Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound containing saccharide radical
Reexamination Certificate
1999-09-28
2002-08-20
Brusca, John S. (Department: 1631)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound containing saccharide radical
C435S006120, C536S025300
Reexamination Certificate
active
06436675
ABSTRACT:
COPYRIGHT NOTIFICATION
Pursuant to 37 C.F.R. 1.71(e), Applicants note that a portion of this disclosure contains material which is subject to copyright protection. The copyright owner has no objection to the facsimile reproduction by anyone of the patent document or patent disclosure, as it appears in the Patent and Trademark Office patent file or records, but otherwise reserves all copyright rights whatsoever.
FIELD OF THE INVENTION
The present invention relates to methods of providing shuffling libraries that include codon-varied oligonucleotide sequences. Codon-varied oligonucleotides can be synthesized using trinucleotide or mononucleotide phosphoramidite sequences, and can be derived from homologous or non-homologous nucleic acid sequences, or combinations of such sequences. In turn, codon-varied oligonucleotide sequences can be utilized for recombination in various methods of artificial evolution.
BACKGROUND OF THE INVENTION
The use of trinucleotide phosphoramidites in solid-phase DNA synthesis was previously thought to be unfeasible, as only marginal yields could be achieved. Sondek, J. and Shortle, D. (1992)
J. Immunol.,
149, 3903-3913. These poor results were attributed to the steric bulk of the trinucleotide molecules. Id. However, it has since been shown that trinucleotide phosphoramidites representing codons for all 20 amino acids can be successfully used to introduce entire codons into oligonucleotides in automated, solid-phase DNA synthesis and thus can function as excellent reagents for the synthesis of mixed oligonucleotides for random mutagenesis. Virnekäs, B., et al., (1994)
Nucleic Acids Res.,
22, 5600-5607. Other references involving the synthesis of trinucleotide phoshoramidites, their subsequent use in oligonucleotide synthesis, and related issues are described in, e.g., Kayushin, A. L. et al., (1996)
Nucleic Acids Res.,
24, 3748-3755, Huse, U.S. Pat. No. 5,264,563 “PROCESS FOR SYNTHESIZING OLIGONUCLEOTIDES WITH RANDOM CODONS,” Lyttle et al., U.S. Pat. No. 5,717,085 “PROCESS FOR PREPARING CODON AMIDITES” Shortle et al., U.S. Pat. No. 5,869,644 “SYNTHESIS OF DIVERSE AND USEFUL COLLECTIONS OF OLIGONUCLEOTIDES,” Greyson, U.S. Pat. No. 5,789,577 “METHOD FOR THE CONTROLLED SYNTHESIS OF POLYNUCLEOTIDE MIXTURES WHICH ENCODE DESIRED MIXTURES OF PEPTIDES,” and Huse, WO 92/06176 “SURFACE EXPRESSION LIBRARIES OF RANDOMIZED PEPTIDES.”
The inventors and their co-workers have developed various rapid artificial evolution techniques for creating improved industrial, agricultural, and therapeutic genes and encoded proteins including via oligonucleotide-mediated recombination. These methodologies and related aspects are described in a variety of sources, e.g., Stemmer et al., (1994) “Rapid Evolution of a Protein”
Nature
370:389-391, Stemmer (1994) “DNA Shuffling by Random Fragmentation and Reassembly: in vitro Recombination for Molecular Evolution,”
Proc. Natl. Acad. USA
91:10747-10751, Crameri et al., (1996), “Construction And Evolution Of Antibody-Phage Libraries By DNA Shuffling”
Nature Medicine
2(1):100-103, Stemmer U.S. Pat. No. 5,603,793 “METHODS FOR IN VITRO RECOMBINATION,” Stemmer et al., U.S. Pat. No. 5,830,721 “DNA MUTAGENESIS BY RANDOM FRAGMENTATION AND REASSEMBLY,” Stemmer et al., U.S. Pat. No. 5,811,238 “METHODS FOR GENERATING POLYNUCLEOTIDES HAVING DESIRED CHARACTERISTICS BY ITERATIVE SELECTION AND RECOMBINATION,” Stemmer et al., (1998) U.S. Pat. No. 5,834,252 “End Complementary Polymerase Reaction,” Minshull et al., U.S. Pat. No. 5,837,458 “Methods and Compositions for Cellular and Metabolic Engineering,” and U.S. Provisional Patent Applications, Ser. Nos. 60/118,813 and 60/141,049 “Oligonucleotide Mediated Nucleic Acid Recombination,” filed Feb. 5, 1999 and Jun. 24, 1999, respectively, each of which is incorporated by reference in its entirety for all purposes. Additional details regarding DNA shuffling can also be found in WO95/22625, WO97/20078, WO96/33207, WO97/33957, WO98/27230, WO97/35966, WO98/31837, WO98/13487, WO98/13485 and WO989/42832, each of which is also incorporated by reference in its entirety for all purposes.
