Interference No. 105,188
Short v. Punnonnen
level of random point mutations introduced at each cycle
would be much lower if existing, already proven, point
mutations or combinations could be permuted by
recombination.
5
(Exh. 2052, p. 550, col. 2, paragraph bridging cols. 1-2);
Since recombination can occur even with “naked” DNA,
“it would seem that it would be easy to employ recombination
10 techniques in order to evolve complex sequences-or even
whole genomes-at the benchtop. But in reality this has not
been the case. To the surprise of most researchers,
recombination methods for mutagenesis, selection, and
replication have been extremely difficult to develop in the
15 laboratory.
(Exh. 2052, pp. 550-551, bridging para.);
So far, a technique called sexual PCR, or DNA
20 shuffling, comes closest to mimicking natural recombination
by allowing the in vitro homologous recombination of DNA.
(Exh. 2052, p. 551, col. 1, first full para.);
25 This method offers practical and theoretical advantages
over existing recursive mutagenesis methods, such as error-
prone PCR or recursive oligonucleotide directed mutagenesis.
By recombining point mutations and wild-type sequences,
[3]
followed by selection for function, sexual PCR will
30 rapidly fine tune the mutation load present in different
parts of a protein. Over several cycles, those areas that
tolerate a high mutation load accumulate more diversity than
those areas that are less permissive to modification.
35 (Exh. 2052, p. 551, cols. 1-2, bridging para.);
Sexual PCR’s advantages for searching sequence space
can be further explained by understanding how it employs
poolwise and pairwise recombination strategies.
40 Recombination by sexual PCR is, in principal, poolwise-more
than two parental sequences can contribute to form the
3
This resembles the procedure used in the Freeman PCT
(Exh. 2040, pp. 22-23, bridging para.).
-20-
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