HUP9903760A2 - Recombination of polynucleotide sequences using random or specific primers - Google Patents

Abstract

The subject of the invention is a method for the in vitro artificial genesis and recombination of polyligase-catalyzed polyligase-catalyzed chain termination of polyligase sequences. According to the solution according to the invention, the chain shortening of the template reaction time(s) is initiated with chain initiators with a random sequence or a defined sequence, which creates a mixture of DNA fragments containing few point mutations. The DNA fragments are denatured, followed by amyhybridization and further enzyme-catalyzed DNA polymerization. This process, repeated in a sufficient number of cycles, leads to the creation of full-length genes containing mutations compared to the original template cell time. Genes are optionally amplified and cloned in an expression vector. The solution according to the invention is suitable for the development of recombinant proteins, proteins and enzymes with advantageous needle properties. ŕ

Description

Representative:

Danube

PUBLICATION COPY

Patent and Trademark Office Ltd.

Budapest

Polynucleotide sequences. Recombination using random or specific primers

The invention relates generally to in vitro methods for mutagenesis and recombination of polynucleotide sequences. More particularly, the invention relates to a simple and efficient method for performing in vitro mutagenesis and recombination of polynucleotide sequences, which is based on polymerase-catalyzed chain extension of primer oligonucleotides, subsequent gene assembly and, optionally, gene amplification.

The solution according to the invention can be advantageously used for the development of recombinant polynucleotides, proteins and enzymes with advantageous properties.

The publications and other cited materials referred to in the description belong to the description of the technical field closest to the solution according to the invention, and provide detailed information from the point of view of putting the invention into practice. Our file number: 88891-6510/SG

- 2 gal and are incorporated by reference into the teaching. For the sake of clarity, the referenced materials are numbered and listed in the bibliography attached to the description.

Proteins are engineered to improve their performance in practical applications. The desired properties depend on the application. Such properties may include tighter binding to a receptor, increased catalytic activity, increased stability, the ability to accept a wider (or narrower) range of protein substrates, and the ability to function under non-natural conditions (such as organic solvents). Several approaches, including rational protein design and random mutagenesis, have been successfully used to optimize protein function (1). The choice of approach for a given optimization problem depends on the degree of understanding of the relationship between sequence, structure, and function. For example, rationally engineering the catalytic site of an enzyme often requires knowledge of the enzyme structure, the structures of its complexes with various ligands and analogues of reaction intermediates, and the details of its catalytic mechanism. However, this information is available only for a few well-studied systems; Little is known about the vast majority of enzymes of potential interest. The amino acids responsible for the existing functions of the protein, and the identification of those amino acids that

- 3 that may be carriers of new functions remains a challenge that is often impossible to meet. This fact, as well as the increasing acceptance that many protein functions cannot be assigned to just a few amino acids and are not independent of side chains distant from the active site, has led more and more research groups to turn to random mutagenesis or directed evolution to create new proteins (1). Natural evolution has inspired the development of several optimization methods, such as genetic algorithms (2, 3) and evolutionary strategies (4, 5). These methods use mutations that cause small, random changes in the members of a population, such as crossover, which combines the properties of different individuals to achieve a specific optimization goal. Computer simulations of various optimization problems have shown (6, 9) that there is a high degree of interplay between mutation and crossover. Developing efficient and practical experimental methods to mimic these key processes is a scientific challenge. The application of such a method should allow the user to discover and optimize the functions of biological molecules (e.g. proteins and nucleic acids) in vivo or 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 set of mutated molecules. The mutated mo

- 4 The set of genes must be sufficiently diverse to contain a molecule encoding a protein with altered or improved function. Initially, a library of mutant genes is generated. Gene products that show improvement in a desired trait or group of traits are identified by selection or screening. The gene(s) encoding the products can be subjected to further cycles of the process to accumulate beneficial mutations. This evolution can proceed over many or only a few generations, depending on how far one wishes to go and the effect of the mutations typically observed in each generation. This approach has been used to generate new functional nucleic acids (12), peptides and other small molecules (12), antibodies (12), and enzymes and other proteins (13, 14, 16). Directed evolution requires little specific knowledge about the product itself, but it is necessary to be able to evaluate the function to be optimized. These methods are quite tolerant of inaccuracies and noise associated with the evaluation of the function (15).

The genetic diversity required for directed evolution can be generated by introducing novel point mutations using a variety of techniques, including mutagenic PCR (15) and combinatorial site-specific mutagenesis (16). However, the ability to recombine genes adds a significant new dimension to evolutionary processes, as demonstrated by its key role in natural evolution.

- 5 Homologous recombination is an important natural process in which organisms exchange genetic information between related genes, thereby increasing the available genetic diversity within a species. Although they may confer powerful adaptive and diversifiable capabilities on the host, these pathways operate with very low efficiency and often result in insignificant changes in structure and function, even after tens of generations. Thus, while these mechanisms are beneficial to hosts and species over geological timescales, combinatorial processes for reconfiguring the performance of enzymes and other proteins are not strictly related to the intermediary metabolism and survival of organisms.

Several groups have recognized the utility of gene recombination in directed evolution. Methods for recombination of genes in vivo are disclosed, for example, in published international application WO97/07205 and U.S. Patent No. 5,093,257. As discussed above, these in vivo methods are laborious and poorly optimized for rapid evolution of function. Stemmer has disclosed a method for recombining related DNA sequences in vitro in which the parental sequences are cut into fragments (usually using an enzyme such as DNase-I) and then rejoined (17, 18, 19). However, non-random DNA fragmentation caused by DNase-I and other endonucleases biases recombination and limits recombination diversity. Furthermore, this method is only applicable to recombining double-stranded polynucleotides, not single-stranded templates. The method also does not work well for some gene combinations and primers. It is not efficient for short sequences [less than 200 nucleotides], for example. Finally, the method is quite laborious, requiring many steps. An alternative, conveniently performed method is therefore needed for the in vitro generation of new genes by point mutagenesis and recombination.

