AU755415B2 - Recombination of polynucleotide sequences using random or defined primers - Google Patents

Description

28. DEC. 20Q0 18:14 SPRUSON FERGUSON 61 A 2 92615486 SPRUSON FERGUSON NO. 8897 P. S&F Ref. 442546DI

AUSTRALIA

PATENTS ACT 1990 COMELETIE SPECIFICATION FOR A STANDARD PATENT

ORIGINAL

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Name and Address of Appl icant: Actual Inventor(s): Address for Service: Invention Title: California Institute of Technology 1201 E. California Boulevard Pasadena California 91125 United States of America Frances H Arnold Zhixin Shao Joseph A Affholter Huimin Zhao Lorraine J Giver Spruson Ferguson St Martins Towcr 31 Market Street Sydney NSW 2000 Recombination of Polynucleotide Sequences Using Random or Defined Primers The following statement is a full description of this invention, including the best method of performing it known to me/us:- S 845c RECEIVED TIME RECIVD TME 28. DEC. 17:07 PRINT TIME 29DE. 61 29. DEC. 6: u GC. 2000 18:14 SPRUSON FERGUSON 61A 2 92615486 NO. 8897 P. 6 SPRUSON FERGUSON RECOMBINATION OF POLYNUCLEOTIDE SEQUENCES USING RANDOM OR DEFINED PRIMERS Background of the Invention 1. Field of the Invention The present invention relates generally to in vitro methods for mutagenesis and recombination s of polynucleotide sequences. More particularly, the present invention involves a simple and efficient method for in vitro mutagenesis and recombination of polynucleotide sequences based on polymerase-catalysed extension of primer oligonucleotides, followed by gene assembly and optional gene amplification.

2. Description of Related Art The publications and other reference materials referred to herein to describe the background of the invention and to provide additional detail regarding its practice are hereby incorporated by reference. For convenience, the reference materials are numerically referenced and grouped in the appended bibliography.

Proteins are engineered with the goal of improving their performance for practical applications.

Desirable properties depend on the application of interest and may include tighter binding to a receptor, high catalytic activity, high stability, the ability to accept a wider (or narrower) range of substrates, or the ability to function in nonnatural environments such as organic solvents. A variety of approaches, including "rational" design and random mutagenesis methods, have been successfully used to optimise protein functions The choice of approach for a given optimisation problem will 20 depend upon the degree of understanding of the relationships between sequence, structure and function, The rational redesign of an enzyme catalytic site, for example, often requires extensive knowledge of the enzyme structure, the structures of its complexes with various ligands and analogs of reaction intermediates and details of the catalytic mechanism. Such information is available only for a very few well-studied systems; little is known about the vast majority of potentially interesting 25 enzymes. Identifying the amino acids responsible for e RECEIVED TIME 28. DEC. 17 0 PRINT TIME 29. DEC. 6:15 Vt ZUUU 16: 14 SHIUSUN FERGUSON 6I1&2 92615485 NO. 8897 P. 7 ISPRUSON FERGUSON WO 93/4=82 PCT/UiS98/05956 existing protein functions and those which might give rise to new functions remains an often-overwhelming challenge. This, together with the growing appreciation that many protein functions are not con ned to a small number of amino acids, but are affected by residues far from active sites, has S prompted a growing number of groups to turn to random mutagenesis, or 'directed' evolution, to engineer novel proteins Various optimization procedures such as genetic algorithms and evolutionary strategies have been inspired by natural evolution. These procedures employ mutation, which makes small random changes in members of the popu~lation, as well as crossover, which combines properties of different individuals, to achieve a specific optimization goal. There also edst strong interplays between mutation and crossover, as shown by computer simulations of different optimization problems 16-9), Developing efficient and practical experimental techniques to mimic these key processes is a scientific challenge. The application of such techniques should allow one, for example, to explore and optimize the functions of biological molecules such as proteins and nucleic acids, in tzvo or even completely free from the constraints of a.

living system (10.11).

Directed evolution, inspired by natural evolution, involves the generation and selection or screening of a pool of mutated molecules which has sufficient diversity for a molecule encoding a protein with altered or enhanced function to be present therein- It generally begins with creation of a library of mutated genes. Gene products which show improvement with respect to the desired property or set of properties are identified by selection or screening. The gene(s) encoding those products can be subjected to further cycles of the process in order to accumulate beneficial mutations. This evolution can involve few or many generations, depending on how far one wishes to progress and the effects of mutations typically observed in each :generation, Such approaches have been used to create novel functional nucleic acids peptides and other small molecules antibodies as well as enzymes and other proteins (13,14,16). Directed evolution requires little specific knowledge about the product itself, only a means to evaluate the function to be optimized. These procedures are even fairly tolerant to inaccuracies and noise in the function evaluation The diversity of genes for directed evolution can be created by introducing new point mutations using a variety of methods, incluading mutagenic PCR (15) or cornbinatorial cassette mutagenesia The ability to recombine genes, however, can add an important dimension to the evolutionary process, at; evidenced by its key role in 'natural evolution.

Mimosa 12;57:20 RECEIVED TIME 28. DEC. 17:07 PITTM 9 E. 61 PRINT TIME 29.DEC, 6:15 Homologous recombination is an important natural process in which organisms exchange genetic information between related genes, increasing the accessible genetic diversity within a species. While introducing potentially powerful adaptive and diversification competencies into their hosts, such pathways also operate at very low efficiencies, often eliciting insignificant changes in pathway structure or function, even after tens of generations. Thus, while such mechanisms prove beneficial to host organisms/species over geological time spans, in vivo recombination methods represent cumbersome, if not unusable, combinatorial processes for tailoring the performance of enzymes or other proteins not strongly linked to the organism's intermediary metabolism and survival.

Several groups have recognized the utility of gene recombination in directed evolution. Methods for in vivo recombination of genes are disclosed, for example, in published PCT application WO 97/07205 and US Pat. No. 5,093,257. As discussed above, these in vivo methods are cumbersome and poorly optimized for rapid evolution of function. Stemmer has disclosed a method for in vitro recombination of related DNA 15 sequences in which the parental sequences are cut into fragments, generally using an enzyme such as DNase 1, and are reassembled (17,18,19). The non-random DNA fragmentation associated with DNase 1 and other endonucleases, however, introduces bias into the recombination and limits the recombination diversity. Furthermore, this method is limited to recombination of double-stranded polynucleotides and cannot be used on single-stranded templates. Further, this method does not work well with certain combinations of genes and primers. It is not efficient for recombination of short sequences (less than 200 nucleotides for example. Finally, it is quite laborious, requiring several steps. Alternative, convenient methods for creating novel genes by point S•mutagenesis and recombination in vitro are needed.

Summary of the Invention The present invention provides a new and significantly improved approach to creating novel polynucleotide sequences by point mutation and recombination in vitro of a set of parent sequences (the templates). The novel polynucleotide sequences can be useful themselves (for example, for 35 DNA-based computing), or they can be expressed in recombinant organisms for directed evolution of the gene products.

According to a first embodiment of the invention, there is provided a method of evolving a polynucleotide toward acquisition of a desired property, comprising: [I:\DayLib\LIBFF]63629spec.doc:gcc 4 a) contacting at least one template polynucleotide with a set of defined-sequence primers, the set of defined-sequence primers comprising a plurality of both forward and reverse primers; b) conducting a multi-cycle polynucleotide extension reaction on the at least one template polynucleotide and the set of defined-sequence primers, wherein: in at least one cycle, the primers anneal to the at least one template polynucleotide and prime replication of the at least one template polynucleotide thereby generating a pool comprising overlapping fragments which are shorter in length than the at least one template polynucleotide and which overlap to span the at least one template 0o polynucleotide; and (ii) in at least one subsequent cycle, the overlapping fragments generated in a previous cycle are denatured to single-stranded fragments, which anneal in new combinations forming annealed fragments, whereby one strand of an annealed fragment primes replication of the other to form a further pool of overlapping fragments; 15 whereby the multi-cyclic polynucleotide extension reaction is continued for sufficient cycles until the further pool of overlapping fragments includes variant forms of the at least one template polynucleotide; and c) screening or selecting the variant forms of the at least one template polynucleotide, or expression products thereof, for an altered or enhanced property 20 relative to the at least one template polynucleotide or an expression product thereof.

There is disclosed herein priming the template gene(s) with random-sequence oligonucleotides to generate a pool of short DNA fragments. Under appropriate reaction conditions, these short DNA fragments can prime one another based on complementarity and thus can be reassembled to form full-length genes by repeated thermocycling in the presence of thermostable DNA polymerase. These reassembled genes, which contain point mutations as well as novel combinations of sequences from different parental genes, can be further amplified by conventional PCR and cloned into a proper vector for expression of the encoded proteins. Screening or selection of the gene products leads to new variants with improved or even novel functions. These variants can be used as they are, or they can serve as new starting points for further cycles of mutagenesis and recombination.

There is further disclosed priming the template gene(s) with a set of primer oligonucleotides of defined sequence or defined sequence exhibiting limited randomness

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r to generate a pool of short DNA fragments, which are then reassembled as described above into full length genes.

[I:\DayLib\LIBFF]63629spec.doc:gcc 4a There is further disclosed herein a novel process we term the 'staggered extension' process, or StEP. Instead of reassembling the pool of fragments created by the extended primers, full-length genes are assembled directly in the presence of the template(s). The StEP consists of repeated cycles of denaturation followed by extremely abbreviated annealing/extension steps. In each cycle the extended fragments can anneal to different templates based on complementarity and extend a little further to create "recombinant cassettes." Due to this template switching, most of the polynucleotides contain sequences from different parental genes are novel recombinants). This process is repeated until full-length genes form. It can be followed by an optional gene amplification step.

The different embodiments of the invention provide features and advantages for different applications. In the most preferred embodiment, one or more defined primers or defined primers exhibiting limited randomness which correspond to or flank the 5' and V 3' ends of the template polynucleotides are used with StEP to generate gene fragments which grow into the novel full-length sequences. This simple method requires no 0 0 s15 knowledge of the template sequence(s).

00 Still further disclosed multiple defined primers or defined primers exhibiting limited randomness are used to generate short gene fragments which are reassembled into fulllength genes. Using multiple defined primers allows the user to bias in vitro recombination frequency. If sequence information is available, primers can be designed to *0 20 generate overlapping recombination cassettes which increase the frequency of recombination at particular locations. Among other features, this method 0* [I:\DayLib\LIBFF]63629spec.doc:gcc 28. DEC. 2000 18:15 SPRUSON FERGUSON 61 A 2 92615486 NO. 8897 P. SPRUSON FERGUSON WO9/42832 PUrS98o59g6 introduces the fleiubility to take advantage of aadable structural and functional Information as well as information accumulated through previous generations of mutagenesis and selection (or screening).

In addition to recombination, the different embodimen~ts of the primer.

based recombination process will generate point mutations. it is desirable to know and be atble to control this point mutation rate, which can be done by manipulating the conditions of DNA Synthesis and gene reassembly. Using the defined-pimer approach, specific point mutations can also be directed to specific positions in the sequence through the use of ntutagenic primers.

The various primer-based recombination methods in accordance with this invention have been shown to enhance the activity of Actirwplanes utaherisjs ECB deacylase over a broad range of pH values and in the presence of organic solvent and to improve the therrnostability of Badilus subtilis subtilisjn E. DNA scquencing confirms the role of point mutation arnd recombination in the generation of novel sequences. These protocols have been found to be both simple arnd reliable- *The above discussed and meany other features and attendant advantages will become better understood by reference to the following detailed description when taken in conjunction with the accompanying drawings.

D~RIEP DESCITN OPTE R WNGS FIG. I depict recombination in accordance with the present invention .*using random-seunc primers and gene reassembly. The steps shown are: a) Synthesis of single-stranded DNA fragments using mesophilic or thermophilic polymerase with random-sequence oligonucleotides as primers (primers not shown)- b) Removal of templates- c) Reassembly with therrnophilic DNA polyrnerase; d) Amnpliflc-ation with thermostable polynterase(e), e) Cloning and Screening (optional); and 0 Repeat the process with selected gene(s) (optional).

FIG. 2 depicts recombination in accordance with the present invention using defined primers. The rnethod is illustrated for the recombination of two genes, where x -mutation. The steps digamdae )The genes are primed with defined primers in PCR reactions that can be done separately (2 primers per reaction) or combined (multiple primers per reaction); c) Initial products are formed until defined primers are exhausted. Template is removed foutional); d) Initial fragments prime and extend themelves in further cycles of PCR with nio addition of exteral primers. Assembly Mimosa 12:57:20 RECEIVED TIME 28. DEC. 17:07 PITTM 9 E. 61 PRINT TIME 29, DEC, 6 2.DC 1U00 18: lb SPRUSON FERGUSON 61A 2 92615486 NO, 8897 P.

SPRUSON FERGUSON WO W42832 P/UW 6 continues until full-length genes are formed; e) (ontiomnafl pull-length genes are amplified in a PCR reaction~ with ecternal primers,; q) kilt0onal)1 Repeat the Process with selected gene(s).

FIG- 3 depicts recomnbination in accordance with the present invwention using two defined flaniting primers and StEP. Only one primer and two single strands from two templates are shown here to illustrate the recombination process. The outlined steps are. a) After denaturation, template genes are primed with one defined primer; b) Short ftrgents are produced by primer extension (or a short time; c) In the next cycle of SEEP, fragments are randomly primed to the templates and exended further: d) Denaturation and annealing/extension is repeated until full-length genes are made (visible on an agarose gel),- e) Full-length genes are purified, or amplified in a PCR reaction with external primers loptional); Q) (optional) Repeat the process with selected is gene(s).

FIG. 4 is a diagrammatic representaion of the results of the recominration of two genes using two flanking primers and staggered extension in accordance with the present invention. DNA sequences of five chosen from the recombined library arc indicated, where x is a mnu~tation present in the parental genes, and the triangle represents a new point mutation.

FIG. 5 is a diagrammratic representation of the sequences of the pNB esterase genes described in Examnple 3- Template genes 2-13 and S-1312 were recombined using the defined primer approach. The positions of the primers **..arec indicated by arrows, and the positions where the parental sequences differ froin one another are indicated by x's. New point mutations are indicated by triangles. Mutations identified in these recombined gnsare listed (only zi0 positions which differ in the parental sequences are listed). Both 6E6 and 6HI are recombination products of the template genes.

S FIG, 6 shows the positions and sequences of the four defined internal 5: primers used to generate recombined genes from template genes R1 and R2 by interspersed primer-based recombination. Primer PSOF contains a mutation at base position 598) which aimultaneously eliminates a.

HindilI restriction site and adds a new unique NheI site. Gene R2 also Mimosa 12:57:20 RECEIVED TIME 28. DEC. 17:07 PITTM 9 E. 61 PRINT TIME 29. DEC. 6 28. DEC. 2069 18:16 SPRUSON FERGUSON 61' 2 92615486 NO. 8897 P. 12 SPRUSON FERGUSON WO 95/42932 PMU9S605 contains a mutation A-+G at the samne base position, which eliminates the Hindill site.

FIG. 7 is an electrophoresis gel which shows the results of the restriction-digestion analysis of plasmids from the 40 clones.

