell Robert M.
Schellenberger Volker
Genencor International Inc.
Genencor International, Inc
Horlick Kenneth R.
Strzelecka Teresa
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