The solution according to the invention is briefly summarized below.

The invention provides a novel and significantly improved approach for generating novel polynucleotide sequences by in vitro point mutation and recombination of a set of parental sequences (the templates). The novel polynucleotide sequences can be used alone (e.g., DNA-based computations) or expressed in recombinant organisms for the directed evolution of gene products. In one embodiment of the invention, a set of short DNA fragments is generated by priming the template gene(s) with random sequence oligonucleotides. Under appropriate reaction conditions, these short DNA fragments can prime each other due to complementarity and thus reassemble to form full-length genes during repeated thermal cycling in the presence of a thermostable DNA polymerase. These reassembled

- 7 assembled genes, which contain point mutations and new sequence combinations from different parental genes, can be further amplified by conventional PCR and then cloned into conventional vectors for expression of the encoded proteins. Screening or selection of gene products leads to new, improved or even functional variants. These variants can be used as produced or can serve as starting points for further cycles of mutagenesis and recombination.

In another embodiment of the invention, the template gene(s) is primed with a set of primers of defined sequence or of limited random but essentially defined sequence, thereby generating a set of short DNA fragments which are then reassembled into full-length genes as described above.

A third embodiment of the invention is a novel process, which we have named the staggered extension process (StEP). Instead of assembling a set of fragments produced by extended primers, full-length genes are assembled directly in the presence of the template(s). The StEP process consists of repeated cycles of denaturation followed by extremely shortened annealing and extension steps. In each step, the extended fragments can hybridize to different templates based on their complementarity and can extend slightly beyond them, creating recombinant units.

Due to this template shift, most oligonucleotides contain sequences from different parental genes (i.e., new recombinants). This process is repeated until full-length genes are generated. The steps of the process are optionally followed by a gene amplification step.

Different embodiments of the invention are advantageous for different applications based on their specific characteristics. In the most preferred embodiment, one or more specific primers or limited random specific primers (flanking or corresponding to the 5' and 3' ends of the template polynucleotides) are used in a StEP process, thereby generating gene fragments that grow into new, full-length sequences. This simple process does not require knowledge of the template sequence or sequences.

In another preferred embodiment, multiple specific primers or limited random specific primers are used to generate short gene fragments that are then assembled into full-length genes. The use of multiple specific primers allows the user to bias the in vitro recombination frequency. If sequence information is available, the primers can be designed to generate overlapping recombination units that increase the recombination frequency at a given site. Among other features, this method is flexible in that it takes advantage of available structural and functional information, as well as information accumulated during previous generations of mutagenesis and selection (or screening).

In addition to recombination, various embodiments of the primer-based recombination process generate point mutations. Advantageously, we know and can control this point mutation rate, which can be achieved by influencing the conditions of DNA synthesis and gene assembly. Using the defined primer-based approach, specific point mutations can be directed at specific sites in the sequence using mutagenic primers.

The various primer-based recombination methods of the invention have been shown to increase the activity of ECB deoxylase from Actinoplanes utahensis over a wide pH range and in organic solvents, and to improve the thermal stability of subtilisin E enzymes from Bacillus subtilis. DNA sequencing confirms the role of point mutations and recombinations in the generation of new sequences. These protocols have been found to be both simple and robust.

The above-described and numerous other features and advantageous effects of the solution according to the invention are described in more detail below using the accompanying drawings. They are briefly described below.

Figure 1 shows recombination using random sequence primers and gene assembly according to the invention. The steps shown are for the

- 10 steps: a) synthesis of single-stranded DNA fragments with random sequence oligonucleotides as primers, using mesophilic or thermophilic polymerases (primers not shown); b) removal of templates; c) reassembly of the gene with a thermophilic DNA polymerase; d) amplification with a thermostable polymerase or polymerases; e) cloning and (optionally) screening; and f) repetition of the process with a selected gene or genes (optionally.

Figure 2 shows the recombination of the invention, carried out with specific primers. The method is illustrated by the recombination of two genes, x represents a mutation. The steps depicted are: a) the genes are primed in PCR reactions with specific primers, which can be performed separately (per two primer reactions) or in combination (per multiple primer reactions), c) initial products are produced until the specific primers are exhausted, the template is optionally removed, d) additional PCR cycles are performed, during which the initial fragments are primed and extended on their own without the addition of external primers, continuing to assemble the genes until full-length genes are obtained, e) optionally, the full-length genes are amplified by PCR using external primers, and f) optionally, the process is repeated with the selected gene or genes.

- 11 Figure 3 shows the recombination according to the invention - performed with two defined flanking primers and the StEP method. Only one primer and two strands, one from each of the two primers, are shown in the figure to illustrate the recombination process. The steps presented are as follows: a) after denaturation, the template genes are primed with a single specific primer, b) short fragments are produced by short-term chain extension, c) in the next cycle of StEP, the fragments are randomly hybridized to the templates as primers and further extended, d) denaturation, further heat treatment (hybridization) and chain extension are repeated until full-length genes are produced (these can already be seen in an agarose gel), e) the full-length genes are purified or amplified in a PCR reaction with external primers (if applicable), f) if applicable, the process is repeated with the selected gene or genes.