FIG. 8 shows the results af sequencing ten genes from the defined primer-based recombination library. Lines represent 986.bp of subtiliamn E gene including 45 nt of its prosequence, the entire mature sequence and 113 nt after the stop codon. Crosses indicate positions of mutations from parent gene R1. and R2, while triangles indicate positions of new point mutations introduced during the recombiniation procedure. Circles represent the mutation introduced by the rnutagenic primer FIG. 9 depicts the results of applying the random-sequence primer recombination method to the gene for Actinoplrznes; utahensis EC5 deacylase.

The 2.4 kb ECB deacylase gene was purified from an agarose gel. The size of the random priming products ranged from 100 to 500 bases. (c) Fragments shorter than 300 bases were isolated- The purified fragments S 20 were used to reassemble the full-length gene with a smear background. A single PCR product of the samne size as the ECB dcacylase gene was obtained **~after conventional PCR with the two primers located at the start and stop regions of this gene. After digestion with Xho I and Psh Al, the PCR product was cloned into a modified pIJ702 vector to form~ a mutant library. (g) Introducing this library into Streptomyces litidans TK23 resulted in approximately 71% clones producing the active ECB deacylase.

FIG, 10 shows the specific activity of the wild-type ECB deacylase and mutant M16 obtained in accordance with the present invention.

FIG. 11 shows pH profiles of activity of the wild-type ECB deacylase and mutant M 16 obtained in accordance with the present invention.

FIG. 12 shows the DNA sequence analysis of 10 clones randomly chosen from the libraxy/Kienow. Lines represent 986-bp of subtilisin E gene including nt of its prosequence, the entire mature sequence and 113 at after the stop codon. Crosses indicate positions of mutations from R1 and R2, while Mimosa 12:57:20 RECEIVED TIME 28. DEC. 17:07 PITTM 9 E. 61 PRINT TIME 29. DEC. 6 Z. UJ. -UUU 18:16 SPRUSON FERGUSON 61 2 92615486 NO. 8897 P. 13 SPRUSON FERGUSON WO 98/42932 PCr/LS9105956 triangles indicate positions of new point mutations introduced during the random-piiing recombination process.

F70.13 Thermostability index profiles of the screened clones from the five libraries produced using different polymerases: a) libraxy/lenow, b) llbrary/T4, c) library/Sequenase, d) librairy/Stoffel and e) library/Pfu.

Normalized residual activity (Ar/Ai) after incubation at 65*C was used as an index of the enzyme therrnostability. Data were sorted and plotted in descending order.

DETAILED DESCRIPTION OF THE INVENTION In one preferred embodiment of the present invention, a set of primers with all possible nucleotide sequence combinations (dp(N)L where L primer length) is used for the primer-based recomnbination. It has been known for years that oligodeoxynucleotides of different lengths can serve as primers for initiation of DNA synthesis on single-stranded templates by the Kienow fragment of Lecoti polymnerase 1 Although they are smaller than the size a: of a normal PCR primer (Le. less than 13 bases), oligomers as short as hexanucleotides can adequately prime the reaction and are frequenitly used in labeling reactions The use of random primers to createc a pool of gene :fragments followed by gene reassembly in accordance with the invention is shown in FIG. 1. The steps include generation of diverse "breeding blocks" from the single-stranded polynucleotide templates through random priming, reassembly of the full-length DNA from the generated short, nascent DNA fragments by thermocycling in the presence of DNA polymerase and nucleotides, and amplification of the desired genes from the reassembled products by conventional PCR for further cloning and screening. This procedure introduces new mutations mainly at the priming step but also during other steps. These new mutations and the mutations alrcady present 0: 30 in the template sequences are recombined during reassembly to create a library of novel. DNA sequences. The process can be repeated on the selected sequences, if desired.

:To carry out the random priming procedure, the template(s) can be single- or denatured double-stranded polynucleotide(s) in linear or closed circular form. The templates can be mixed in equimolar amounts, or in amounts weighted, for example, by their functional attributes. Since, at least in some cases, the template genes are cloned in vectors into which no additional mutations should be introduced, they are usually first cleaved with Mimosa 12:57:20 RECEIVED TIME 28. DEC. 17:07 PITTM 9 E. 61 PRINT TIME 29.DEC. 6:14 DEC. 2000 18 :16 SPRUSON FERGUSON 61 2 92615486 NO. 8897 P. 14 SPRUSON FERGUSON Wo 98/42"32 PCT(1598/)5956 restriction endonuclease(s) and purified orn the vectors. The resulting linear DNA molecules are denatured by boiling, annealed to random-sequence oligodeoxynucleoticjes and incubated with DNA polymerase in the presence of an appropriate amount of dNTP&. Hexanucleotide primers are preferred, s although longer random primers (up to 24 bases) may also be used, depending on the DNA poiyrnerase and conditioning used during random priming synthesis. Thus the oligonucleotides prime the DNA of interest at various positions along the entire target region and are extended to generate short DNA fragments complementary ro each strand of the template DNA. Due to events such as base mie-incorporationa and misprixning, these short DNA fragments also contain point mutations. Under routinely establiahed reaction conditions, the short DNA fragments can prime one another based on homnology and be reassembled into full-length genes by repeated therrnocycling in the presence of therinostable DNA polymerase. The resulting rull-length genes will have diverse sequences, most of which, however, still resemble that of the original template DNA. These sequences can be further amplified by aL conventional PCR and cloned into a vector for expression.

Screening or selection of the ervpressed mutants should lead to variants with improved or even new specific functions. These variants can be immediately used as partial solutions to a practical problem, or they can serve as new starting points for further cycles of dlirectcd evolution.

:Compared to other techniques used for protein optimization, such as combinatorial cassette and oligonucleor-ide-directed nu tagenesis (24,2S,26), error-prone PCR (27, 28), or DNA shuffling (17,18,19), some of the advantages 2S of the randomn-primer based procedure for in vitro protein evolution are summaized as follows: 1. The template(s) used for random priming synthesis may be either single- or- double-stranded polynucleotides- In contrast, error-prone PCR and the DNA shuffling method for recomabination (17,18,19) necessarily employ 30 only double-stranded polynucleotides.. Using the technique described here, mutations and/or crossovers can be introduced at the DNA level by using different DNA-dependent DNA polyrnerases, or even directly from mRNA by using different RN~A-dependent DNA polyrnerases. Recombination can be performed using single-stranded DNA templates.

2. In contrast to the DNA shuffling procedure, which requires fragmentation of the double-stranded DNA template (generally done with DKAse 1) to generate random fragments, the technique described here employs random priming synthesis to obtain DNA fragments of controllable size as "breeding blcs for further reassembly (FIG. One immediate advantage is Mimosa 12.57:20 RECEIVED TIME 28.DEC. 17:07 RECEVED IME 8. DC. 1:07PRINT TIME 29. DEC. 6:14 Zd, DEC. 2000 18: 16 SPRUSON FERGUSON 6IA 2 92615486 NO, 8897 P. SPRUSON FERGUSON WO 98142932 PMFUS98WO5956 that two sources of nuclease activity (DNase I and exonuclease) are eliminated, and this allows easier control over the size of the final reassembly and amplification gene fragments.

3. Since the random primers are a population of synthetic oligoniucleotides that contain all four bases in every position, they are uniform in their length and lack a sequence bias. The sequence heterogeneity allows them to form hybrids with the template DNA strands at many positions, so that every nucleotide of the temiplate (except, perhaps, those at the extreme terminus) should be copied at a similar frequency into products. In this way, both mutations and crossover may happen more randomly than, for example, with error-prone PCR or DNA shuffling.

4. The random-primed DNA synthesis is based on the hybridization of a mixture of hexanuclcotides to the DNA templates, and the complementary strands are synthesized from the 3'-OH termini at the randomn hexAnucleotide is primer using polymerase and the four deoxynuclec tide triphosphates. Thus the reaction is independent of the length of the DNA template. DNA fragments of 200 bases length cant be primed equally well as linearized plasmid or X DNA This is particularly useful for engineering peprldes, for example.

S_ Since DNase I is an endonuclease that hydrolyzes double-stranded DNA preferentially at sites adjacent to pyrixnidine nucleotides, its use in DNA shuffling may result in bias (parcicularly for genes with high G+C or high AiT ~*content) at the step of template gene digestion- Effects of this potential bias on the overall mutation rate and recombination frequency may be avoided by using the random-priming approach. Bias in random priming due to preferential hybridization to GC-rich regions of the template DNA could be overcome by increasing the A and T content in the random oligonucleotide library.

An important part of practicing the present invention is controlling the average size of the nascent, single-strand DNA synthesized during the random priming process. This step has been studied in detail by others. Hodgaon and Fisk (30) found that the average size of the synthesized single-strand DNA is **an inverse function of primer concentration: length -0k//ne where Pc is the primer concentration. The inverse relationship between primer concentrationt and output DNA fragment size may be due to steric hindrance. Based on this guideline, proper conditions for random-priming synthesis can be readily set for individual genes of different lengths.

Mimosa 12:57;20 RECEIVED TIME 28. DEC. 17:07 PRINT TIME 29DE. 61 29. DEC. 6 14 Z, UU UUU I d: 1'I FXU6UN PHUUMN bIa 2 92615485 NO. 8897 P. 16 SPRUSON FERGUSON WO 98/42832 PCTUS98/05956 Since dozens of polyrneruses al-e currently available, synthesis of the short, nascent DNA fragments can be achieved in a variety of fashions. For exarrple, bacteriophage T4 DNA polymerase (23) or T7 sequenase version DNA polyrnerase (31,32) can be used for the random priming synthesis.

For single-stranded polynucleotide templates (particularly for RNA templates), a reverse tranacriptase is preferred for random-priming synthesis.

Since this enzyme lacks exanuclease activity, it is rather prone to error.

In the presence of high concentrations of dNTPs and Mn 2 about I base in every 500 is misincorporated (29).

By modifying the reaction conditions, the PCR can be adjusted for the random priming synthesis using themostable polymerase for the short, nascent DNA fragments. An important consideration is to identify by routine experimentation the reaction conditions which ensure that the short random primers can anneal to the templates and give suafficient DNA amplification at is higher temperatures. We have found that random primers as short as dp(N)12 can be used with PCR to generate the extended primers. Adapting the PCR to the random priming synthesis provides a convenient method to make shor-t, nascent DNA fragments and makes this random priming recomnbination technique very robust.

In many evolution scenarios, recombination should be conducted :between oligonucleatide sequences for which sequence information is available for at least some of the template sequences. In such scenarios, it is often possible to define and synthesize a series of primers which are interspersed between the various mutations. When defined primers are used, they can be between 6 and 300 bases long. In accordance with the present invention, it was discovered that by allowing these defined primers to initiate a series of overlapping primer extension reactions (which may be facilitated by thermocycling), it is possible to generate recombination cassettes each containing one or more of the accumulated mutations, allelic or isotypic differences between templates. Using the defined primers in such a way that overlapping extension products are generated in the DNA polymerization reactions, exhaustion of available primncr leads to the progressive cross- :hybridization of primer extended products until complete gene products are generated. The repeated rounds of annealing, extension and denaturation assure recombination of each overlapping cassette with every other.

A preferred embodiment of the present invention involves methods in which a set of defined oligonucleotide primers is used to prime DNA synthesis.

FIG. 2 illustrates an exemplary version of the present invention in which Mimosa 12:57:20 RECEIVED TIME 28. DEC. 17:07 PITTM 9 E. 61 PRINT TIME 29.DEC. 6:14 UC, 2000 18:17 SPRUSON FERGUSON 61 2 92615486 N.89 .1 SPRUSON FERGUSON WO 98/42832 PCT/US9N/0596 42defined pimers are used. Careful design and positioning of oligonucleotide primers facilitates the generation of non-random extended recombination primers and is used to determine the major recombination (co-segregation) events along the length of homologous temrplates.

Another embodiment of the present invention is an alternative approach to primer-based gene assembly and recombination in the presence of template. Thus, as illustrated in FIG. 3, the present invention includes recombin~ation in which enzyme-catalyzed DNA polymerization is allowed to proceed only briefly (by limiting the time and lowering the temperature of the extension step) prior to denaturation. Denaturation is followed by random annealing of the extended fragments to template sequences and continued partial extension- This process is repeated multiple times, depending on the concentration of primer and template, until full length sequences are made.

Th~is process is called staggered extension, or StEP. Although random primers can also be used for StEP, gene synthesis is not nearly as efficient as with defined primers. Thus defined primers are preferred.

In this method, a brief annealing/ extension step(s) is used to generate the partially extended primer. A typical annealing/ extension step is done *under conditions which allow high fidelity primer annealing (Tneaing greater 20 than Tm- 2 5 but limit the polymnerization/ extension to no more than a few :seconds (or an average extension to less than 300 nts). Minimum extensions az-c preferably on the order of 20-50 nts. It has been demonstrated that therrnoatablc DNA polymerases typically exhibit maximal polymerization rates of 100-150 nucleotides /second/ enzymne molecule at optimal temperatures, 25 but follow approximate Arenius kinetics at temperatures approaching the optimum temperature (Topt). Thus, at a temperature of 550C, a thermiostable polymerase exhibits only 20-25% of the steady state polymerization rate that it o ~exhibits at 72*C (Topt), or 24 rts/second At 376C and 22 0 C. Taq oroo polymerase is reported to have extension activities of 1.5 and 0.25 nts/second, respectively Both time and temperature can be routinely oo o altered based on the 'desired recombination events and knowledge of basic polymerase kinetics and biochemistry.

The progress of the staggered extension process is monitored by removing aliquots from the reaction tube at various time points in the primer extension and separating DNA fragments by agarose gel electrophoresis- Evidence of effective primer extension is seen from the appearance of a low Mimosa 12:57:20 RECEIVED TIME 28. DEC. 17:07PRN TIE 2 DC. 61 PRINT TIME 29. DEC, 6 14 Z. ut Mu 18: 1 SPRUSON FERGUSON 61'A 2 92615486 NO. 8897 P. 18 SPRUSON FERGUSON WO 99/42932 PCr/US9woz"s molecular weight 'Smear' early in the Process which in~creases in molecular weight with increasing cycle number.

Unlike the gene amplification Process (which generates new DNA eXPOnentially), StEP generates new DNA fragments in an additive manmer in S its early cycles which contain DNA segments corresponding to the different template gen~es. Under non-amplirying conditions, 20 cycles of StEP generates a maximal molar yield of DNA of approimxately 40 times the initial template concentration. In comparison, the idealized polymerase chain reaction process for gene ampliflcation is multiplicative throughout, giving a maxtimal molar yield of approximately I x 1O'-fold through the same number of steps, In practice, the difference between the two processes can be observed by PCR, giving a clear 'band' after only a few (less than 10) cycles When starting with template at concenrations of less than 1 ng/ul and primers at 10-500-fold excess (vs. 1 0 6 -fold excess typical of gene amnplification), Under similar is reaction conditions, the StEP would be expected to give a less visible 'smear', which increases in molecular weight with increasing number of cycles. When significant numbers of primer extended DNA molecules begin to reach sizes of greater than~ 1/2 the length of the full length gene, a rapid jump in molecular weight occurs, as half-extended forward and reverse strands begin to cross- 20 hybridize to generate fragments, nearly 2 times the size of those encountered **to that point in the process. At this point, consolidation of the smear into a discrete band of the appropriate molecular weight can occur rapidly by either continuing to subject the DNA to StEP, or altering the therinocycle to allow complete extension of the primed DNA to drive exponential gene amplification.

