Recursive nucleic acid recombination (“shuffling”) provides for the rapid evolution of nucleic acids, in vitro and in vivo. This rapid evolution provides for the generation of encoded molecules (e.g., nucleic acids and proteins) with new and/or improved properties. Proteins and nucleic acids of industrial, agricultural and therapeutic importance can be created or improved through DNA shuffling procedures.
A number of publications by the inventors and their co-workers describe DNA shuffling. For example, 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; 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;” and Stemmer et al., U.S. Pat. No. 5,811,238 “METHODS FOR GENERATING POLYNUCLEOTIDES HAVING DESIRED CHARACTERISTICS BY ITERATIVE SELECTION AND RECOMBINATION” describe, e.g., a variety of shuffling techniques.
Many applications of DNA shuffling technology have also been developed by the inventors and their co-workers. In addition to the publications noted above, Minshull et al., U.S. Pat. No. 5,837,458 METHODS AND COMPOSITIONS FOR CELLULAR AND METABOLIC ENGINEERING provides for the evolution of new metabolic pathways and the enhancement of bio-processing through recursive shuffling techniques. Crameri et al. (1996), “Construction And Evolution Of Antibody-Phage Libraries By DNA Shuffling” Nature Medicine 2(1):100-103 describe, e.g., antibody shuffling for antibody phage libraries. Additional details regarding DNA shuffling can also be found in various published applications, such as WO95/22625, WO97/20078, WO96/33207, WO97/33957, WO98/27230, WO97/35966, WO98/31837, WO98/13487, WO98/13485 and WO989/42832.
A number of the publications of the inventors and their co-workers, as well as other investigators in the art also describe techniques which facilitate DNA shuffling, e.g., by providing for reassembly of genes from small fragments of genes, or even oligonucleotides encoding gene fragments. In addition to the publications noted above, Stemmer et al. (1998) U.S. Pat. No. 5,834,252 “END COMPLEMENTARY POLYMERASE REACTION” describe processes for amplifying and detecting a target sequence (e.g., in a mixture of nucleic acids), as well as for assembling large polynucleotides from fragments.
Review of the foregoing publications reveals that DNA shuffling is an important new technique with many practical applications. Thus, new techniques which facilitate DNA shuffling are highly desirable. In particular, techniques which reduce the number of physical manipulations needed for shuffling procedures would be particularly useful. The present invention provides significant new DNA shuffling protocols, as well as other features which will be apparent upon complete review of this disclosure.
USPTO Backgrounds
USPTO Backgrounds