Figure 4 is a diagrammatic representation of the results of recombination of two genes using two flanking primers and the wandering extension of the invention. The DNA sequences of five genes selected from the recombined library are shown, x represents the mutation present in the parental genes and the triangle represents a new point mutation.

Figure 5 is a diagrammatic representation of the sequences of the pNB esterase genes described in Figure 3. The 2-13 and 5B12 template genes were recombined using the defined primer approach. The positions of the primers were determined by

- 12 lac are indicated, and positions where the parental sequences differ from each other are indicated by x. New point mutations are indicated by triangles. We have listed the mutations identified in the recombinant genes (only those positions that differ in the parental sequences are listed). Both 6E6 and 6H1 are recombination products of template genes.

Figure 6 shows the positions and sequences of four specific internal primers used to generate recombinant genes from the R1 and R2 template genes by interspersed primer-based recombination. The P50F primer contains a mutation (A -> T at base 598) that simultaneously eliminates a HindIII restriction site and creates a new, unique NheI site. The R2 gene also contains an A -> G mutation at the same base position that eliminates the HindIII site.

Figure 7 shows an electrophoresis gel showing the results of restriction digest analysis of plasmids from 40 clones.

Figure 8 shows the results of sequencing 10 genes from the recombination library based on the defined primers. The lines represent 986 base pairs of the subtilisin E gene, including 45 nucleotides of its prosequence, the complete mature sequence and 113 nucleotides after the stop codon. The crosses indicate the locations of mutations from the parental R1 and R2 genes, while the triangles indicate the new sequences introduced during the recombination process.

- 13 point mutation sites. The circles indicate the mutation introduced with the mutagenic P50F primer.

Figure 9 shows the results of the random primer recombination procedure for the ECB deacylase gene of Actinoplanes utahensis. (a) The 2.4 kb ECB deacylase gene was purified from agarose gel. (b) The sizes of the products produced by random priming ranged from 100 to 500 bases. (c) Fragments shorter than 300 bases were isolated. (d) The purified fragments were used to reassemble the full-length genes with a smeared background. (e) A single PCR product of the same size as the ECB deacylase gene was obtained after conventional PCR with two primers located at the start and stop regions of the gene. (f) After digestion with XhoI and PshAI, the PCR product was cloned into a modified plJ702 vector, generating a mutant library. (g) Introduction of the library into Streptomyces lividans strain TK23 resulted in approximately 71% of clones producing active ECB deacylase.

Figure 10 shows the specific activity of wild-type ECB deacylase and the specific activity of the M16 mutant produced by the method of the invention.

Figure 11 shows the pH profile of wild-type ECB deacylase and the M16 mutant produced by the method of the invention.

Figure 12 shows DNA sequence analysis of ten clones randomly selected from the Klenow library. The lines represent 986 base pairs of the subtilisin E gene,

- 14 which includes 45 nucleotides of the gene prosequence, the complete mature sequence and the 113 nucleotides after the stop codon. The crosses indicate the positions of the mutations in the R1 and R2 genes, the triangles indicate the positions of the new point mutations introduced during the random primer recombination procedure.

Figure 13 shows the thermostability index profiles of screened clones from five libraries prepared using different polymerases. The five libraries are: a) Klenow library, b) T4 library, c) sequence library, d) Stoffel library, and e) Pfu library. The normalized residual activity (A _r /A i ) measured after incubation at 65°C was used as an index of the thermostability of the enzyme. The data were organized and presented in descending order.

The solution according to the invention is described in detail below.

In a preferred embodiment of the invention, a primer set containing all possible nucleotide sequence combinations [dp(N) _L , where L = primer length] is used for primer-based recombination. It has been known for years that oligodeoxynucleotides of different lengths can serve as primers for the initiation of DNA synthesis on single-stranded templates by the Klenow fragment of E. coli polymerase I (21). Oligomers of hexanucleotide length, although smaller than normal PCR primers (13 bases), can also adequately initiate the reaction and are often used as markers.

- 15 in ligation reactions (22). Figure 1 illustrates the use of random primers to generate a set of gene fragments, followed by assembly of the genes according to the invention. In carrying out the process, a variety of parent blocks are generated by random priming from single-stranded polynucleotide templates, the resulting short, nascent DNA fragments are assembled into full-length DNA using temperature cycling in the presence of DNA polymerase and nucleotides, and the desired genes are amplified from the assembled products by conventional PCR for further cloning and screening. This process introduces new mutations mainly during the priming step, but this also occurs during the other steps. These new mutations, and mutations already present in the template sequences, recombine during gene reassembly, thus creating a library of new DNA sequences. If desired, the procedure can be repeated with the sequences obtained by selection.

In order to carry out the random priming procedure, the template(s) can be/single-stranded or denatured double-stranded polynucleotide(s)/ in linear or closed, circular form. The templates can be mixed in equimolar amounts or, for example, in a weighted ratio according to their functional characteristics. Since - at least in some cases - the template genes are available in a cloned form in a vector and no further mutations should be introduced into the vectors, the genes are first cut with a restriction endonuclease or

- 16 endonucleases are used to excise the vector and then purified. The resulting linear DNA molecules are denatured by boiling, annealed to oligodeoxynucleotides of random sequence by heat treatment, and incubated with DNA polymerase in the presence of an appropriate amount of dNTP. Hexonucleotide primers are preferably used, although longer random primers (up to and including 24 bases) can also be used, depending on the DNA polymerase and the conditions used in random priming synthesis. Thus, the oligonucleotides initiate the synthesis of the DNA in question at different sites along the entire length of the targeted region, and during chain extension, short DNA fragments are generated, some of which are complementary to one strand of the template DNA and some to the other. Due to events such as base mismatching and primer mismatching, these short DNA fragments also contain point mutations. Once the procedure is routine and the reaction conditions are set accordingly, the short DNA fragments can be initiated to synthesize each other's chains based on their homology, and then assembled into full-length genes by repeated temperature cycles in the presence of a thermostable DNA polymerase. The sequences of the resulting full-length genes are diverse, but most of them still resemble the original template DNA. These sequences can then be further amplified by conventional PCR methods and then cloned into a vector for expression. Screening or selection of the expressed mutants leads to variants with improved properties or even new, specific functions.