Following gene assembly (and, if necessary, conversion to double stranded form) recombined genes are amplified (optional), digested with suitable restriction enzymes and ligated into expression vectors for screening of the expressed gene products. The process ca~n be repeated if desired, in order to accumulate sequence changes leading to the evolution of desired functions.

The staggered extension and homologous gene assembly process (StEP) **represents a powerful1, fle-dble method for recombining similar genes in a *random or biased fashion. The process can be used to concentrate *.*recombination within or away from specific regions of a known series of sequences by controlling placement of primers and the time allowed for annealing/ extension steps. It can also be used to recombine specific cassettes of homologous genetic information generated separately or within a single reaction. The method is also applicable to recombining genes for which no Mimosa 12:57:20 RECEIVED TIME 28.DEC. 17:07 RECEVED IME 8. DC. 1:D7PRINT TIME 29. DEC. 6:14 Dt Ub, ZUUU 16:18 bRUSON FERGUSON 61A 2 92615486 NO. 8897 P. 19 SPRUSON FERGUSON WO 96/42832 PCT/US9S/0Sg56 sequence information is available but for which functional S' and 3' amplification primers can be prepared. Unlike other recombination methods, the staggered extension process can be run in a single tube using conventional procedures without complex separation or purification steps.

Some of the advantages of the defined-primer embodiments of the present invention are summarized as follows: 1. The StEP method does not require separation of parent molecules from assembled products.

2. Defined primers can be used to bias the location of recombination events.

3. StEP allows the recombination frequency to be adjusted by varying extension times.

4. The recombination process can be carried out in a single tube.

is 5. The process can be carried out on single-stranded or double-stranded polynucleotides.

6. The process avoids the bias introduced by DNase I or other endonucleases.

.o a: 7. Universal primers can be used.

20 8. Defined primers exhibiting limited randomness oan be used to increase :the frequency of mutation at selected areas of the gene.

As will be appreciated by those skilled in the art, several embodiments of the present invention are possible, Exemplary embodiments include; 2S 1. Recombination and point mutation of related genes using only defined flanking primers and staggered extension.

2. Recombination and mutation of related genes using flanking primers and a series of internal primers at low enough concentration that exhaustion of the primers will occur over the course of the thermocycling, forcing the overlapping gene fragments to cross-hybridize and extend until recombined synthetic genes are formed.

3- Recombination and mutation of genes using random-sequence primers at high concentration to generate a pool of short DNA fragments which are reassembled to form new genes.

Mimosa 12:57:20 RECEIVED TIME 28. DEC. 17:07 PRINT TIME 29, DEC, 6:14 2b T{ UUU 18: 18 SPRUSON FERGUSON 61A 2 92615486 NO. 8897 P. SPRUSON FERGUSON *WO 98/42832 PCTAUS9S/05956 4. Recombination and mutation of genies using a set of defined primers to generate a pool of DNA fragments which are reassembled to form new genes.

Recombination and mutation of single-stranded polyniucleotides using one or snore defined primers and staggered extension to form new genes.

6. Recombinationi using defined primers with limited randomness at more than 30% or more than 60% of the nucleotide positions within the primer.

Examiples of practlice showing use of the primer-based recomabination method are as follows.

EXAMPL9 1 Use of defined flanking primers and staggered extension to is recombine and enhance the therinostability of subtillein B This example shows how the defined primer recombination method can be used to enhance the therm ostability of subtilisin E by recombination of two genes known to encode subtilisin X variantz with thermostabilities exceeding that of wild-type subtilisin E. This example demonstrates the general method outlined in FIG. 3 utilizing only two primers corresponding to the 5' and 3' :ends of the templates.

outlined in FIG. 3, extended recombination primers are first generated by the staggered extension process (StEP), which consists of repeated cycles of denaturation followed by extremely abbreviated annealing/ extension step(s). The extended fragments are reassembled into full-length genes by thermocycling-asvisted homologous gene assembly in the presence of a DNA polymrerase, followed by an optional gene amplification step.

Two thermostable subtilisin E mutants RI and R2 were used to test the defined primer based recombination technique using staggered extension.

The positions at which these two gene differ from one another are shown in Table 1. Among the ten nucleotide positions that differ in R1 and R2, only those mutations leading to amino acid substitutions Asn 181-Asp (NI81D) and Asn 218-Ser (N218S) confer thennostability. The remaining mutations are neutral with respect to their effects on thermostability The half-lives at of the single variants N181D and N218S are approximately 3-fold and 2fold greater than that of wild type subtilisi& 9, respectively, and their melting temperatures, Tm. are 3.70C and 3.2'C higher than that of wild type enzyme, Mimosa 12:57:20 RECEI VED TIME 28. DEC. 17:07 PITTM 9 E. 61 PRINT TIME 29. DEC. 6 14 O.u-ZH luu i:d iU ?U6UN ftfU'U bl&Z 9 2615485 NO. 8897 P. 21 SPRUSON FERGUSON W O 9 9 4 2 9 3 2P C T U S 9 8 0 5 9 5 6 respectvel. Random recombination events that Yield sequences containing both these functional mutation, will give rise to ertzyines whose half lives at are approximately 8-fold greater than that Of wild type aubtilisiri

E,

Provided no new deleterious mutations are introduced into these genes during the recoznbinatian process_ Furtherrze, the Overall Point mutagenes3is rate associated with the recombination process can be estimated from the catalytic Activity profle of a small sampling of the recombined variant library. If the Point mutagenesis rate is zero, 25% Of the Population should exchibit wild type.

like activity, 25% of the Population should have double muatanit (1BlD11+N218).]ike Activity and the remaining 50% should have single mutant (141811) or N218S).like activity. Finite point mutagenesis increases the fraction of the library that encodes enzymes with wild-type like (or lower) aictivity. This fraction can be used to estimate the Point mutagenesis rate.

is TA13LE I DNA and amino acid substtutiona in thermostabi, subtilin E mutants R1 and R2.

Base- Position Ano acid 9Gene Base Substitution in codon Amino acid substitution *780 A- G 219Asa--.)Ser *.Ri 1107 A- G 2 218 Asa--)Ser %:1141 A- T 3 229 synonymous 15 233 synonymous 484 A-4G 3 J,0 synionymnous 520 A +T3 22 synonymous 598 A-sG 3 48 synonymnous 731 G-sA 1 93 Val-+sIle R2 745 T-sC 3 97 synonymous 780 A 2 109 Asn-4Ser 995 A-G 1 181 Asl-*Asp 1189 A- G 3 245 snonymous 99Mutations listed are relative to wild type subtilisin E with base substitution at 780 in common.

Mimsa 2:5:2 RECEIVED TIME 28,DEC. 17:07 RECEVED IME 8. DC. 1:D7PRINT TIME 29. DEC.' 6:14 2. D1EC. 2Q00 18:18 SPRUSON FERGUSON 61 2 92615486 NO. 8897 P. 22 SPRUSON FERGUSON WO 9W7.3 PCTAUS9S/0956 Materials and Methods Procedure for defthed primer based recombinatifon using tw.o flanking primer, Two defined primers, P5N (5'-CCOAG CGVFG CATAT TGGA AG-3' (SEQ. ID. NO: underlined sequence is NdeI restriction site) and P35 COACT CTAGA GQGATC -CGATT C-3- (SEQ. ID. NO: underlined sequence is Bamlfl restriction site), corresponding to 5' and 3' flanking primers, respectively, were used for recomabination. Conditions (100 u] final voh~re): 0. 15 pmol plasraid DNA containing genes R1 and 2R2 (mixed at 1- 1) were used ns template, 15 pmol of each flanking primer, 1 times Taq buffer, 0.2 mM of each dNTP, 1.5 mM MgCI 2 and 0.25 U Taq polymerase. Program:, 5 minutes of 80 cycles of 30 seconds 94*C, 5 seconds 55 0 C. The product of correct size (approximately 1kb) was cut from an 0.8% agarose gel after electrophoresis and purified using QIAEX Hl gel extraction kit. This piurified product was digested with Ndel and 8arnHI and subcloned into pBE3 shuttle vector. This gene library was amplified in coli HB101 and transferred into B. subtilis DB428 competent cells for expression and screening, as described elsewhere DNA sequencing .me. 20 Genes were purified using QlAprep spin plasmid miniprep kit to obtain 0*sequencing quality DNA. Sequencing was done on an ABI 373 DNA :Sequencing System using the Dye Terminator Cycle Sequencing kit (Perkin- Elmer, Branchburg, NJ).

Results The progress of the staggered extension was monitored by removing aliquots (10 ul) from the reaction tube at various tim~e points in the primer extension process and separating DNA fragments by agal-ose gel electrophoresis. Gel electrophoresis of primer extension reactions revealed that annealing/ extension react~ions of 5 seconds at 55vC resulted in the 0 occurrence of a smear approaching 100 bp (after 20 cycles), 400 bp (after cycles), 800 bp. (after 60 cycles) and finally a strong approximatelyil kb band 00*0*within this smear. This band (mixture of reassembled products) was gel purified, digested with restriction enzyme BamnM and NdeI, and ligated with vector generated by Bamnffl-Ndel digestion of the E. colif/ B. subtilis pBE,3 shuttle vector. This gene library was amplified in E. coli HB101 and transferred into B. subtilis D5428 competent cells for expression and screening Mimosa 12:57:20 RECEIVED TIME 28. DEC. 17:07 PITTM 9 E. 61 PRINT TIME 29. DEC. 6 14 Zd-vt .UU Id: lY SPRUSON FERGUSON 1'2 92615486 N.89 .2 SPRUSON FERGUSON WO 98/42832 PC7/TJS9/O95 6 The tliermostability of enzyme variants was determined in the 96-wel Plate format described previously About 200 clones were screened, and approximately 25% retained subtilisin act.ivity. Among these active clones, the frequency of the double mutant-lie phenoiype (high therrnostability) was approximuately 23%, the single mutant-like phenotype was approxiately 42%, and wiild type-hice phenotype was approximately 34%_ This distribution is very close to the values expected when the two thermostable mutations N218S and N 18 1D can recombine with each other completely freely.

Twenty clones were randomly picked from E. woli H1 4110 gene library.

Their plasmnid DNAs were isolated and digested with Nde) and BantHl. Nine out of 20 had the inserts of correct size (approximately 1 kb). Thus, approximately 55% of the above library had no actiavity due to lack of the correct subtilisin E gene. These clones are niot members of the subtilisin library and should be removed from our calculations. Taking into account this factor, we find that 55% of the library (25% active clones/45% clones with correct size insert) retained subtillain activity. This activity profile indicates a point mutagenesis rate of less than 2 miitations per gene Five clones with inserts of the correct size were sequenced. The results are summarized in FIG.

4. AU ive genes are recombination products with minimum crosuovers varying from I to 4. Only one new point mutation was found in these ive genes.

EXAMPLEI 2 Use of defined flanking primers and staggered extension to recombine pilE esterase mnutants The two-primer recomination method used here for pNB esterace is 0055 analogou~s to that described in Example I for su.btilsin E. Two template pNB 9000 esterase mutant genes that differ at 14 bases are used. Both templates (61IC7 .00. :and 404) are used in the plasmaid form. Both target genes are present in the 0 30 extension reaction at a concentration of I Flanking primers (RM1A and 0 RM2A, Table 2) are added at a final concentration of 2 ng/ul (approxdiately 200-fold molar excess over template).

*0.

Mimosa 12:57:20 RECEIVED TIME 28.DEC. 17:07 RECEVED IME 8. DC. 1:07PRINT TIME 29. DEC. 6:13 u O yt .UUU 1d:l I' -?RUSUN FEN(USUN bI2 292515486 N.89 .2 SPRUSON FERGUSON WO 99/42832 PCTAUS9SiO5956 TABLE 2 Priner used in the recombination of the pNB esterase genes Primer Sequence RM lA GAG CAC ATC AGA TCT ATT AAC (SEQ. ID. NO: 3) RM2A GOA GTG OCT CAC AGT CO TOG (SEQ. ID. NO: 4) Clone 61C7 was isolated based on its activity in organic solvent and contains 13 DNA mutations vs. the wild-ye sequence. Clone 4G4 was isolated for thermostability and contains 17 DNA niutaxiono when compared with wild-type. Eight mutafions are shared between them, due to common ancestry. The gene product from 4G4 is significantly more thermostable than the gene product from 6 1C7. Thus, one measure of recombination between the genes is the co-segregation of the high solvent activity and high thermostability or the lose of both proper-ties in the recombined genes. In addition, recombination frequency and mutaenic rate can be ascertained by sequencing random clones- For the pNB esterase gene, primer extension proceeds through is rounds of extension with a thermocycle consisting of 30 sconds at 94*C followed by 15 seconds at 55*C. Aliquots (10 Wd) are removed following cycle 20, 40, 60, 70, 80 and 90. Agarose gel electrophorcsis reveals the formation of a low molecular weight 'smear' by cycle 20, which increases in average size *and overall intensity at each successive sample point. By cycle 90, a 20 pronounced smear is evident extending from 0.5 kcb to 4 kb, and exhibiting maximalj aignal intensity at a size of approximately 2 kb (the length of the full length genes). The jump from half-length to full length genes appears to occur between cycles 60 and The intense smear is amplified through 6 cycles of polymnerase chain 25 reaction to more clearly define the full length recombined gene population. A minus-primer control is also amplified with flanking primners to determine the background due to residual template in the reaction mix. Band intensity from the priner extended gene population exceeds that of the control by greater than 10-fold, indicating that amplified, non-recombined template comprise only a small fraction of the amplified gene population.

The amplified recombined gene Fool is digested with restriction .*.enzymes Xbal and SamHI and ligated into the pNB1O6R expression vector described by Zock et al. Transformation of ligated DNA into E. coi strain TG1 is done using the well characterized calcium chloride transformation Mimosa 12:57:20 RECEIVED TIME 28.DEC. 17:07 RECEVED IME 8. DC. 1:D7PRINT TIME 29, DEC. 6:13 ZUUU I:I9 SVFUbUN FEMGUSOUN bl Z 92 15486 SPRUSON FERGUSON NO, 8897 P. WO 98/42832 PCTUS98/059s6 ;4 procedure. Transformed colonies are selected on LB/agar plates containing lpg/mrl tetracycline.

The MUrntagenic rate of the process is determined by measuring the percent of clones expressing an active esterase In addition, colonies picked at random are sequenced and used to define the mutagenic frequency of the method and the efficiency of recombination.

ZXAKPIXE3 Recombination of pB esterase genes using interspered Internal defined primers and staggered extaslon This example demonstrates that the interspersed defined primer recombination technique can produce novel sequences through point mutagenesis and recombination of mutations present in the parent sequences.

Experimental design and background Information Two pNB esterase genes (2-13 and 5-B12) were recombined using the defined primer recombination technique, Gene products from both 2-13 and 5-B12 are measurably more thermostable than wild-type. Gene 2-13 contains 9 mutations not originally present in, the wild-type sequence, while gene B12 contains 14. The positions at which these two genes differ from one another are shown in FIG. Table 3 shows the sequences of the eight primers used in this example.

Location (at the 5' end of the template gene) of oligo annealing to the template 2S genes is indicated in the table, as is primer orientation (F indicates a forward primer, R indicates reverse). These primers are shown as arrows along gene 2-13 in FIG. oO.

