Methods for forming recombined nucleic acids

Details Methods for forming recombined nucleic acids Methods for forming recombined nucleic acids

2000-05-08

2003-03-18

Jones, W. Gary (Department: 1655)

Chemistry: molecular biology and microbiology

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

Preparing compound containing saccharide radical

C435S091200, C435S006120

Reexamination Certificate

active

06534292

ABSTRACT:

FIELD OF THE INVENTION
This invention is related to the generation of recombined nucleic acid molecules, and more particularly to providing a novel method including the generation of random fragments and the assembly of a recombined nucleic acid molecule using template fragments which have non-extendable ends.
BACKGROUND OF THE INVENTION
Recombining nucleic acids has useful applications such as finding sequences which produce products having improved or desired characteristics. In particular, it is useful to develop a method for the production of mutant proteins which method allowed for the development of large libraries of mutant nucleic acid sequences which were easily searched.
A variety of in vitro DNA recombination methods exist. Examples include those described in U.S. Pat. No. 5,605,793 and in U.S. Pat. No. 5,965,408. Generally, recombination methods depend on a step of making fragments, and a step of recombining the fragments. For example, U.S. Pat. No. 5,605,793 generally relies on 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.
Generally, existing methods generate DNA fragments with functional 3′ ends, which can be readily extended in the presence of complementary sequences. Thus, in one of the examples described above, a consequence of this feature is that fragments in the assembly or recombination step are independent from each other. The fragments can anneal to their complementary counterparts and get extended by DNA polymerase irrespectively of their position in the primary sequences of the gene—extension of different fragments occurs in parallel.
Although, a number of methods exist, it would generally be desirable to provide novel methods of recombining nucleic acids. Moreover, it would be desirable to provide a method of forming recombined nucleic acid molecules which took into account the position of a fragment relative to the primary or initial sequence from which it was fragmented. In particular, it would be desirable to provide methods of forming recombined nucleic acid molecules wherein a sequence was fragmented, and wherein the fragments were used as templates only, rather than as templates and primers for extension.
SUMMARY OF THE INVENTION
The present invention provides methods of forming recombined nucleic acid molecules. Generally, the methods involve providing or generating fragments, and using the fragments to form a recombined nucleic acid molecule. Preferably, the fragments come from at least two different initial sequences, and the recombined nucleic acid molecule has a sequence which differs from either of the initial sequences. As such, a large number of recombined nucleic acids can be formed which can be screened to identify a sequence which provides a molecule having a desired characteristic.
In one aspect of the invention, template fragments of at least one sequence, sometimes called the initial sequence, are provided. Preferably, the template fragments have non-extendable 3′ ends. In a preferred embodiment, the template fragments are generated as described below. Moreover, the method comprises providing at least one primer, and reacting said primer and said template fragments under conditions to extend said primer to form said recombined nucleic acid molecule. In preferred embodiments herein, the method is repeated until the recombined nucleic acid has been assembled to have the length of the sequence from which the fragments were formed.
In one embodiment, at least two sequences which differ from each other are used. Preferably, the two sequences which differ from each other are homologs of one another. In one embodiment, the sequences are variants of the same naturally-occurring sequence. In preferred embodiments, the recombined nucleic acid has at least one fragment from each sequence such that the recombined nucleic acid molecule has a sequence which differs from either of said at least one sequence.
Additionally, vectors, host cells, recombinant nucleic acids, proteins, pools of nucleic acid molecules and screening assays are provided herein.


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