- 17 These variants can either be used immediately, for example as a partial solution to a practical problem, or they can serve as a starting point for further directed evolutionary cycles.

Compared to other methods used for protein optimization, such as combinatorial cassette and oligonucleotide-directed mutagenesis (24, 25, 26), error-prone PCR (27, 28), or DNA shuffling (17, 18, 19), some of the advantages of the random priming-based in vitro protein evolution method can be summarized as follows:

1. The template(s) used for random primer synthesis can be either single-stranded or double-stranded polynucleotides. In contrast, error-sensitive PCR and DNA shuffling recombination methods (17, 18, 19) can only be performed on double-stranded polynucleotides. Using the method described herein, mutations and/or crossovers can be introduced at the DNA level using various DNA-dependent DNA polymerases, or even directly from mRNA using various RNA-dependent DNA polymerases. Recombination can be performed using single-stranded DNA templates.

2. In contrast to the DNA shuffling process, which requires fragmentation of a double-stranded DNA template, usually with DNase I, to produce random fragments, the method described herein utilizes random primer synthesis to generate DNA fragments of controllable size as parental or original blocks.

- 18 for further assembly (Figure 1). One immediate advantage is that two sources of nuclease activity (DNaseI and 5’-3’ exonuclease activity) have been eliminated, allowing us to more easily control the size of the gene fragments generated during final assembly and amplification.

3. Because random primers are populations of synthetic oligonucleotides that contain all four bases at all positions, are uniform in size and free from sequence errors. The heterogeneity of the sequence allows them to hybridize at many locations on the template DNA strand, so that all nucleotides of the template (except perhaps those at the very 5'-terminus) are copied into the products with similar frequency. In this way, both mutation and crossover can occur more randomly than in error-sensitive PCR or DNA shuffling.

4. Random-primed DNA synthesis is based on the hybridization of a mixture of hexonucleotides to a DNA template, and the complementary strands are synthesized starting from the 3’-OH terminus of the random hexonucleotide primers using a polymerase and the four deoxynucleotide triphosphates. The reaction is therefore independent of the length of the DNA template. The synthesis of DNA fragments of 200 bases in length can be initiated equally well using the DNA fragment as a linearized plasmid or as λ-DNA (29). This property is particularly advantageous in the genetic engineering of peptides, for example.

5. Since DNase I is an endonuclease that cleaves double-stranded DNA mostly adjacent to pyrimidine nucleotides, its use in DNA shuffling may introduce bias (especially for genes with high G+C or A+T content) in the template gene digestion step. The random priming approach avoids the effects of this potential bias on the overall mutation rate and recombination frequency. The bias due to the higher probability of hybridization to GC-rich regions of the template DNA during random priming can be avoided by increasing the A and T content in the random oligonucleotide primer.

An important element in the implementation of the present invention is the control of the average size of the nascent single-stranded DNA synthesized during the random priming process. This step has been studied in detail. Hodgson and Fisk (30) found that the average size of the synthesized single-stranded DNA is an inverse function of the primer concentration: chain length = k/ ^InPc, where _Pc is the primer concentration. The inverse relationship between the primer concentration and the size of the resulting DNA fragment is probably due to spherical inhibition. With this in mind, it is not difficult to set up the appropriate conditions for random priming synthesis for individual genes of different lengths.

Since dozens of polymerases are currently available, the synthesis of short, nascent DNA fragments can be achieved in a variety of ways.

- 20 can be implemented. For example, T4 bacteriophage DNA polymerase (23) or T7 sequence version 2.0 DNA polymerase (31, 32) can both be used for random chain initiation synthesis.

For single-stranded polynucleotide templates (especially RNA templates), reverse transcriptase is preferred for random primer synthesis. Because these enzymes lack 3'—>5' exonuclease activity, they are error-prone. At dNTP and Mn ²⁺ concentrations, one base in every 500 is incorrectly incorporated (29).

By modifying the reaction conditions, PCR can be adapted for random primer synthesis using a thermostable polymerase to generate short, nascent DNA fragments. It is important to ensure that the reaction conditions that ensure that the short, random primers hybridize to the template and allow adequate DNA amplification at high temperatures are determined by routine experimentation. We have found that short, random primers of dp(N) length can be used to generate chain-extended primers by PCR. Adapting the PCR process for random primer synthesis provides a convenient method for generating short, nascent DNA fragments and makes this random primer recombination method very reliable.

According to many evolutionary scenarios, recombination must occur between oligonucleotide sequences, since they provide sequence information.