°oooo *o :*00 00 0..

o *ooe Mimosa 12:57:20 RECEIVED TIME 28. DEC, 17:07 PRINT TIME 29, DEC. 6:13 0- vt ZUUU 16: ly 6FRUSON FERGUSON 51& 2 92615486 ISPRUSON FERGUSON NO, 8897 P. 26 WO 9842 TABLt3 Bequences of primers used in this example Dame orientation location RMlA F -76 RM2A R -4454 S2 F 400 F 1000 87 F 1400 Sa R 1280 810 R 8a0 S13 R 280 sequence GAGCACATCAGATCTA'ITAAC (E.I.N:3 GOAGGCTCACAGTCOOTGG (SEQ. 11D. NO: 4) TTGAACTATCGOCTaGGCGO (SEQ. ID. NO:- TrACTAGGGAAOCCGCTGGCA (SEQ. IDl. NO: 6) TCAGAGATfACGATCGAAAAC (SEQ. It). NO: 7) GA'rGTATCGTGTGAGAAAG ISEQ. MD. NO. 8) AATCGCCGGAAGCAGCCCCt-rC (SEQ. ID. NO- 9) CACOACAGGAAGmTGACT (S)EQ. 1D. NO: 4* 9* Materials and Methods 3 Defined-primrer based recomrbination 1 Preparation of genes to be recombined. Plasmids containing the genes to be recombined were purified from transformed TOI cells using the Qiaprep kit (Qagen, Chatsworth, CA). Plasmids were quantitated by UJV absorption and mixed 1:1 for a final concentration of 50 ng/uI.

2. Staggered extension PCR and reassembly. 4 pil of the plasmid mixture was used as template in a 100 0± standard reaction (1.5 mM MgClg, 50 mM lCI, 1.0 mM Tris.HCI pH 9.0, 0. 1% Triton X-I100, 0.2 mM dNTPs, 0.25 U Thq polynierase (Promnega, Madison, WI)) which also contained 12.5 ng of each of the 8 primers. A control reaction which contained no primers was also i5 assembled. Reactions were thermocycled through 100 cycles of 949C, seconds; 55*C, 1.5 seconds. Checking an aliquot of the reaction on an agarose gel at this point showed the product to be a large smear (with no visible product in the no primer control)- 3. .OpnI digestion of the templates. 1 i from the assembly reactions was then digested with Dp&I to remove the template plasrnic. The 10 0~ Dprdl digest contained 1 x NEBuffer 4 and 5 U .Dprl (both obtained from New England Biolabs, Beverly, MA) and was incubated at 37'C far 45 minutes, followed by incubation at 70*C for 10 minutes to heat kill the enzyme.

4. PCR amplification of the reas9sembled products. The 10 pil digest was 25 then added to 90 ol of a standard PCR reaction (as described in step 2) containing 0,4 ;AM primers Sb (ACTTA.ATCTAGAGGGTA*IA) (SEQ. ID. NO: 11) and 3b (AGCCTCGCGGOATCCCCGGG) (SEQ. 11). NO; 12) specific for the ends of the gene. After 20 cycles of standard PCR (94 0 C. 30 seconds: 484C, Mimosa 12:57:20 RECEIVED TIME 28. EC. 17:07 PITTM 9 E. 61 PRINT TIME 29.DEC.

6 13 vt. ML UU 18: 19 SPRUSON FERGUSON 61JA 2 92615486 NO. 8897 P. 27 SPRUSON FERGUSON WO 9&(4282 P'CF/UsS9956 seconds, 72'C, 1 minute) a strong band of the correct size (2 kb) was visible when the reaction was checked on an agarose gel, while only a very faint band was visible in the lane from the no-primer control. The product band was purified and cloned back into the expression plaamid pNB106R and ransformed by electroporation into TOl Icells- Results Four 96 well plates of colonies resulting from this transformation were assayed for pNB esterase initial activity and therinostability. Approximately 60% of the clones exhibited initial activity and thermostabilty within 20% of the parental gene vales. Very few of the clones were inactive (less than of parent initial activity values). These results suggest a low rate of muragenesis. Four mutants with the highest thermostability values were sequenced. Two clones (6E6 and 61-11) were the result of recomabination between the parental genes (FIG. One of the remaining two clones contained a novel point mutation, and one showed no difference fromn parent 5512. The combination of mutations T99C and C204T in mutant 6E6 is evidence for a recombination event between these two sites. In addition, mutant 6111 shows the loss of mutation A1072G (but the retention of mutations C1038ST and T1310C), which is evidence for two recombination events (one between sites 1028 and 1072, and another between 1072 and 1310). A total of five new point mutations were found in the four genes sequenced.

25 EXAMPLE 4 Recombination of two thermostablo subtilisin E variants using internal defined primers and staggered extenzion This example demonstrates that the defined primer recombination technique can produce novel sequences containing new combinations of mutations present in the parent sequences. It further demonstrates the utility of the defined primer recombination technique to obtain fu.rther improvements in enzyme per-formance (here, thermostability). This example further shows that the defined primers can bias the recombination so that recombination appears most often in the portion of the sequence defined by the primers *.35 (inside the primers). Furthermore, this example shows that specific mutations :can be introduced into the recombined sequences by using the appropriate defined primer sequence(s) containing the desired mutation(s), Mimosa 12:57:20 RECEIVED TIME 28. DEC. 17:07 PITTM 9 E. 61 PRINT TIME 29. DEC. 6 13 Dtb. Z UUU 18 :21i SPRUSUN HHRUSON 618 2 92615486 NO. 8897 P. 28 SPRUSON FERGUSON WO 98/42932 PCT/Us9a10l5956 Genes encoding two thernostsable subtilisin E variants of Example I (RI andi R2) were recombined using the defined primer recomnbination procedure with internal priners. FIG. 6 shows the four deflned internal primers used to generate recombined progeny genes from template genes RI and R2 in this example. Prumer PSOF contains a mutation (A-e.T at base position 598) which eliminates a Hindu!l restriction site and simultaneously adds a new unique Nhel site. This primer is used to demonstrate that specific mnutations can also be introduced into the population of recombined sequences by specific design of the defined primer. Gene R2 also contains a multation A->0 at the same base position, which eliminates the Hindill site.

Thus restriction analysis (cutting by INhe! and HindIMi of random clones sampled from the recombined library will indicate the efficiency of recombination and of the introdunction of a specific mutation via the mutagenic primer. Sequence analysis of randomly-picked (unscreened) clones provides is further information on the recombination and mutagenesis ev.ents occurring during defined primer-based recombination.

Materials and Methods Defined-primer based reombvination A version of the defined primer based recombination illustrated in FIG.

was carried out with the addition of StEP.

1I. Preparation of genes to be recombined. About 10 ug of plasrnids containing RI and R2 gene were digested at 37*C for 1 hour with Ndel and 25 Baml-f (30 U each) in 50 jpl of lx: buffer B (Boehringer Mannheim, Indianapolis, IN). Inserts of approximately 1 kb were purified from 0.8% *...preparative agarose gels using QIAF.X H gel extraction kit. The DNA inserts were dissolved in 10 mM Tris-HCI (pH The DNA concentrations were estimated, and the inserts were mixed 1.-1 for a concentration of 50 ng/ul.

2. Staggered extension PCR and reassemrbly. Conditions (100 ul final volume): about 100 ng inserts were used as template, 50 ng of each of 4 internal primers, lx: Faq buffer, 0,2 mM of each dNTP, 1.-5 MM MgC1 2 and U Taq polymerase. P~rogram: 7 cycles of 30 seconds at 94'C, 15 seconds at a 5S'C, followed by another 10 cycles of 30 seconds at 94*C, 1S seconds at 5 seconds at 72'C (staggered extension), followed by 53 cycles of we seconds at 94'C, 15 seconds at 55*C, 1 minute at 72*C (gene assembly).

1 pnl digestion of the templates. 1 al of this reaction was diluted up to 9- with dH2O and 0.5 I of DpnI restriction enzyme was~ added to digest the Mimosa 12:57:20 RECEIVED TIME 28. DEC. 17:07 PITTM 9 E. 61 PRINT TIME 29.DEC. 6:13 Zd. DEC. 2000 18: 20 SPRUSON FERGUSON 61' 2 92615486 NO, 8897 P. 29 SPRUSON FERGUSON WO 98/42832 PCT/US98/05956 DNA template for 45 minutes, followed Iincubation at 70'C for 10 minutes and then this 10 ul was used as template in a 10-cycle PCR reaction.

4. PCR amplification of reassembled products. PCR conditions (100 P1i inal volume): 30Opmol of each outside primer P5N' and P313, Ix Taq buffer, 0.2 S mM of each dNTP and 2.5 U of Taq polyrnerase. PCR program: 10 cycles of seconds at 94*C, 30 seconds at 55*C, 1 minute at 72*C. This program gave a single band at the correct size. The product was purified and subcloned into p8Ri3 shuttle vector. This gene library was amplified in E. coli H8101 and transferred into B. subtilis DB428 competent cells for expression and screening, as described elsewhere Thermostability of enzyme variants was determined in the 96-well plate format described previously (33).

.DNA sequencing Ten E. coh H8101 transformants; were chosen for sequencing. Genes were purified using QlAprep spin plasrnid miniprep kit to obtain sequencing quality DNA. Sequencing was done on an ABI 373 DNA Sequencing System using the Dye Terminator Cycle Sequencing it (Perkin-Elmner, Branchburg,

NJ).

Resulti 1) restriction ranalylsis.

Forty clones randomly picked from the recombined library were digested 'with restriction enzymes NheI and BamMHI. In a separate experiment the same forty plasmids wer-e digested with Hindu!l and BamHl. These reaction products were analyzed by gel electrophoresis. As shown in FIG. 7, 25 eight out of 40 clones (approximately 20%) contain the newly introduced NheI restriction site, demonstrating that the inutagenic primer has indeed been able to introduce the specified mutation into the population.

DNA sequence aznalysis The first ten randomly picked clones were subjected to sequence analysis, and the results are summarized in FIG. 8. A minimum of 6 out of S. 4 the 10 genes have undergone recombination. Among these 6 genes, the miinial crossover events (recombination) between genes RI and R2 vary from I to 4. AJI visible crossovers occurred within the region defined by the four primers. Mutations outside this region are rarely. if ever, recombined, as shown by the fact that there is no recombination between the two mutations at base positions 484 and 520. These results show that the defined primers can bias recombination so that it appears most often in the portion of the sequence defined by the primers (inside the primers). Mutations very close Mimosa 12.57:20 RECEIVED TIME 28. DEC. 17:07 PITTM 9 E. 61 PRINT TIME 29.DEC, 6:13 ZUUU 1:2 U SFRUSON FERGUSON 61 2 92615486 N0. 8897 P. SPRUSON FERGUSON WO 98/42832 PCT/US98/0S956 together also tend to remain together (for example, base substitutions 731 and 745 and base substitutions 1141 and 1153 always remain as a pair).

However, the sequence of clone 7 shows that two mutations as dclose as 33 bases apart can be reeombined (base position at 1107 and 1141).

s Twenty-three new point mutations were introduced in the ten genes during the process. This error rate of 0.23% corresponds to 2-3 new point mutations per gene, which is a rate that has been determined optimal for generating mutant libraries for directed enzyme evolution The mutation types are listed in Table 4. Mutations are mainly transitions and are evenly distributed along the gene.

TA3LE 4 New point mutations identified In ten recombined genes Transition requency Transversion Frequency A--T

I

A-G 4 A-C 1 C-T 3 C-A

I

T- C 5 C G 0 1 G G T 0 T- A 3 G 0 A total of 9860 bases were sequenced. The mutation rate was 0.23% s15 4 Phenotypic analysis Approximately 450 B. subrilis DB428 clones were picked and grown in SO nediurl n supplemented with 20 ug/rml kananmycin in 9 6 -well plates.

pproximately 56% of the clones expressed active enzymes. From previous experience, we know that this level of inactivation indicates a mutation rate on the order of 2-3 mutations per gene Approximately 5% clones showed S"double mutant (N181D+N218S).likc phenotypes (which is below the expected value for random recombination alone due primarily to point rnutagenesis). (DNA sequencing showed that two clones, 7 and 8, from the ten **-*.randomly picked clones contain both N218S and NII81D mutations.) 25 Mimosa 12:57:20 Mimosa 12:57:20 RECEIVED TIME 28. DEC, 17:07 PRINT TIME 29, DEC, 6 13 vt. Zb UUU I6 ZU 6FRUSON FERUMN b1l 2 92515486 NO. 8897 P. 31 SPRUSON FERGUSON WO 98/42832 PCT/US98/05956 EXAMPLZ 6 Optimizadon of the ActbwapkLrwns utahenzzis DCD deacylase by the xandom-pulming recombination metho4 In this example, the method is used to generate short DNA fragments from deniatured, linear, double- stranded DNA restriction fragments purified by gel electrophoresls; 22). The purified DNA, mixed with a Molar excess of primers, is denatured by boiling, and synthesis is then carried out using the Kienow fragment of E. coli DNA polymeraae IL This enzyme lacks exonuclease activity, so that the random priming product is synthesized exclusivrely by primer extension and is not degraded by exonuclease. The reaction is carried out at pH 6.6, where the exonuclease activity of the enzyme is much reduced These conditions favor random initiation of synthesis.

is The procedure involves the following steps: 1. Cleave the DNA of interest with appropriate restriction endonucleasels) and purify the DNA fragment of interest by gel electrophoresis using Wizard PCR Prep Kit (PromeMa Madison, WI). As an example, the Adinoplanes utahensis ECB deacylase genie was cleaved as a 2.4 kb-long Xho I-Psh Al fragment from the recombinant plasmid pSHP100. It was essential to linear-ize the DNA for the subsequent denaturation step. The fragment was purified by agarose gel electrophoresis using the Wizard PCR Prep Kit (Promega, Madison, WI) (FIG,9, step Get puriication was also essential in .order to remove the restriction endonuclease buffer from the DNA, since the 25 Mg 2 ions make it difficult to dcnature the DNA in the next step.

2. 400 ng (about 0.51 prnol) of the double-stranded DNA dissolved in H 2 0 was mixed with 2.75 pg (about 1.39 nrnol) of dp(N)6 random primers.

After immersion in boiling water for 3 minutes, the mixture was placed immediately in ani ice/ethaniol bath.

The size of the random priming products is an inverse function of the concentration of pimer The presence of high concentrations of primer is thought to lead to steric hindrance. Under the reaction conditions described here the random priming products are approximnately 200-400 bp, as determined by clectrophoresis through an alkaline agarose gel (FIG. 9 step b).

*S 3. Ten pi of 10 x reaction buffer flOX buffer- 900 mM HEPES, pH1 0.1 M magnesium chloride, 10 mnM dithiothreitol, and 5 mMd each dATP, dCTP, dGTP and dTTP) was added to the denatured sample, and the total volume of the reaction mixture was brought up to 95 sil wvith Mimosa 12:57:20 RECEIVED TIME 28. DEC. 17:07 PITTM 9 E. 61 PRINT TIME 29.DEC, 6:13 vt U. ZUUUId 1: ZI SFUUN FLEUSON b1a 2 Y26b485 NO. 8897 P. 32 SPRUSON FERGUSON WO 98/42632 PCTUS9SIOS9Sd /1 4. Ten units (about 5 i4) of the MGenow fragment of B&coli DNA polYmerase I was added. All the components werec mixed by gently tapping the outside of the tube and were centrifuiged at 12,000 g for 1-2 seconds in a JMCrOELuge to move all the liquid to the bottom. The reaction was caried out at 220C for 35 minutes.

