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METHODS OF PRODUCING MUTANT POLYNUCLEOTIDES

IP.com Disclosure Number: IPCOM000132419D
Publication Date: 2005-Dec-14
Document File: 91 page(s) / 3M

Publishing Venue

The IP.com Prior Art Database

Abstract

The present invention relates to methods of producing mutants of a polynucleotide and to mutant polynucleotides and artificial variants encoded by the mutant polynucleotides

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Attorney Docket No. 10666.504-WO

METHODS OF PRODUCING MUTANT POLYNUCLEOTIDES

Background of the Invention

5

Field of the Invention

     The present invention relates to methods of producing mutants of a polynucleotide and to mutant polynucleotides and artificial variants encoded by the mutant polynucleotides.

10

Description of the Related Art

        The diversity necessary for screening in directed evolution of proteins is often created by error prone mutagenesis to find mutations or positions influencing enzyme activity. Although error prone mutagenesis in principle mutates all base pairs randomly, 15 the outcome of the mutagenesis is rather limited for two main reasons: (A) a given amino acid codon is typically mutated to only 6 or 7 other residues (from one substitution per codon, two or three substitutions are very unlikely), and (B) the mutation rate is biased towards A-T base pairs. Typically 75% of the mutated base pairs are A-T pairs, leaving only 25% of mutated G-C pairs, and the resulting mutation is also biased 20 towards certain bases. Also, additional mutations are normally included to overcome silent mutations, which enhance the chance of hitting destructive mutations due to error in folding, maturation, secretion, etc.

        Transposons are segments of DMA that can move around to different positions in the genome of a single cell. They can cause mutations and/or an increase (or 25 decrease) in the amount of DNA in the genome. These mobile segments of DMA are sometimes called "jumping genes".

        Many transposons move by a "cut and paste" process. The transposon is cut out of its location and inserted into a new location. This process requires a transposase that is encoded within some transposons. Transposase binds to both ends of the 30 transposon, which consists of inverted repeats which are identical sequences reading in opposite directions, and to a sequence of DNA that makes up the target site. Some transposases require a specific sequence as their target site while others can insert the transposon anywhere in the genome. The DNA at the target site is cut in an offset manner, like the "sticky ends" produced by some restriction enzymes. After the 35 transposon is ligated to the host DNA, the gaps are filled in by Watson-Crick base pairing, which creates identical direct repeats at each end of the transposon.

Often transposons lose their gene for transposase, but as long as there is a

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   transposon in the cell that can synthesize the enzyme, their inverted repeats are recognized and they, too, can be moved to a new location. Alternatively, if it desirable that the transposon remains stably integrated in the same place, the transposase may be provided transiently in trans, which is often the case when in vitro transposition is 5 carried out.

        Transposons have proven to be invaluable genetic tools for molecular geneticists. Several uses of transposons include mutagenesis fo...