- 21 of them, or more precisely, at least part of the template sequences. In such a scenario, it is often possible to define and synthesize a series of primers “interspersed” along the gene. If specific primers are used, they can be between 6 and 100 bases in length. In the course of developing the present invention, it was recognized that by allowing these specific primers to initiate overlapping chain extension reactions (which can be enabled by temperature cycling), it became possible to produce recombination units (cassettes) that each contain one or more accumulated mutations or allelic or isotypic differences between the templates. By using the specific primers in such a way that overlapping chain extension products are generated during the DNA polymerization reaction, the depletion of available primers leads to progressive cross-hybridization of the chain extension products, which continues until the complete gene products are formed. The repeated hybridization, extension and denaturation cycles ensure that each overlapping unit recombines with all the others.

A preferred embodiment of the invention is a method in which a set of specific oligonucleotide primers is used to initiate DNA synthesis. Figure 2 illustrates an example of an embodiment of the invention in which specific primers are used. Careful design and positioning of the oligonucleotide primers facilitates non-random

- 22-like chain-extended recombination primers are produced and are suitable for determining the main recombination (cosegregation) events along the entire length of homologous templates.

Another embodiment of the invention is in fact an alternative approach to primer-based gene assembly and recombination in the presence of a template. Accordingly, as illustrated in Figure 3, the invention provides a recombination in which enzyme-catalyzed DNA polymerization is allowed to proceed only briefly before denaturation. This is achieved by reducing the temperature of the extension step and limiting the reaction time. After denaturation, the extended fragments are hybridized to the template sequences and partial extension is continued. This process is repeated several times, depending on the concentration of the primer and template, until full-length sequences are obtained. This process is called staggered extension (StEP). Although random primers can be used during StEP, gene synthesis is not nearly as efficient as with specific primers, so specific primers are preferred.

In this method, short hybridization and/or chain extension step(s) are used to produce partially extended primers. Typically, the hybridization/chain extension step is performed under conditions that allow for high fidelity.

- 23 the primer hybridization (T _{hybridization S} should be greater than T _m ' ²⁵ ) is _allowed to proceed, but the polymerization/chain extension step is not allowed to proceed for more than a few seconds (on average, one chain extension should allow the incorporation of less than 300 nucleotides). The minimum chain growth is preferably in the order of 20-50 nucleotides. We have shown that the typical maximum polymerization rate of thermostable DNA polymerases is 100-150 nucleotides/second/enzyme molecule under optimal temperature conditions, and as the temperature approaches the optimum (T _opt ) temperature, it follows approximately Arrhenius kinetics. Therefore, at 55 C°, a thermostable polymerase provides only 20-25% of its steady state polymerization rate, which is achieved at 72 C° (T _opt ). This is only 24 nucleotides/second (40). At 37°C and 22°C, Tag polymerase has been reported to have chain extension activities of only 1.5 nucleotides/second and 0.25 nucleotides/second, respectively (24). Both time and temperature can therefore be routinely varied according to the desired recombination events, given the knowledge of the kinetics and biochemistry of the polymerase.

The progress of the wandering chain extension process can be monitored by removing aliquots from the reaction vessel at different times during the primer chain extension and separating the DNA fragments by agarose gel electrophoresis. Efficient primer chain extension is indicated by the appearance of a smeared signal in the low molecular weight range at the beginning of the process,

- 24 which shifts to the higher molecular weight range with increasing cycle number.

Unlike the gel amplification process (which generates new DNA exponentially), StEP produces new DNA fragments additively in the early cycles. These fragments contain DNA segments corresponding to different template genes. Under non-amplifying conditions, 20 StEP cycles can achieve a maximum polar yield of about 40 times the initial template concentration. In comparison, an ideal polymerase chain reaction process, in which the gene is amplified multiplicatively throughout, would yield a polar yield of about ^1x106 after the same number of steps. In practice, the difference between the two methods can be observed when performing PCR, which gives a clear band after only a few (less than 10) cycles if the template concentration is less than 1 ng/μΐ, while using primers in a 10- to 500-fold excess (compared to the 10 ⁶ -fold excess typically used in gene amplification). Under similar reaction conditions, StEP is expected to produce a less visible smear, which shifts towards the higher molecular weight range with increasing number of cycles. When a significant number of chain-elongated DNA molecules start to reach a size greater than half the total length of the gene, a rapid jump in molecular weight occurs as the half-length forward and reverse strands synthesized start to cross-hybridize, resulting in the flow

- Fragments reaching this point of 25 mat produce fragments approximately twice as long. At this point, rapid transformation of the smeared band into a sharply separated band of appropriate molecular weight can occur either by continuing the DNA treatment to the StEP procedure or by changing the temperature cycle, allowing full chain elongation of the primed DNA by exponential gene amplification.

Following gene assembly (and, if necessary, conversion to double-stranded form), the recombined genes are optionally amplified, digested with appropriate restriction enzymes, and ligated into expression vectors for screening of expressed gene products. The process can be repeated, if desired, to accumulate sequence changes leading to the evolution of the desired function.

The wandering extension and homologous gene assembly (StEP) process is an efficient and flexible method for recombining similar genes in a random or intentional manner with systematic error. The process can be used to focus recombination events at or away from specific regions of a known sequence by controlling the location of primers and the time allowed for the hybridization/extension steps. The process can also be used to recombine specific units of homologous genetic information produced separately or in a single reaction. The process can be used to recombine genes whose sequences are not known.

- 26 information, but for which functional 5'- and 3'-end amplification primers can be produced. Unlike other recombination methods, the wandering chain extension method can be performed in a single tube using conventional procedures without complex separation and purification steps.