The rate of the extension depends upon the concentrations of the template and the four nucleotide precursors. Because the reaction was carried out under conditions that minimize exonucleolytic digestion, the newly synthesized products were not degraded to a detectable extent.

S. After 35 minutes at 229C, the reaction was terminated by cooling the sample to 0 0 C on ice. 100 gil of ice-cold H20 W.as added to the reaction mixture.

6. The random primed products were purified by passing the whole reaction mixture through Celitricon-1OG (to remove the template and proteins) is and Ceritricon- 10 filters (to remove the primers and fragments less than bases). successively. Centricon filters are avaiable from Amnicon Inc (Berverly, MA). The retentate traction (about 85 pl in volume) was recovered from This fraction contained the desired random priming products a (FIG. 9, step c) and was used for whole gene reassembly.

Reassembly of the whole gene was accomplished by the following stps 1. For reassembly by PCR, 5 i of the random-primed

DNA

fragments from Centricon-lO, 20 iii of 2x PCR pre-mix (5-fold diluted cloned Pfu buffer, 0.5 mM each dNTP,*0.lU/.d cloned Pftu polymerase (Stratagene, La Jolla, 8 p1l of 30% glycerol anid 7 Pil Of H 2 0 were mixed on ice. Since the concentration of the random..pried DNA fragments used ror reassembly is the most important variable, it is useful to set up sevreral separate reactions with different concentrations to establish the preferrxed concentration.

2. After incubation at 96 0 C for 6 minutes, 40 thermocycles were performed, each with 1.5 minutes at 950C, 1.0 minutes at 559C and :minutes 5 second/cycle at 720C, with the extension step of the last cycle proceeding at 720C for 10 minutes, in a DNA Engine PTC-200 (MJ Research Inc., Watertown, MA) apparatus without adding any mineral oil.

3. 3 p1 aliquots at cycles 20, 30 and 40 were removed from the 35 reaction mixture and analyzed by agarose gel electrophoresi3. The *reassembled PCR product at 40 cycles contained the correct size product in a smear of larger and smaller sizes (see FIG. 9. step d).

Mimosa 12:57:20 RECEIVED TIME 28. DEC. 17:07 PITTM 9 E. 61 PRINT TIME 29. DEC, 6 13 vt Y ZHUU I1d: Z1 6RU60N HRUUSN 5Il 2 92615485 NO. 8897 P. 33 SPRUSON FERGUSON WO 99/42932 PCTIV/U98I5g6 The Correctly reassembled product Of this first PCR was further amplified in a second PCR reaction which contained the PCR primers complementary to the ends of the template DNA. The amplification procedure wag as follows: 51. 2.0 9iI of the PCR reassembly aliquots were used as template in 1OO-AI standard PCR reactions, which contained 0.2 =mM each primers of XhoF28 CQTAGAGCGAGTCTCGAGiGGGAGATC 3 (SEQ. ID, NO: 13) and pshR22 AGCCGGCGTGACOTGGGCAOC (SEQ. ID. NO: 14), 1.5 mM MgC1a, 10 mM Thia-HCl [pH 9.0J, 50 mM MCI, 200 jiM each of the four dNTP&, 6% glycerol, 2.5 U of Taq polymerase (Promega, Madison, WI) and 2.5 U of Pftt polyrnerase (S tratagene, La Jolla, CA).

2. After incubation at 96*C for 5 minutes, 15 therrnocycles were performed, each with 1.5 minutes at 95 0 C, 1-0 minutes at 5S0C and minutes at 72*C, followed by additional 15 thermocycles of 1.5 minutes at 9S'C, 1.0 miinutes at 550C and 1.5 minutes S second/cycle at 729 with the extcension step of the last cycle proceeding at 72*C for 10 minutes, in a DNA Engine PTC-200 (MJ Research Inc., Watertown, MA) apparatus without adding any mizneral oil.

3. The amnplification resulted in a large amount of PCR product with the correct size of the ECB deacylase whole gene (FIG. 9, step e).

Cloning was accomplished as follows: The PCR product of ECS deacylase gene was digested with )Clw I and .Psh Al restriction enzymes, and cloned into a modified pI, 17 02 vector.

2. S. lividans TK23 protoplasts were transformied with the above ligation mrixture to form a mutant library.

In itugcreenng th-e ECL3 deacvlase mutants Each transformant within the S. tividans TH23 library obtained as described above was screened for deacylase activity with an in situ plate assay :method using ECD as substrate. Tr'ansformed protoplasta were allowed to regenerate on R2YE agar plates by incubation at 30 0 C for 24 hours and to develop in the presence of thiostrepton for further 48-72 hours. When the colonies grew to proper size, 6 Ml of 451C purified-agarose (Sigma) solution 3s containing 0.5 mg/nil ECB in 0. 1 M sodiumi acetate buffer (pH 1 5.5) was S. poured on top of each R2YE-agar plate and allowed to further develop for 18- 24 hours at 370C. Colonies surrounded by a clearing zone larger than that of a control colony containing wild-type recombinant Plasraid pSHP1SO..2 were Mimosa 12:57:20 RECEIVED TIME 28,DEC. 17:07 RECEVED IME 8. DC. 1:07PRINT TIME 29. DEC. 6:12 Zb. DEU. -HUU 18:21 SPRUSON FERGUSON 61JA 2 92615486 NO. 8897 P. 34 SPRUSON FERGUSON WO 98/42832 P U9/55 PC14-eO~~ indicative of more efficient ECB hydrolysis resulting from improved en~yme properties or improved enzyme expression and secretion level, and were chosen as potential Positive mutants. These colonies were picked for subsequent preservation and manipulation.

NPFLC asegy of the F-CB deacylase mutants Single Positive tranhformants were inoculated into 20 ml fermentation medium containing 5 iig/mi thiostrepton and allowed to grow at 30 0 C for 48 hours. At this step, all cultures were subjected to IXPLC assay using ECB as substrate. 100 ;ii of whole broth was used for an HPLC reaction at 30-C for minutes in the presence of 0.1 M NaAc: (pH 10% (tr/v) NMeOH and 200 jig/mI of ECB substrate. 20 jil of each reaction mixture was loaded onto a PolyLC POlYhYdroxyethyl aspartamide columnn (4.6 x 100 mm) and cluted by acetorgtz-ile gradient at a flow rate of 2.2 mI/min. The ECB-nucleus was detected at 225 nm.

Prxnflcation heECBdaclsmtat After the HPLC assy, 2.0 ml pre-cultures of all potential positive mutants were then used to inoculate 50-mi fermentation medium and allowcd to grow at 300C, 280 rpm for 96 hours. These 50-mi cultures were then centrifuged at 7,000 g for 10 minutes. The supernatants were re-centrifuged at 16,000 g for 20 minutes. The supernatants containing the ECB deacylase mutant cnzymes were stored at -200C.

The supernaant from the positive mutants were further concentrated 9* 25 to 1/30 their original volume with an Arnicon filtration unit with molecular ***.weight cutoff of 10 kD. The resulting enzyme samples were diluted with an equal volume of 50 mM KH- 2 p0, (pH 6.0) buffer and 1.0 ml was applied to Hi- Trap ion exchange column. The binding buffer was 50 MM KH 2 P0 4 (pH1 and the elution buffer was 50 mM KH 2

PQ

4 (pHi 6.0) and 1.0 M Nad.- A linear gradient from 0 to 1.0 M NaCI was applied ini 8 column volumes with a flow rate of 2.7 mI/min. The ECB deacylaae mutant fraction eluted at 0.3 M NaCI .9 and was concentrated and buffer exchanged into 50 mM KH 2 PO4 (pH 6.0) in Ainicon Centricon-10 units. Enzyme purity was verified by SDS-PAGE, and the concentration was determined using the Blo-Rad Protein Assay.

9. jug of each purified ECB deacylase mutant was used for the activity assay at 3000 for 0-60 minutes in the presence of 0.1 M NaAc (pH Mimosa 12:57:20 RECEIVED TIME 28. DEC. 17:07 PITTM 9 E. 61 PRINT TIME 29, DEC. 6 12 Hb J~,2UU 18 :22 SPRUSON FERGUSON 61' 2 92615486 NO. 8897 P. SPRUSON FERGUSON WO 90/42832 C/z9/ss MeOH and 200 Mig/ml of ECB substrate. 20 ol of each reaction mixture was loaded onto a PolyLC polyhydroxyethyl aspartamide column (4.6j x 100 Mm) and eluted with an acetonitrile gradient at a flow rate of 2.2 mI/mim. The reaction products were monitored at 225 nsm and recorded On an IBM PC data acquisition system. The ECB nucleus peak wase numerically integrated and used to calculate the specific activity of each mutan.

As shown in FIG. 10, after only one round of applying this randompriming based technique on the wild-type ECB deacylase gene, one mutant (M16) from 2,012 original transfomants was found to possess 2.4 tines the specific activity of the wild-type enzyme. FIG I1I shows that the activity of M1 6 has been increased relative to that of the wild-type enzyme over a broad pH range.

EXAMIPLE 7 Improving the thermostability Ba cillus subtflis subtfisj 3 using the random-sequence primer recoahbinatjan method This example demonstrates the use of various DNA polyrnerases for primer-based recomnbination. It fur-ther demoxnstrates the stabilization of subtilisin E by recombination.

Genes RI and R2 encoding the two thermostable subtilisin E variants described in Example 1 were chosen as the templates for recombination.

Target gene preparation Subtilisin E therniostable mutant genes Rl and R2 (FIG-1 1) were subjected to random primed DNA synthesis. The 986-bp fragment including 4S nt of subtilisin E prosequence, the entire mature sequence and 113 nt after the stop codon were obtained by double digestion of plasmid pBE3 with Sarm Hl and Nde I and purified from a 0.8% agarose gcl using the Wizard PCR Prep Kit (Promega, Madison, WI). It was essential to linearize the DNA for the subsequent denaturation step. Gel purification was also essential in order to :remove the restriction endonuclease buffer from the DNA, since the Mg 2 ions make it difficult to denature the DNA in the next step, Random prirned DNA synthesis Random primed DNA synthesis used to generate short DNA fragments ~from denatured, linear, double-stranded DNA. The purified subtilis subtiuisin E rnutant genes, mixed with a molar excess of primrers, were denatured by boiling, and synthesis was then cax-zied out using one of the Mimosa 12:57:20 RECEIVED TIME 28.DEC. 17:07 RECEVED IME 8. DC. 1:07PRINT TIME 29. DEC. 6:12 Zd UUU 18 :22 SPRUSON FERGUSON 61A 2 92615486 NO. 8897 P. 36 SPRUSON FERGUSON WO 98142932 C S9/56 following DNA polymerases: the Kienow fragment of coli DNA polymerase 1, bacteriophage T4 DNA polymerase and V1 sequenaae version 2.0 DNA polymerase.

Under its optimal performance conditions bae riophage T4 DNA 3 polymerase gives similar synthesis results as the Ki3enow fragment does. When '17 sequenase version 2.0 DNA polymerase (31, 32) is used, the lengths of the synthesized DNA fragments are usually larger. Some &Mount Of MnCI 2 has to be included during the synthesis in order to control the lengths of the synthesized fragments within 50-400 bases.

Short, nascent DNA fragments can also be generated with PCR using the Stoffel fragment of Tagq DNA polymerasne or JPfi DNA polymerase. Ana important consideration is to identify by routine experimentation the reaction conditions which ensure that the short random primers can anneal to the templates and give sufficient DNA amplification at higher termperatures. We is have found that random primcrs; as short as dp(N) 12 can be used with PCR to generate fragments.

2.1 Random primed DNA synthesis with the Klenow fragment The Kienow fragment of E. coli DNA polyrneraze I lacks exonuclease activity, so that the random priming product is synthcsized exclusively by primer extension and is not degraded by exonuclease- The reaction was carried out at pH 6.6, w~here the exonuclease activity of the enzyme is much reduced These conditions favor random initiation of synthesis.

25 1. 200 ng (about 0.7 pnmol) of RI DNA anid equal amnount of R2 DNA .*..dissolved in H20 was mixed with 13.25 jig (about 6.7 nmoI) of dp(N) 6 random primers. After ixnzcrsion in boiling water for 5 minutes, the mixture was placed immediately in an ice/ethanol bath.

The -size of the random priming products is an inverse function of the concentration of primer The presence of high concentrations of primer iii :thought to lead to steric hindrance. Under the reaction conditions described here the random priming products are approximately SO-SOO bp, as determined by agarose gel electrophoresis.

2- Ten P1 of 10 x reaction buffer [lOx buffer: 900 mM HEPES, pH 6.6; 0.1 M magnesium chloride, 20 mM dithiothreitoi, and 5 mM each dA'rF, dCTP, a a dOT? and dll'P) wan added to the denatured sample, and the total volume of the reaction mixture was brought up to 95jul with Mimosa 12;57:20 RECEIVED TIME 28, DEC. 17:07 PITTM 9 E. 61 PRINT TIME 29.DEC, 6:12 28. DEC. 2000 18:22 SPRUSON FERGUSON 612 292615486 N.89 .3 SPRUSON FERGUSON WO 92142832 3. Ten units (about 5 pl) of the iKlenow fragment of Zcoli DNA polymerase I (Doehringer Mannheim, Indianapolis, IN) wag added. All the components were mixed by gently tapping the outside of the tube and were centrifuged at 12,000 g for 1-2 seconds in Q microfuge to move all the liquid to the bottom- The reaction was carried out at 22-C for .3 hours.

The rate of the extension depends upon the concentrations of the template and the four nucleotide precursors. Because the reaction was Carried out under conditions that minimize exonucleolytic digestion, the newly synthesized products were not degraded to a detectable extent.

4. After 3 hours at 22 9 C, the reaction was terminated by cooling the sample to 0IC on ice. 100 Ul Of ice-COldI

H

2 0 Was added to the reaction tnixture.

The random primed products were purified by passing the whole reaction mixture through Mierocon- 100 (Axnicon, Beverly MA) (to remove the template and proteins) and Microcon..10 filters (to remove the primers and fragments less than 40 bases). successively. The retentate fraction (about iii in volumue) was recovered from the Microcon-lO. This fraction containing the desired random priming products was buffer-exchlanged against PCR reaction buffer with the ncw Microcon-1O further use in whole gene reassembly.

2.2 Random primed DNA synthesis with bacteriophage T4 DNA polymerase Bacteriophage T4 DNA polymerase and the Kienow fragment of Rcoli DNA polynierase I are similar in that each possesses a polyrnerase activity and a 3-5' exonuclease activity. The exonucleases activity of 2S bacteriophage T4 DNA polymerase is more than 200 times that of the Kienow fragment. Since it does not displace the short oligonucleotide primers from 3ingle-strailded DNA templates the efficiency of mutagenesia is different from the Kienaw fragment.

1 200 ng (about 0.7 pmol) of R1 DNA and equal amount or R2 DNA 30 dissolvedj in 1- 2 0 was mixed with 13.25 u~g (about 6.7 nmol) of dp(N) 6 random primers. After immersion in boiling water for 5 minutes, the mixture was placed imm~ediately irt an ice/ethanol bath. The presence of high concentra-.

tions of primer is thought to lead to steric hindrance.