Recently, the use of oligonucleotides for “family” shuffling was described by the inventors and their co-workers in U.S. Provisional Patent Applications, Ser. Nos. 60/118,813 and 60/141,049, supra. Additional oligonucleotide shuffling methods would be desirable. The present invention provides new codon-based oligonucleotide mediated shuffling methods and related compositions, as well as a variety of additional features which will become apparent upon review of the following description.
SUMMARY OF THE INVENTION
The present invention provides recombination methodologies in which codon-varied oligonucleotides are shuffled to provide recombined nucleic acid populations. Codon-varied oligonucleotides are synthesized, e.g., utilizing codon- or trinucleotide-based phosphoramidite coupling chemistry. This approach affords extensive flexibility to shuffling processes, as codon-varied oligonucleotides can be based upon homologous or non-homologous nucleotide sequences, or even combinations of such sequences.
In a first aspect, the present invention is directed to a method of recombining codon-varied oligonucleotides. It includes synthesizing, hybridizing, and elongating a set of overlapping codon-varied oligonucleotides to provide a population of recombined nucleic acids. In one embodiment, this method can include selecting at least first and second nucleic acids to be recombined, where the set of codon-varied oligonucleotides includes a plurality of codon-varied nucleic acids which correspond to the first and second nucleic acids. The first and second nucleic acids can be homologous or non-homologous.
In one embodiment, the sythesizing step of this method is a trinucleotide synthesis format that includes providing a substrate sequence having a 5′ terminus and at least one base, both of which have protecting groups thereon. The 5′ protecting group of the substrate sequence is then removed to provide a 5′ deprotected substrate sequence, which is then coupled with a selected trinucleotide phosphoramidite sequence. The trinucleotide has a 3′ terminus, a 5′ terminus, and three bases, each of which has protecting groups thereon. The coupling step yields an extended oligonucleotide sequence. Thereafter, the removing and coupling steps are optionally repeated. When these steps are repeated, the extended oligonucleotide sequence yielded by each repeated coupling step becomes the substrate sequence of the next repeated removing step until a desired codon-varied oligonucleotide is obtained. This trinucleotide synthesis format can optionally include coupling together one or more of: mononucleotides, trinucleotide phosphoramidite sequences, and oligonucleotides.
The synthesizing step is optionally a “split-pool” synthesis format that includes providing substrate sequences, each having a 5′ terminus and at least one base, both of which have protecting groups thereon. The 5′ protecting groups of the substrate sequences are removed to provide 5′ deprotected substrate sequences, which are then coupled with selected trinucleotide phosphoramidite sequences. Each trinucleotide has a 3′ terminus, a 5′ terminus, and three bases, all of which have protecting groups thereon. The coupling step yields extended oligonucleotide sequences. Thereafter, the removing and coupling steps are optionally repeated. When these steps are repeated, the extended oligonucleotide sequences yielded by each repeated coupling step become the substrate sequences of the next repeated removing step until extended intermediate oligonucleotide sequences are produced.
Additional steps of the split-pool format optionally include splitting the extended intermediate oligonucleotide sequences into two or more separate pools. After this is done, the 5′ protecting groups of the extended intermediate oligonucleotide sequences are removed to provide 5′ deprotected extended intermediate oligonucleotide sequences in the
Gustafsson Claes
Minshull Jeremy
Ness Jon
Stemmer Willem P. C.
Welch Mark
Brusca John S.
Kim Young
Kruse Norman J.
Maxygen Inc.
Quine Intellectual Property Law Group P.C.
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