The advantages of the embodiment of the solution according to the invention using specific primers can be summarized as follows:

1. The StEP process does not require the separation of the starting molecules from the assembled products.

2. Specific primers can be used to influence the location of recombination events.

3. StEP allows us to adjust the recombination frequency by varying the chain extension durations.

4. The recombination process can be performed in a single test tube.

5. The method can be performed on both single-stranded and double-stranded polynucleotides.

6. The method avoids the systematic error otherwise introduced by DNaseI or other endonucleases.

7. Any primer can be used.

8. Limited random, defined primers can be used in selected regions of the gene to increase mutation frequency.

- 27 As will be apparent to those skilled in the art, the solution according to the invention can be implemented in a number of ways. Some exemplary embodiments are as follows:

1. Recombination of related genes and implementation of point mutations in them using specific flanking primers and roving strand extension.

2. Recombination and mutation of related genes using a series of flanking primers and internal primers, applied at concentrations low enough that the primers are depleted during temperature cycling, forcing the overlapping gene fragments to cross-hybridize with each other and continue to grow until the recombined synthetic genes are formed.

3. Gene recombination and mutation using high concentrations of random sequence primers, resulting in a pool of short DNA fragments that reassemble to create new genes.

4. Recombination and mutation of genes using a specific set of primers, resulting in a set of DNA fragments that are then assembled to create new genes.

5. Recombination and mutation of single-stranded polynucleotides using one or more specific primers and roving chain extensions, resulting in the formation of new genes.

6. Recombination using limited random, defined primers with respect to more than 30 or more than 60% of nucleotides within the primer.

Practical examples demonstrating the application of the primer-based recombination method are presented below:

Example 1

Use of specific flanking primers and roving chain extension to enhance the thermostability of subtilisin E by recombining the enzyme

This example demonstrates how a specific primer-based recombination method can be used to enhance the thermostability of subtilisin E by recombining two genes known to encode subtilisin E variants with greater thermostability than wild-type subtilisin E. This example illustrates the general procedure depicted in Figure 3 using only two primers corresponding to the 5' and 3' ends of the template.

As shown in Figure 3, extended recombination primers are first generated by a wandering extension procedure (StEP), which consists of repeated cycles of denaturation followed by extremely shortened hybridization/extension step(s). The extended fragments are assembled into full-length genes by assembly of homologous genes using temperature cycling in the presence of DNA polymerase, optionally followed by gene amplification. Two thermostable subtilisin E mutants, R1 and R2, were used for the specific primer-based recombination.

- 29 methods for testing, using wandering chain extension. The sites where these two genes differ are shown in Table 1. Of the 10 nucleotides at which R1 and R2 differ, only the mutations that result in the exchange of asparagine 181 for aspartic acid (N181D) and asparagine 218 for serine (N218S) confer increased thermostability. The remaining mutations are indifferent in their effect on thermostability (33). The half-lives of the variants containing only the N181D and N218S mutations at 65°C are approximately three and two times, respectively, that of wild-type subtilisin E, and their melting temperatures ( _Tm ) are 3.7 and 3.2°C higher than those of the wild-type enzyme, respectively. Random recombination events that contain both functional mutations are expected to produce enzymes with half-lives at 65°C that are approximately 8 times the half-life of wild-type subtilisin E, provided that no new deleterious mutations are introduced into the genes during the recombination process. The average point mutation rate associated with a recombination process can also be estimated from the catalytic activity profiles of small samples of the library of recombinant variants. If the point mutagenesis rate is zero, 25% of the population should exhibit wild-type-like activity, 25% should exhibit activity corresponding to the double mutant (N181D+N218S), and the remaining 50% should exhibit activity corresponding to the single mutant (N181D or N218S). For a finite value of the rate, mutagenesis increases the fraction of the library that

- 30 l, which encodes enzymes with wild-type activity or less. This fraction can be used to estimate the rate of point mutagenesis.

Table 1

DNA and amino acid substitutions in the heat-stable R1 and R2 mutants of subtilisin E

Gene Base Base substitution Codon position Amino acid Amino acid substitution 780 The - > G 2 109 Asn Ser RÍ 1107 The - * G 2 218 Asn - -> Cheese 1141 The - > T 3 229 synonym 1153 The - > G 3 233 synonym 484 The - > G 3 10 synonym 520 The - > T 3 22 synonym 598 The - > G 3 48 synonym 731 G - > The 1 93 With - * Ile R2 745 T - > C 3 97 synonym 780 The - > G 2 109 Asn - > Cheese 995 The - > G 1 181 Asn - > Asp 1189 The - * G 3 245 synonym

The reference for the listed mutations is wild-type subtilisin E. The mutation at position 780 is identical in the two mutants.

- 31 Materials and methods

Primer-based recombination method using two flanking primers

Two specific primers, P5N [5'-CCGAG CGTTG CATAT GTGGA AG-3, SEQ ID NO: 1; the underlined sequence corresponds to the NdeI restriction site], and P3B (5'-CGACT CTAGA GGATC CGATT C-3', sequence given in ID No. 2; the underlined sequence corresponds to the BamHI restriction site] were used for recombination. The primers correspond to the 5'- and 3'-end flanking primers. The reaction conditions were as follows (based on a volume of 100 μΐ): 0.15 pmol of plasmid DNA containing the R1 and R2 genes in a 1:1 ratio was used as template, and 15 pmol of both flanking primers, single Taq buffer, 0.2 mM of each dNTP, 1.5 mM MgCl ₂ and 0.25 units of Taq polymerase. The program was as follows: 5 min at 95°C, 80 cycles of the following two steps: 30 s at 94°C, and 5 s at 55°C. The product of the appropriate size (approximately 1 kb) was excised from the 0.8% agarose gel after electrophoresis and purified using the QIAEX II Gel Extraction Kit. This purified product was digested with NdeI and BamHI and subcloned into the pBE3 shuttle vector. The resulting gene library was amplified in E. coli HB101 and then transferred into B. subtilis DB428 competent cells for expression and screening as previously described (35).