2- Ten jii of 10 xc reaction buffer [lOx buffer. 50O mM Tris-MCI, pH 8-8; 150 mM (NH4)2So 4 70 mM magnesium chloride, 100 mM 2 -niercaptoethanol, 0.2 mg/ml bovine serum albumin and 2 mM each dATP, dCTP, dGTP and dTTP) wvas added to the denatured sample, and the total volume of the reaction mixture was brought up to 90 pal with Mimosa 12:57:20 RECEIVED TIME 28,DEC. 17:07 RECEVED IME 8. DC. 1:07PRINT TIME 29. DEC. 6:12 V UUU 18:22 SPRUSON FERGUSON 612 292615486 NO. 8897 P. 38 SPRUSON FERGUSON WO 96/42a32 PCT/US9we595 3. Tn uits(abut 1 jA oftheT4 DNA polyrnerase I (Boeliringer Msarihein, Indianapolis, IN) Was added. All the components were mixed by gently tapping the outside of the tube and were centrifuged at 12,000 g for 1-2 seconds in a microtuge to move anl the liquid to the bottom. The reaction was carried out~ at 37gC for 30 minutes. Under the reaction conditions decrizbed here the randor. priming products ame approximately 50-500 bp.

4. After 30 minujtes at 37 0 C, the reaction was terminated by cooling the sample to 0'C on ice- 100 jil of ice-cold H20 was added to the reaction mixtuire.

5. The random primed products were purified by passing the whole reaction mixture through Microcon- 100 (to remove the template and proteins) arid Microcon- 10 filters (to remove the primers and fragments less than bases), successively. The reteritate fraction (about 65 jAI in volume) was recovered from the Microcon-10. This fraction containing the desired raendom priming products was buffer-exchanged against PCR reaction buffer with the new Microcon-10 further use in whole gene reassembly.

2.3 Random primed DNA synthesis with the 17 sequenase v2.0 DN~A polymnerase Since the 17 sequenase v2.0 DNA polyrnerase lacks exonuclease activity and is highly processive, the average length of DNA synthesized is greater than that of DNAs synthlesized by the Kienow fragment or T4 DNA polymerase. But in the presence of proper amount of MnC12 in the reaction, thesiz ofthesynthesized fragments can be controlled to less than 400 bps.

2S 1. 200 ng (about 0-7 pnnol) of RI DNA and equal amount of R2 DNA dissolved in H 2 0 was mixed with 13.25 jug (about 6.7 nmol) of dp(N) 6 random primers. After immersion in boiling water for 5 minutes, the mixture was placed immediately in an ice/ethanol bath- The presence of high concentrations of primer is thought to lead to steric hindrance.

2. Ten pl. of 10 x reaction buffer [1OX buffer: 400 mM Tris-HCI, pHI :200 mMW magnesium chloride, 500 mM NaCi, 3 mM MnCl 2 and 3 m?4 each dATP, dCTP, dGTP and dTTP) was added to the denatured sample, and the total volume of the reaction mixture was brought up to 99.2 p1 with H 2 0.

3. Ten units (about 0.8 jil) of the 17 Sequenase v2.0 (Amersham Wfe 35 Science, Cleveland, Ohio) was added. All the components were mixed by gently tapping the outside of the tube and were centrifu.ged at 12,000 g for 1-2 seconds in a microfuge to move all the liquid to the bottom. The reaction was Mimosa 12:57:20 RECEIVED TIME 28.DEC. 17:07 RECEVED IME 8. DC. 1:D7PRINT TIME 29. DEC. 6:12 2b. DEC. 200 18:23 SPRUSON FERGUSON 61 2 92615486 NO. 8897 P. 39 SPRUSON FERGUSON WO 99/42s32 carried out at 22&C for 15 mainutes. Tinder the reaction conditions described here the random priming products are approximately 50-400 bps.

4. Alter 15 nminutes at 220C, the reaction was terminated by cooling the sample to 0 0 C on ice. 100 wl of ice-cold H 2 0 WAS added to the reaction mixture.

The random primed products were purified by pasaing the whole reaction mixtu~re through Microcon-100 (to remove the template and proteins) and Microcon-1 0 filter-s (to remove the primers and fragments less than bases), successively. The retentate fraction (about 65 Pal in volume) was recovered from the Microcon-10. This fraction containing the desired random priming products was buffer-exchanged against PCR reaction buffer with the new Microcort- 10 further use in whole gene reassembly.

2.4 Random primed DNA synthesis with PCR using the Stoffel fragment of is Taq DNA polymerase Similar to the Kienow fragment of E. coli DNA polymnerase 1, the Stoffel fragment of Taq DNA polymerase lacks S' to 3' exonuclease activity. It is also more therrnostable than Taq DNA polyrnerasc. The Stoffel fragment has low processivity, extending a primer an average of only 5-10 nucleotides before it dissociates- As a result of its lower procesivity, it may also have improved fidelity.

1. 50 ng (about 0.175 psuol) of R1 DNA and equal amount of R.2 DNA dissolved in H 2 0 Was mixed with 6.13 tg (about 1.7 nmol) of dp(N) 12 random 0*0 plrmers.

25 2. Ten ml of lOx reaction pre-mix [lOx reaction pre-mix: 100 mM Tris-HCI, pH 8.3; 30 raM ma,6nesium chloridc, 100 rnM KCI, and 2 mM each dATP, dCTP, dGTP and dTTP) was added, and the total volume of the reaction mixture was brought up to 99.0 gl with H 2 0.

3. After incubation at 96*C for 5 minutes, 2.5 units (about 1.0 of the Stoffel fragment of Tag DNA polymerase (Perkin-Elrner Corp., Norwalk, C'1) was added. Thirty-five thermnocycles were performed, each with 60 seconds at 00 951C, 60 seconds at 55 0 C and S0 seconds at 72 0 C, without the extension step 0*0***of the last cycle, in a DNA Engine PTC-200 (MJ R~esearch Inc., Watertown,

MA)

apparatus. Under the reaction conditions described here the random priming 0* 35 products are approximately 50-500 bp.

4. The reaction was terminated by cooling the sample to 0*C on ice. 100 pil *0 of ice-cold H20 was added to the reaction mixture.

Mimosa 12:57:20 RECEIVED TIME 28.DEC. 17:07 RECEVED IME 8. DC. 1:07PRINT TIME 29. DEC. 6:12 0- ut ZHU I d J 6FRUSON FERGUSON 51a 2 92615486 SPRUSON FERGUSON NO. 8897 P. 40 WO 99/42832 PCT/1S98/o595M The r'andom Primed products were Purified by passing the whole reaction Ixture through hfcocon-1C0 (to remove the template and proteins) and Microcon-10 filters (to remove the primers and fragments less than bases). suZccesively. The retentate fraction (about 65 pl in volume) was recovered from the Microcon-10. Thia fraction containing the desired4 random Priming products was buffer-exchnged against PCR reaction buffer Vwith the new Microcon-10 further use in whole gene reassem~bly.

Random primed DNA synthesis with PCR using P114 DNA polymeras 10Pfi4 DNA polymerase is extremely thermnostable, and the enzymue possesses an inherent 3' to 5' exonuclease activity but does not possess a exonuclease activity. its base substitution fidelity has9 been estimiaed dbe 2 x 10-6.

1. 50 ng (about 0.17S pinol) of R1 DNA and equal amount of R2 DNA .13 dissolved in 140wa mnixed with 6.13mg (about 1.7 nrnol) of dp(N) 12 random primecrs.

2. PiftY Jul of 2 X reaction Pre-nux (2 x; reaction pre-znax: 5-fold diluted cloned Pfu buffer (Stratagene, L~a Jolla, CA), 0.4 mM each dNTP], was added.

and the total volume of the reaction mixturc wae brought up to 99.0 ji tvith a a a a a.

a.

220 a a a.

a a.

a.

a. a.

3. After incu~bation at 960C for 5 minutes, 2.5 units (about 1.0 pl) Of pjfu DNA Polymnerase (Stratagene, La Jolla, CA) was added. Thirty-five the,,,ocycles were Performed, each with 60 seconds at 95 0 C, 60 seconds at 55-C and 50 seconds at 72-C, Without the extension step of the last cycle, in a DNA 25 Engie 1'rC-200 (MJ Research Inc., Watertown. MA) apparatus. Under the reaction conditions described here the major random priming products are approximately 50-500 bp.

4. The reaction was terminated by cooling the sample to O'C on ice. 100 ji of ice-cold

H

2 0 was added to the reaction mixture, 30 5. The random primed products were purified by passing the whole reaction mixture through Microcon-00 (to remove the template and proteins) and Microcon.10 filters (to remove the primers and fragmients less than bases), successively. The retentate fraction (about 65 jil in volume) was recovered from the Microcon. 10. This fraction containing the desired random 35 priming products was buffer-exchanged against PCR reaction buffer with the new Mirrocon- 10 further use in whole gene reassembly, Mimosa 12:57:20 RECEIVED TIME 28.DEC. 17:07 RECEVED IME 8. DC. 1:07PRINT TIME 29. DEC. 6:12 .tZuuu 16: 3 SPRUSON FERGUSON 61A 2 92615486 NO. 8897 P. 41 SPRUSON FERGUSON WO 98/42332 PCT/USqaO59q% Reassembly of the whole gene 1 For reassembly by PCR, 10 W. of the randomn-primxed DNA fragments from. Micrecon-lO, 20 p1I of 2 X PCR pre-mix (S-fold diluted cloned Pfu buffer, mM each dNTIp, 0. IU/g.d cloned Pfiu polymerase (Stratagene, La Jolla, CA)), 15 p1l of H20 were mixed on ice.

2. After incubation at 96'C for 3 minutes, 40 therrnocycles were perfornied, each with 1.0 minute at 95'C. 1.0 minute at 55*C and 1.0 mirlute 5 second/cycle at 720C, with the extension step of the last cycle proceeding at 72*C for 10 minutes, in a DNA Engiric PTC-200 (MJ Research Inc., Watertown, MA) apparatus without adding any mineral oil.

3. 3 p1 allquots at cycles 20, 30 and 40 were removed from the reaction rnixturc and analyzed by agarose gel electrzophoresis. The reassembled

PCR

product at 40 cycles coritaincd the corre-ct size product in a Syncar or larger and smaller sizes.

Amplification The correctly reassemnblcd product of this first PCR wats further ampifedina ecndPCR reaction which contained the PCR primers cormplementaxy to the ends of the template DNA.

1. 2.0 pl of the PCR reassembly aliquots were used as template in 1OO-pl standard PCR reactions, which contained 0.3 MM each primers of P1 (S' 9CCGAGCGrrGC ATATGTGGAAG (SEQ. ID. NO.- 15) and P2 CGACTCTAGAGGATCCOA.IC 3-1 (SEQ. ID. NO: 16), 1.S mM MgCI 2 10 mM Tris-HC1 [pH 9.01, 50 raM KCI, 200 MM each of the four dNTPs, 2.5 u of Taq 25 polymerasc (Prornega, Madison, WI, USA) and 2.5 .U of Pfu polyrrerasc (Stratagenc, LaJolla, CA).

2. After incubation at 96*C for 3 minutes, 15 thermocycles were performed, each with 60 seconds at 959C, 60 seconds at 55*C and 50 seconds at 72'C, followed by additional 15 therinocycles of 60 seconds at 95*C, 9*9 30 seconds at 551C and 50 seconds 5 second/cycle) at 72'C with the extension :step of the last. cycle proceeding at 72*C for 10 minutes, in a DNA Engine PTC- 200 (MJ Research Inc-, Water-town, MA) apparatus without adding any mineral oil.

3. The amplification resulted in a large amount of PCR product with the 35 correct size of the subtilisin E whole gene.

Cloning Mimosa 12:57:20 RECEIVED TIME 28,DEC. 17:07 RECEVED IME 8. DC. 1:07PRINT TIME 29. DEC. 6:12 Mu. Iu du 1: J VFRU60N ftfiUSN b1 5 2 926lb485 NO. 8897 P, 42 SPRUSON

FERGUSON

WO 98/42832 PCT/US98/O5956 317 Since the short DNA fr-agments were generated with five different

DNA

polymerases, there were five Pools of final PCR amnplified reassembled Produ~cts. Each of the DNA pool was used for constructing the Corresponding atzbtiljsjn E mutant library.

1. The PCR amplified reassembled product was purified by Wizard DNA.

CleanUp kit (Promega, Madison, WI), digested with Bin HI and Nde 1, electrophoresed in a 0.8% agarose gel. The 986-bp product was cut frorm the gel and purified by Wizard PCR Prep ldt (Prornega, Madison, WI). Products were ligated with vector generated by Brn HI-Nde I digestion of the pBE3 shuttle vector.

2. E. MRl HB 10 competent cells were transformed with the above ligation Mixture to form a mutant library. About 4,000 transfornants from this library were pooled, and recombinant plasmid mixture was isolated from this pool.

3. subtifhs DB428 competent cells were transformed with the above isolated plasrnid mixture to form another library of the subtilisin E variants.

4. Based on the DNA polymernec used for random priming the short, nascent DNA fragments, the five libraries constructed here were named, library/MKenow, library/T4, library/Sequenase, library/Stoffel and librai-y/ Pfu.

About 400 tranformants from each library were randomly picked and subjected to screening for thermostability [see Step Random clone sequencfng Ten random clones fromn the B. subtirfs DB428 library/Kienow was chosen for DNA sequence analysis. Recombinant plasmids were individually 25 purified from B. subtilis D13428 using a QlAprep spin plasmid roiniprep it *.*.(QIAGEN) with the modification that 2 mg/mIl lysozyme was added to P1 buffer and the cells were incubated for 5 minutes at 37*C, retransforned into competent E- coi HB 1101 and then purified again using QiAprep spin plasm-id mnriprep kit to obtain sequencing quality DNA. Sequencing was done on an 30 ABI 373 DNA Sequencing System using the Dye Termuinator Cycle Sequencing kit (Perkin-Elmer Corp., Norwalk,

CT).

Screening for thermostabilfty About 400 transfornants; from each of the five libraries described at 5 Step were subjected to screening. Screening was based on the assay *described previously (33. 35), using sucnlAaAaPoPepntonld ID. NO- 25) as substrate. R. aubtilis DB428 containing the plasmid library were grown on LB/kanamycin (20 lig/m) plates. After 18 hours at Mimosa 12:57:20 RECEIVED TIME 28.DEC. 17:07 RECEVED IME 8. DC. 1:G7PRINT TIME 29. DEC. 6:12 0. VUM ZUUU I1d: Z4 FXUSON FERGUSON 51'?2 92615486 NO, 8897 P. 43 SPRUSON FERGUSON WO 98/42832 PCTR.1698,956 37*C single colonies were picked into 96-well plates containing 100 Isl SO/kanarnycin medium per well. These plates were shaken and incubated at 370C for 24 hours to let the cells to grow to saturation. The cells were spun down, and the superniatants were sampled for the thermnostabilit y assay.