- 32 DNA sequencing

The genes were purified using the QIAprep spin plasmid miniprep kit, resulting in sequencing-grade DNA. Sequencing was performed on an ABI373 DNA sequencing system using the Dye Terminator Cycle Sequencing kit (PerkinElmer, Branchburg, N.J., USA).

Results

The process of wandering chain extension was monitored by removing aliquots (10 μΐ) from the reaction tube at different times during the chain extension process and separating the DNA fragments by agarose gel electrophoresis. Gel electrophoresis of the primer chain extension reactions showed that the five-second hybridization/chain extension reactions at 55°C resulted in a smeared band of approximately 100 bp (after 20 cycles), then 400 bp (after 40 cycles), then 800 bp (after 60 cycles), and finally a strong band of approximately 1 kb within the smear. This band (the mixture of reassembled products) was gel purified, digested with the restriction enzymes BamHI and NdeI, and amplified using BamHI and NdeI-digested E. coli/B. subtilis pBE3 transfer vector. This gene library was amplified in E. coli HB101 cells and then transferred to B. subtilis DB428 competent cells for expression and screening (35).

The thermal stability of the enzyme variants was determined as previously described using a 96-well plate setup.

- 33 meg (33) . About 200 clones were screened, of which about 25% retained subtilisin activity. Among the active clones, the frequency of the double mutant-like phenotype (high thermostability) was approximately 23%, that of the single mutant phenotype approximately 42%, and that of the wild type phenotype approximately 34%. This distribution is very close to the value expected in the case of completely free recombination of the two thermostable mutations, N218S and N181D.

Twenty clones were randomly selected from the E. coli HBlOl gene library. Their plasmid DNAs were isolated and digested with NdeI and BamHI. Nine of the twenty (45%) contained inserts of the correct size (approximately 1 kb). Thus, approximately 55% of the contents of the above library were definitely inactive due to the lack of the correct subtilisin E gene. These clones are not members of the subtilisin library and thus should not be considered in our calculations. Taking this factor into account, we found that 55% of the library (25% active clones/45% clones containing the correct size insert) retained their subtilisin activity. This activity profile indicates a point mutagenesis rate of less than two mutations per gene (36). Five clones containing the correct size insert were sequenced. The results are summarized in Figure 4. All five genes are recombination products with a small amount of crossover (1-4). Only one new point mutation was found in the five genes.

Example 2

Use of defined flanking primers and wandering chain length for recombination of pNB esterase mutants

The two-primer recombination procedure used in this example for pNB esterase is analogous to the procedure described in Example 1 for subtilisin E. Two mutant pNB esterase genes, differing by 14 bases, are used as templates. Both templates (61C7 and 4G4) are used in the form of plasmids. Both target genes are present in the chain extension reaction at a concentration of 1 ng/μΐ. The flanking primers (RM1A and RM2A, Table 2) are added to the reaction mixture at a final concentration of 2 ng/μΐ (approximately 200-fold molar excess over template).

Table 2. Primers used for recombination of pNB esterase genes

Chain starter Sequence RM1A GAG CAC ATC AGA TCT ATT AAC (sequence given in ID number 3) RM2A GGA GTG GCT CAC AGT CGG TGG (Sequence given in ID No. 4)

- 35 Clone 61C7 was isolated based on its activity in organic solvents. This clone contains 13 DNA mutations compared to the wild-type sequence. Clone 4G4 was isolated based on its thermal stability, this clone contains 17 DNA mutations compared to the wild-type. The two mutants share 8 mutations due to their common ancestor. The gene product of 4G4 is significantly more thermally stable than the gene product of 61C7. Thus, one measure of recombination between genes is the high solvent activity and high thermal stability, or the loss of both properties in the recombinant genes. In addition, the recombination frequency and mutagenic ratio can be estimated from the sequencing of random clones.

In the case of the pNB-esterase gene, chain elongation takes place over 90 temperature cycles, with a cycle program of 30 seconds at 94°C, followed by 15 seconds at 55°C. After the 20th, 40th, 60th, 70th, 80th and 90th cycles, aliquots (10 μΐ) of the reaction mixture were taken as samples. By the end of the 20th cycle, agarose gel electrophoresis showed a low molecular weight smear that shifted towards the higher molecular weight range and increased in intensity in each successive sample. By the end of the 90th cycle, the smear clearly extends from 0.5 kb to 4 kb, with maximum signal intensity occurring at approximately 2 kb (corresponding to the length of the full-length gene). The jump in half the length of the entire gene in this case occurs between the 60th and 70th cycles.

The resulting intense, smeared band is amplified through 6 cycles of polymerase chain reaction to clearly identify

- 36 determine the full-length recombinant gene population. A primerless control is also amplified with flanking primers to determine the background due to remaining template in the reaction mixture. The signal intensity of the extended gene population is more than 10-fold higher than that of the control, indicating that the amplified, non-recombinant template comprises only a small fraction of the amplified gene population.

The pooled amplified recombinant genes were digested with XbaI and BamHI restriction enzymes and ligated into the pNM106R expression vector [Zock et al., (35)]. Ligation of the ligated DNA into E. coli TG1 strain was performed using the well-characterized _CaCl2 transformation procedure. Transformed colonies were plated on LB/agar plates in the presence of 20 pg/ml tetracycline.