Three replica 96-well assay plates were duplicated for each growth plate, with each well containing 10 ML of supernatant- The subtilisin activities were then measured by adding 1.00 ml of activity assay solution (0.2 mM succinyl-Aja.- Ala-Pro-Phe-p-nitroanilide (SEQ. ID. NO; 25), 100 mM Tris-HCI, 10 mM CadJ 2 pH 8.0, 37 Reaction velocities were measured at 405 nm over 1.0 min. in a ThermoMax microplate reader (Molecular Devices, Sunnyvale CA). Activity measured at room temiperature was used to calculate the fraction of active clones (clones with activity less than 10% of that of wild type were scored as inactive). Initial activity (Ai) was measured after incubating one assay plate at 650C for 10 minutes by immediately adding 100 jil of prevarined (370C) assay is solution (0.2mM succinyl-Ala-AIa.Pro-Ph'e-p-ntoanilide (SEQ. ID. N~O: 100 mM Tris-HCI, pHI 8.0, 10 mM CaCd 2 into each well. Residual activity (Ar) was measured after 40 minute incubation.

Sequence Analysis After screening, one clone that showed the highest thermostability es within the 400 transformants from the library/IKlenow was re-streaked on LB/kananycin agar plate, and single colonies derived from this plate were 9. inoculated into tube cultures, for glycerol stock and plasmid preparation. The too recombinant plasinid was purified using a QlAprep spin plasmid miniprep kit 00 25 (QIAGEN) with the modification that 2 mg/nil lysozyme was added to P1 buffer *too and the cells were incubated for S minutes at 37*C, retransformed into competent E. coli HR 10 1 and then purified again using QlAprep spin plasmid to..

0 miniprep kit to obtain sequencing quality DNA. Sequencing was done on an AB! 373 DNA Sequencing System using the Dye Termiinator Cycle Sequencing kit (Perkin-Elmer Corp., Norwalk, CT).

Results 0 1. Recomnbination frequency and efficiency associated with the randomsequence recombination.

35 The random primed process was carried out as described above. The 0O*.process is illustrated in FIG. 1. Ten clones from the mutant library/Kienow were selected at random and sequaenced. As aumnmarized ini FIG. 12 and Table S, allJ clones were different from the parent genes. The frequency of occurrence Mimosa 12:57:20 RECEIVED TIME 28,DEC. 17:07 RECEVED IME 8. DC. l:D7PRINT TIME 29. DEC. 6:11 a 3 To, transversion. Some nucleotides are more mutablc than others. One one and one C-IA transversjons were found within the 10 sequenced clones.

These mutations were generated very rarely during the error-prone

PCR

mutagenesis of subtilisin Random-~priming process may allow access to a greater range of amino acid substituitions than PCR-based point mutagenesis- It Is interesting to note that a short stretch of S' C GOT ACO CAT GTA GCC GGT ACO 3' (SEQ. ID. NO: 16) at the position 646-667 in parents R1 and is R2 was mutated to 5' C GOT ACO AlT GCC 0CC GG? ACG 3' (SEQ. ID. NO- 17) in random clonc C#6. Since the stretch contains two short repeats at the both ends, the newly introduced mutations may result from a uplipped-st-and raispairing process instead of point-mutation only process. Since there is nio frame-shift, this ldrnd of slippage may be usefuil for domain conversion.

Mimosa 12:57:20 RECEIVED TIME 23. DEC. 17:07 PRINT TIME 29. DEC. 6:11, ZUUU I d: 24 'SFRUSON FEGUSON 51A 2 92515486 NO. 8897 P. 44 SPRUSON FERGUSON WO 99/42032 PCSI956% of a particular point mutation from parfnt R1 or R2 in the recombined genes ranged from 40% to 70%, fluctuating around the expected value of 50O/6- This indicates that the two parent genes have been nearly randomly recombined with the random primer technique. FIG. 12 also shows that all ten mutations s can be recombined or dissected, even those that are only 12 bp apart.

We then estimated the rates of subtiniz thermoinactivation at 650C by analyzing the 400 random clones from each of the five libraries constructed at Step The thermostabilities obtained from one 96-well plate are shown in F1O.13, plotted in descending order. Approximately 21% of the clones exhibited ther-nostability comparable to the mutant with the N181D and N218S double mutations. This indicates that the NISID mutation from lRC2 and the N216S mutation fromt RCI have been randomly recombined.

Sequence analysis of the clone exhibiting the highest thermostability among the screened 400 transiformants from the library/Kienow showed the mutation is 181D and N218S did exist.

2. Frequency of newly introduced mutations during the random priming process Approximately 400 transformants from each of the five B.su~blilits DS428 libraries [see Step (Sfl were picked, grown in SG medium supplemented with 20 ug/mi Icanoxnyciri in 96-well plates and subjected to subtilisin E activity screening. Approximately 77-84% of the clones expressed ~.active enzymes, while 16-23% of the transformants were inactive, presumably as a result of newly introduced mutations. From previous experience, we 2 r know that this rate of inactivation indicates a mutation rate on the order of 1 to 2 mutations per gene As shown in FIG. 12, 15 new point mutations were introduced in the process. Thiu error rate of 0.18% corresponds to 1-2 new point mutations per gene, which is a rate that has been determined from thr- inactivation curve.

Mutations are nearly randomly distributed along the gene.

trnvrso. Soeceitte r Page(s) were not lodged with this application Zb. EC. 2000 18:24 SPRUSON FERGUSON 61 A 2 92615486 NO. 8897 P. SPRUSON FERGUSON WO 99/42M32 PC'FPJ9593 956 3. Comparison of different DNA polymnerase fidelity in the random-priming process During random-priming recombinatioN, homologous DNA sequences are nearly randomly recombined and nlew point mutations are also introduced. Though these point mutations may provide useful diversity ror some in utr evolution applications, they are problematic recombination of beneficial mutations already identified previously, especially when the mutation rate is this high. Controlling error rate during random priming process is particularly important for successfully applying this technique to solve in s'iio evolution problems. By choosing different DNA polymerase and modifying the reaction conditions, the random priming molecular breeding technique can be adjusted to generate mutant libraries with different error rates.

The Kienow fragment of B.coli DNA polymerase J, bacteriophagc T4 DNA is polymerase, T7 secjucnase version 2.0 DNA polymerase, the Stoffel fragment of Taq polymerase and Pfu polymerase have been tested for the nascent DNA fragment synthesis. The activity profiles of the resulting five populations [see Step are shown in FIG. 13. To generate these profiles, activities of the individual clones measured in the 96-well plate screening assay are plotted in 2D descending order. The Library/Stoffel and Library/Kienow contain higher percentage of wild-type or inactive subtilisin E clones than that of the Library/Pfu. In all five populations, percentage of the wild-type and inactive clones ranges from 17-30%, EXAMPLE S Use of defined flanking primers and staggered extension to recombine single stranded DNA This example demonstrates the use of the defined primer recombination with staggered extension in the recombination of single stranded DNA.

:Method Description Single-stranded DNA can be prepared by a variety of methods, most easily from plasmids using helper phage. Many vectors in current use are derived from filamentous phages, such as M13znp derivatives. After **35 transformation into cells, these vectors can give rise both to a new doublestranded circles and to a single-stranded circles derived from one of the two strands of the vector. Single-stranided circles are packaged into phage particles, secreted from cells and can be easily purified from the culture supernatant Mimosa 12:67:20 RECEIVED TIME 28. DEC. 17:07 PITTM 9 E. 61 PRINT TIME 29.DEC. 6:11 28. DEC. 2000 18:25 SPRUSON FERGUSON 61 12 292615486 NO. 8897 P. 46 SPRUSON FERGUSON WO 93/42932 PT)9/55 Two defined primers (for example, hybridizing to 5' and 3' ends of the templates) are used here to recombine singe stranded genes. Only one of the primers is needed before the final PCR amplification. Extended recombination primers are first generated by the staggered extension process (StEP), which consists of repeating cycles of denaturaion followed by extemely abbreviated annealing/extension step(s). The extended fragments are then reassembled into full-length genes by therrnocycling-assisted homologous gene assembly in the presence of a DNA polym er-ae, followed by a gene amplification step.

The progress of the staggered extension process is monitored by remnoving aliquots (10 ul) from the reaction tube (100 ul starting volume) at various time points in the primer extension and separating DNA fragments by agarosa gel electrophoresis. P-vidence of effective primer extension is seen as appearance of a low molecular weight 'smear' early in the process which increases in molecular weight with increasing cycle number. Initial reaction is conditions are set to allow template denaturation (for example, 94*C-30 second denaturation) followed by very brief annealing/ extension step(s) (e.g.

55'C-1 to 15 seconds) repeated through 5-20 cycle increments prior to reaction sampling. Typically, 20-200 cycles of staggered extension are required to generate single stranded DNA 'smne&rB' corresponding to sizes 2o greater than the length of the complete gene.

The experimental design is as in Example 1. T1wo thermostable subtijisin E mutants Ri and R2 gene are subcloned into vector Ml3mpl8 by restriction digestion with EcoRi and BamH1. Single stranded DNrA is prepared as dlescribed (39).

T'woflanking prfmer based recombination Two defined primers, P5N (5'-CCGAG CQTTG TAT GTaGA AG-3' (SEQ. ID. NO: 18), underlined sequence is Ndel restriction Site) and P3B CGACT CTA GA GQGATC CGAIT C-3' (SEQ. ID. NO: 19), underlined sequence is 30 5amHI restriction site), corresponding to 5' and 3' flanking primers, respectively, are used for recombination. Conditions (100 ul final vohine): a. *.0.15 pmol single-stranded DNA containing RI and R2 gene (mixed at 1:1) are used as template, 15 pmaol of one flanking primer (either PSN or P35), Ix Taq buffer, 0.2 rnM of each dM11'. 1.5 MM MgC12 and 0.25 L3 Tag polyrnerase.

3.9 Pr ogram: 5 muinutes of 951C, 80-200 cycles of 30 seconds at 94*C, 5 seconds .at 55*C. The single-stranded DNA products of correct size (approximately 1kb) are cut from 0,8% agaro3e gel after electrophoresis and purified using QIAEX 11 gel extraction kit. This purified product is amnplified by a Mimosa 12:57:20 RECEIVED TIME 28.DEC, 17:07 RECEVED IME 8. DC. 1:07PRINT TIME 29. DEC. 6:11 Zb. I) L. UUU 18:25 SPRUSON FERGUSON 61 A 2 92615486 NO. 8897 P. 47 SPRUSON

FERGUSON

WO OW42832 r/9WSG conventional PCR. Condition (100 u, final volime): 1-10 ng of template, 17mo1 Of each flanldng primer, Ix Taq buffer, 0.2 mM of each dNTP, 1.5 mm M9Cb2 an~d 0.25 U Taq polymerase. Program: 5 minutes at 95*C, 20 cycles of seconds at 940C, 30 seconds at 550C, 1 minute at 72 0 C. The PCR product is purified, digested with Ndel and Samnfil and subeloned into pBE3 shuttle vector. This gene library is amplified in F_ coil HB101 and transferred into B.

.suhis DB428 competent cells for expression and screening, as described elsewhere Thermostability of enzyme variants is determined in the 96- Well plate format described previously (33).

This pr-otocol resulits in the generation of novel sequences containin~g novel combinations of mutations from the parental sequences as well as novel point mutations. Screening allows the identification of enzyme variants that are more thermostable than the parent enzymes, as in Examnple 1.

As Is apparent from the above examples. primer-based recombination may be used to explore the vast space of potentially useful catalysts for theiroptimal performance in a wide range of applications as well as to develop or evolve new enzymes for basic structure-function studies.

While the present specification describes using DNA-dependent

DNA

polymerase and single-stranded DNA as temrplates, alternative protocols Eire also feasible for using sigesrne RNA as a template. By using specific protein mRNA astetemplate and RNA-dependent DNA polymerase (reverse transcriptase) as the catalyst, the methods described herein may be modified to introduce mutations and crossovers into cDNA clones and to create molecular diversity directly from the mRIJA level to achieve the goal of 5 optimizing protein functions. This would greatly simplify the ETS (expressiontagged strategy) for novel catalyst discovery.

In addition to the above, the present invention is also useful to probe proteins from obligate intracellular pathogens or other systems where cells of interest cannot be propagated (38).

Having thus described exemplary embodimnents of the present inven- .:tion, it should be noted by those skilled in the art that the within disclosuires are exemplary only and that various other alternatives, adaptations, and modifications may be made within the scope of the present inlvention- Accordingly, the present invention is not limited to the specific embodiments 35 as illustrated herein, but is only limited by the following claims.

Mimosa 12:57:2D RECEIVED TIME 28. DEC. 17:07PITTIE 2.DE. 61 PRINT TIME 29.DEC, 6:11 28. DEC. 2Q00 18:25 SPRUSON FERGUSON 61' 2 92615486 NO. 8897 P. 48 SPRUSON FERGUSON WO 98/4232 PCT/US98,o5956

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WO 98/42832 PTU9/59 18. Stefmmer, W. P. C. 1994b. DNA shuffing by random fragmentation anti reassembly -in vitro recombination for molecular evolution. Proc. Nail.

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Moore, J. H-M. Jin, 0. Kuchner and F. H. Arnold. 1997. Strategies for the in vitro Evolution of Protein Function: Enzyme Evolution by Rand~rn-corbination of Emproved Sequences,j. Molecular Biology, in 21. 4enow. H. and I. lKenningsen. 1970. Selective elimination of the exoriliclease activity of the deoxyribonucleic acid polymerase from Escherichis coli B by limited proteolysis. Proc. Nat]. Acad. Sci, 65:168 22. Feinberg, A. and Vogeistein, a. 1983. A technique for radiolabeiing DNA restii cdt endonuclease fragments to high specific activity. Anal.

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Genr-at~ion of large libraries of random mutants in Bacillus subtilis by PCR-based plasinid multimerization. Biotechniques, in press.

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Innis, M. A. et al., 1988, Proc. Nat]. Acad. Sci. 85.9436-9440.

*0 .0 0 Mimosa 12:57:20 RECEIVED TIME 28-DEC. 17:07 RECEVED IME 8. DC. 1:07PRINT TIME 29. DEC. 6:11 28. DEC. 2060 18:26 SPRUSON FERGUSON 61 2 292615486 NO, 8897 P. 51 SPRUSON FERGUSON WO 98/42832 PTUPW956 SEQUENCE LISTING GENRAL INFORMATION: APPLIAT~i FraZICeS K. Arnold zhijdn Shao Joseph A. Affholter Huimink Zhao Lori Giver (ii) TITLE OF INVENTION; Recombination of Po3.ynuclectide Sequences Using Defined or Random Primier Sequ1ences (iii) NUMBER OF SECUENCES: (iv) CORRESPONDENCE ADDRESS: ADDRESSER: Oppetheimer Pains Smith STREET: 2029 Century Park East, Suite 3800 IC) CITY: Los Angeleo STATE. CA COTDMrY! USA ZIP. 90067 COMPUTER~ R=AABLE FORM: KEDIU TYPE: Floppy disk~ COMPUTER: IBM PC compatible OPERATING SYSTEM: "indows SOFTWAR.PE Microsoft Word CURRENT APPLICATION DATA; APPLICATION NUMBER: FILING DATE:

CLASSIFICATION:

*(vii) PRIOR APPLICATION DATA: APPLICATION ZIUMUER: 60/041,666 FILING DATE: March 25, 1997 APPLICATION NUMBER: 60/045,211 FILING DATE: April 30. 1997 APPLICATION NUMBER: 60/046,256 FILING DATE: May 12, 1997 (viii) ATTOVEYAGNT INFORMATION: NAME: Oldenkamp, David J.

REGISTRATION NUMBER: 29,421 REFNEMCE/DOCCET NUMBER; 330187-84

TLCMUIAONINFORMATION:

TELEFAX., (310) 27'7-1297 INFORMATION FOR SEQ ID NO: 1: (i SEQUENCE C11ARACTERISTICS: LENGTH: 22 nuclectides TOPOLOGY: linear (ii) MOLECULE TYPE: oligonucleotide Mimosa 12:57:20 RECEIVED TIME 28. DEC. 17 0 7 RECEVED IME 8. DC. 1:07PRINT TIME 29. DEC. 6:10 28, DEC. 2000 18:26 SPRUSON FERGUSON 61' 2 92615486 NO. 8897 P. 52 SPRUSON FERGUSON WO 98/42832 PCTMU99B/0956 (xi) SEQUENCE DECRPTION: sEQ ID NO: 2.: CCG AGC OTT GCA TAT MT OAA G 22 INFORM4ATION~ FOR SEQ ID NO: 2: 1i) SEQUENCE CHARACTERITICS: LENG"M: 21 nucleotides TY3 fluelcotide TOPOLOGY: linear (ii) MOLjECULE TYPE: oligonucleotide (Xdj SEQUENCE DESCR~IPTION: SEQ ID NO! 2: CGA CTC TAG AMO ATC COA TTC 21 INFORMI4TON FOR SEQ ID NO: 3: SEQUENCE CHARACTRISTICS- LENGTH: 21. nucl.eotidea TYPE: nucleozide TOPOLOGY: linear (ii) MOLECULE TYaPE: oligonucleotide (xi) SEQUENCE DESCRIPTION! SEQ ID NO: 3: GAG CAC ATC AGA TCT AT? APAC 21 INPORJ'ATION FOR SEQ Ip No, 4: Wi SEQUENCE CHARACTERISTICS: LENGTH: 21 nucl.eotides 90 TYPE: wicleotide .00 TOPOLOGY; linear Ui) MOLECULS TYPE: oligonucileotide 00 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4: 99* OA GTG OCT CAC ACT COG TUjG 21 INFORMIATION FOR SEQ ID NO:, S- WI SEQUNCE CHARACTERSTICS: LENGTH: 21 flucleotides TYPE: nucleotide TOPOLOGY: linear MOLECULE TYPE: oligonuelotide Cxi) SEQUENCE DESCRIPTION- SZQ Xr) NO! Mimosa 12:57.:20 RECEIVED TIME 28. DEC. 17 07 PRINT TIME 29. DEC. 6: 28. DEC. 2000 18:26 SPRUSON FERGUSON 61 A 2 92615486 NO, 8897 P. 53 SPRUSON FERGUSON

A

WO 98/42532 PCT/US9"5O9S6 TTG AAC TAT COG CM GOO Coo 21 INFORMATION~ FOR SEQ ID NO: 6! Wi SEQUENCE CHARACTERISTICS; LMTOTHi 21 nuoleotidea TYPE: nucleotide TOPOLOGY: linear (ii) MOLECULE TYPE; oligonucleotide (Xi) SEQUENCE DESCRIMTON: SEQ ID NO: 6; TTA CTA GGG AAG CCG CTG OCA 21 INFORMATION FOR SEQ ID NO: 7;- SEQU.ENCE CHARACTERI~STICS: LENGTH: 21 nucleotides TYPE: nucleotide TOPOLOGY: liniear (ii) MOLECULE TYPE; oligonucleotide (Xi) SEQUENCE DESCRIPTION: BEQ ID NO: 7: -TCA GAG AT? ACG ATC GAA AAC 21 INFORMATION FOR SEQ ID NO: 8; SEQUENCE CHARACTERISTICS- C(A) LENGTH: 21 nucleoti.deg TYPE; nucjleotide TOPOLOGY; linear MOLECULE TYPE; oligonucleotide (Xi) SEQUENCE DZSClt PTION: SEQ ID NO: 8: GOA TTO TAT COT OTG AGA AAG 21 INFORMATION FOR SEQ ID NO; 9: Ws. SEQUJENCE CHARXCTr!RISTICS: LENGTH:-21 nucleotides TYPE: nucleotide TOPOLOGY: linear (ii) MOLECUILE TYPE: oligoflucleotide 0 o.

(xi) SEQUENCE DESCRIPTION: SEQ ID NO; 9: *AAT OCC OA AGC AGC CCC TTC 21 INFORMATION FOR SEQ ID NO; to, Mimosa 12:57:20 RECEIVED TIME 28. DEC. 17:07'PITTM 9 E. 61 PRINT TIME 29.DEC, 6:10 28. DEC. 2000 18:26 SPRUSON FERGUSON 61' 2 92615486 NO. 8897 P. 54 SPRUSON

FERGUSON

WO 98/42832 Wi SEQU1ENCE

CHRCERSIS

LENGTHf: 21 nuclectidea TYPE: nIucleotide

TOPOLOGY

2 linear (i)MOLECULE TYPE: aligonuclootide (xi) SEOUM:CE DESCRIPTION: SEQ ID NO: CAC GAC AGG AAG ATT TG ACT 21 INFORMATION POIR SRQ ID NO: 11: SEQUENCE CHfARACTERISTICS: LENGTH(: 20 nuclectides TYPE: flucleotide TOPOLOGY: li-near (i)MOLECULE TYPE: OligcflUcleotide (x)SEQUESNCE DESCR~prION4 SEQ ID NO:11 ACT TAA TCT AGA COGo TAT TA 2 INFO9pjT-oN pOlt SEQ XD No. 12.

SEQUECE

CHARACTERISTICS:

LENGTH: 20 nucleotides TYPE: nlucleotde TOPOLOGYI; linsar MOLE.~rCL TYPE:. 19ncetd (Xi) SEQUENCE DESCRIPTION: sz0 ID N*O: 12: *.:AOC CTC GCG GGA TCC CCO GG IFO RMATION FOR sEQ ID NO: 13: Wi. SEQUENE

CHRCERSIS

LENGTH: 28 nUcleotidev TYPE-- nCletide TOPOLOGY. linear (Wi MOLECULE TYPE, OligOnueleotide (X-t SZQUENCS DESCRIPTION: SVQ ID, NO: 13: GOT AGA GCG ACT CTC GAG00 Goo x3 ATG C =lFORMATION FOR SEQ ID ZZO: 14: SEQUENCE CHARACTE1ZISTICS: LENGTrH: 22 nualeotides Mimosa 12:57:20 RECEIVED TIME 28. DEC. 17:07 PITTM 9 E. 61 PRINT TIME 29,DEC. 6:10 28. DEC. 2060 18:27 SPRUSON FERGUSON 61 A 2 92615486 NO, 8897 P. SPRUSON FERGUSON WO 98/42832 PcTfl(iS98&O5956 57 TYPE: nuclaotide 1c) TOPOLOGY: linaear (ii) MOLECULE TYPE: oligonucleotide (XI) SEOUENCE DESCRIPTZONi SEQ ID NO: 14: AGC COO COT OAC GTO GGT CAG C 22 INFORMATION F~OR BEQ ID NO: Wi SEQUENCE CHARACTERISTICS- LENGTK: 22 nucleotides TYPEi nucleotide TOPOLOGY: linear (ii) MOLECULE TYPEi oligonucleotide (xi) SEQUENCE DESCRIPTZOb?: SEQ ID NO: CCG AGC GTT OCA TAT OTO OAA G 22 INFORMATZO10 FOR SEQ ID NO-. 16: SEQUENCE CHARACTERISTICS: LENGTH: 21 nucleocideuI TYPE. nucleotide TOPOLOGY: linear (ii) MOLECULE TYPE: oligoziucleocide (Xi) SBQtENCE DE!SCRIPTION: SZQ ID NO; 16: CGA CTC TAG AGO AIC CGA TTC 21 IMPORjiATION FOR SEQ ID NO. 17: Wi SEQUENCE CHARACTERISTCS: LENGTH: 22 nulceotides TYPE: nucleotide TOPOLOGY; linear (ii) MOLECULE TYPE; oligonucleotide (xi) SEQUENCE DESCRIPTION: SEQ ID) NO: 1.7! CGO TAC GCA TOT ACC CO TACO0 22 INFORMIATION FOR SEQ ID NO; 1e; U) SEQUENCE CHARACTERISTICS- LEN M 22 fluclectides TYPE: tueleotide TOPOLOGY: linear Mimosa 12:57:20 RECEIVED TIME 28. DEC. 17:017RN IE 29 E. 61 PRINT TIME 29.DEC. 6:10 28. DEC. 2o0o 18:27 SPRUSON FERGUSON 61 A2 292615486 NO. 8897 P. 56 SPRUSON FERGUSON WO 98/42832 PCT/1JS93s956 (ii) MOLECULE TYPE: o 2 .±gonucleotide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18: CO TAC GAT TGC CGC COG TAC G 2 DM~OR14ATION FOR SEQ IV NO: 19: BEQUENcE CHARACTERISTICS: LENGTH: 22 nucleotidee TYPE: fucleotide TOPOLOGY: liniear (ii) MOLECu= TYPE: oligonucleotide (Xi) SEQUENCE DESCRIPTION, 880 ID NO: 19:- CCG ACC GTT OCA TAT OTC GAA 0 22 INFORM4ATION3 FOR SEQ XD NO: Wi SEQU3ENCE CHARACTERISTICS: LE140TH: 21 nulclOtidee TYPE! nxucleotiade TOPOLOGY: linear (ii) MOLECULE TYPE: oligonucleotide (xi) SQUENCE5 DESCRIP'TION- SEQ It) NO; *0CCA CTC TAG AGG ATC COA TTC 21 INOMTO FOR SEC TO NO; 21: i) SEQUENCE

CHARACTERISTICS:

9e LENGTH: is nul3eocides (W TYPE: nucleotide TOPOLOGY: linear (ii) MOLECULE TYPE: oligonuclaotide (xi) SEQUJENCE DESCRIPTION: SEQ ID NO; 21: GGC GGA GCT AaC TTC OTA is INFORMATION F0OR SEQ ID NO.- 22.' (W SEQUENCE CHIARACTERSTICS- LENGTH: 18 nucleotides TYPE: nucleotide TOPOLOGY: linear MOLVCULB TYPE: oligotnucleotide (xi) SEQUENCE DESCRIPTION: SEQ TD NO: 22: Mimosa 12:57:20 RECEIVED TIME 28.DEC. 17:07 RECEVED IME 8. DC. 1:D7PRINT TIME 29. DEC. 6:10D 28. DEC. 2000 18:27 SPRUSON FERGUSON 61 A 2 92615486 NO. 8897 F. 57 SPRUSON FERGUSON WO "8142532 C/gs0% GAT GTO ATOG CT CCT GGC is INFORMATION FOR aEQ ID NO: 23: soumme Cx Ac zISi-ICS LENGTHI: 18 nucleotides TYPE: nucleotide TOPOLOGY: linear (iMOLECULE TYPE: Oligonucleotide (xi) SEQUJENCE DESCRIPTION: SEQ ID NO: 23! CAG AAC ACc OAT TGA OTT 1 -INFOR14ATION FOR SRIQ ID NO: 24: Wi SEQUENCE

CHARACTRISTICS!

LVNGT~t IS nucleotides TYPE: nutcleotide TOPOLOGY: linear i) MOLECULE TYPB: oligonucleatide (Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 24; 'a AGT OCT TTC TAA ACO ATC 1 INFORM'ATION FOR SEQ ID NO: *see SEQUENCE CHARACTERISTICS: LENGTH 4 amino acids S 0(B) TYPE: peptide @00 0(C) TOPOLOGY: linear *000(ii)MOLECULE TYPE: paptide (Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 0000Ala Ala Pro Phe Mios 12572 RECEIVED TIME 28-DEC. 17:07 RECEVED IME 8. DC. 1:07PRINT TIME 29. DEC. 6:10

Claims (18)

1. A method of evolving a polynucleotide toward acquisition of a desired property, comprising: a) contacting at least one template polynucleotide with a set of defined- sequence primers, the set of defined-sequence primers comprising a plurality of both forward and reverse primers; b) conducting a multi-cycle polynucleotide extension reaction on the at least one template polynucleotide and the set of defined-sequence primers, wherein: in at least one cycle, the primers anneal to the at least one to template polynucleotide and prime replication of the at least one template polynucleotide thereby generating a pool comprising overlapping fragments which are shorter in length than the at least one template polynucleotide and which overlap to span the at least one 0 0" template polynucleotide; and 0 (ii) in at least one subsequent cycle, the overlapping fragments 0 0 15s generated in a previous cycle are denatured to single-stranded fragments, which anneal in new combinations forming annealed fragments, whereby one strand of an annealed fragment primes replication of the other to form a further pool of overlapping fragments; whereby the multi-cyclic polynucleotide extension reaction is continued for sufficient cycles until the further pool of overlapping fragments includes variant forms of 20 the at least one template polynucleotide; and c) screening or selecting the variant forms of the at least one template polynucleotide, or expression products thereof, for an altered or enhanced property relative to the at least one template polynucleotide or an expression product thereof.

2. The method of claim 1, wherein the at least one template polynucleotide is a plurality of different template polynucleotides, and the variant forms comprise recombinant forms of the different template polynucleotides.

3. The method of claim 2, wherein the plurality of different template polynucleotides are allelic variants.

4. The method of claim 1, wherein at least some of the pool of overlapping fragments generated in the at least one cycle of step differ from each other due to priming from a primer lacking perfect complementarity with the at least one template polynucleotide.

The method of claim 1, further comprising amplifying the variant forms of the AISTM- at least one template polynucleotide. [I:\DayLib\LIBFF]63629spec.doc:gcc

6. The method of claim 1, wherein the polynucleotide extension is conducted under conditions of incomplete extension of the primers hybridized to the at least one template polynucleotide.

7. The method of claim 1, wherein the primers hybridized to the at least one template polynucleotide are extended by fewer than 300 nucleotides on average.

8. The method of claim 1, wherein the primers in the set of defined-sequence primers are 6-100 nucleotides in length.

9. The method of claim 1, wherein at least one defined-sequence primer further comprises a random nucleotide at one or more nucleotide positions with the primer.

10. The method of claim 1, wherein the at least one template polynucleotide is removed after the at least one cycle of step

11. The method of claim 1, wherein the at least one template polynucleotide is single stranded.

12. The method of claim 1, wherein the at least one cycle of step is performed 1 5 under conditions of incomplete extension of the primers hybridized to the at least one template polynucleotide, and the method further comprises adjusting the conditions to allow complete extension of the annealed fragments in step (ii).

13. The method of claim 1, wherein the primers in the primer set are exhausted during the polynucleotide extension reaction thereby forcing annealing of the overlapping 20 fragments in step (ii).

14. The method of claim 1, wherein the average length of the overlapping fragments in the polynucleotide extension reaction increases with successive cycles.

15. The method of claim 1, wherein the at least one template polynucleotide is of unknown sequence.

16. The method of claim 1, wherein the variant forms of the at least one template are screened for an enzymatic activity.

17. A method of evolving a polynucleotide toward acquisition of a desired property, substantially as hereinbefore described with reference to any one of the examples. [I:\DayLib\LIBFF]63629spec.doc:gcc 56

18. A polynucleotide evolved in accordance with the method of any one of claims 1-17. Dated 23 September, 2002 California Institute of Technology Patent Attorneys for the Applicant/Nominated Person SPRUSON FERGUSON see* ,*0:0 [1:\DayLib\LIBFF]63629spec.doc:gcc