The mutagenicity of the process is determined by measuring the percentage of clones expressing active esterase (20). In addition, randomly picked colonies are sequenced to determine the mutagenicity of the process and the efficiency of recombination.

Example 3: Recombination of pNB esterase genes using interspersed, defined upper strand primers and roving strand extension

The following example demonstrates how the “scattered, specific primer-based recombination method” is based on point mutagenesis and the mutations present in the original sequences.

- 37 is capable of creating new sequences through recombination of tations.

Experimental design and background information

Two pNB esterase genes (2-13 and 5-B12) were recombined using a primer-based recombination method. The products of both the 2-13 and 5-B12 genes are measurably more thermostable than the wild type. The 2-13 gene contains 9 mutations, while the 5-B12 gene contains 14 mutations that are not originally present in the wild type sequence. The sites at which these two genes differ from each other are shown in Figure 5.

Table 3 describes the sequences of the 8 primers used in the example.

The location of hybridization of cligo to the template genes (at the 5' end of the template gene) is shown in the table, as is the orientation of the primers (F for forward primer, R for reverse primer). These primers are shown as arrows along the 2-13 gene in Figure 5.

Table 3

Sequence of primers used in Example 3

Name Orientation Location Sequence RM1A F -76 GAGCACATCAGATCTATTAAC (sequence given in ID 3) RM2A R +454 GGAGTGGCTCACAGTCGGTGG (sequence given in ID 4) S2 F 400 TTGAACTATCGGCTGGGGCGG (sequence given in ID 5) S5 F 1000 TTACTAGGGAAGCGGCTGGCA (sequence given in ID 6) S7 F 1400 TCAGAGATTACGATCGAAAAC (sequence given in ID 7) S8 R 1280 GGATTGTATCGTGTGAGAAAG (sequence given in ID 8) S10 R 880 AATGCCGGAAGCAGCCCCTTC (sequence given in ID number 9) S13 R 280 CACGACAGGAAGATTTTGACT (10th sequence given under the same reference number)

Materials and methods

Primer-based recombination

1. Production of genes to be recombined

Plasmids containing the genes to be recombined were purified from transformed TG1 cells using the Qiaprep reagent kit (Qiagen, Chatsworth, CA, USA).

- 39 vl. The amount of plasmids was measured by UV absorption and mixed in a 1:1 ratio, with a final concentration of 50 ng/μΐ.

2. Wandering PCR and gene assembly A plasmid mixture of μΐ was used as template in a 100 μΐ standard reaction [1.5 mM MgCl ₂ , 50 mM KC1, 10 mM TrisHC1, pH 9.0, 0.1% Triton X-100, 0.2 mM dNTP, 0.25 units Taq polymerase, (Promega Madison WI, USA)] containing 12.5 ng of each of the eight primers. A control reaction mixture was also prepared that did not contain any primers. The reaction mixture was subjected to 100 temperature cycles, with a cycle program of 94°C for 30 seconds, followed by 55°C for 15 seconds. An aliquot of the reaction mixture was then analyzed in an agarose gel. The product appeared as a large smeared band (no visible product was found in the control without primer).

3. DpnI digestion of templates

1 μl of the gene assembly reaction mixtures was digested with DpnI to remove the template plasmid. The 10 μl DpnI digest contained single concentration NEB buffer-4 and 5 units of DpnI (both obtained from New England Biolabs, Beverly, MA, USA), and was incubated at 37°C for 45 min and then at 70°C for 10 min to heat-inactivate the enzyme.

4. PCR amplification of the reassembled gene products

The 10 μΐ digestion mixture was added to 90 μΐ standard PCR mixture (see step 2) containing 0.4 μΜ of primers 5b (ACTTAATCTAGAGGGTATTA; SEQ ID NO: 11) and 3b (AGCCTCGCGGGATCCCCGGG; SEQ ID NO: 12), which are specific for the two ends of the gene. After 20 cycles of standard PCR (94°C for 30 seconds, 48°C for 30 seconds, 72°C for 1 minute), a single strong band of the correct size (2 kb) was obtained when the reaction mixture was run on an agarose gel, while only a few very faint bands were visible in the control lane without primer. The product band was purified and cloned back into the expression plasmid pNB106R, then transformed into TG1 cells by electroporation.

Results

Four 96-well plate colonies from the transformation were tested for initial pNB esterase activity and thermostability. Approximately 60% of the clones had initial activity and thermostability values less than 20% different from the values obtained for the original genes. A very small proportion of the clones (10%) were inactive (less than 10% of the original initial activity values). These results indicate a low rate of mutagenesis. The genes of four mutants with greater thermostability were sequenced. Two clones (6E6 and 6H1) were the result of recombination between the original genes (Figure 5). One of the remaining two clones contained a novel point mutation, and the other showed no difference from the original 5B12. The combination of T99C and C204T mutations in the 6E6 mutant is evidence of a recombination event between the two sites. In addition, the 6Hl mutant lost the A1072G mutation (but retained the C1038T and T1310C mutations), which is evidence of two recombination events (one between positions 1028 and 1072, and another between positions 1072 and 1310). A total of five novel point mutations were found in the four sequenced genes.

Example 4

Recombination of two thermostable subtilisin E variants with internal defined primers and wandering chain extension

This example demonstrates that the primer-based recombination method is suitable for generating new sequences containing new combinations of mutations present in the original sequences. We also demonstrate the applicability of the primer-based recombination method to further improve enzyme function (in this case, thermal stability). The example also illustrates that primer-based recombination can introduce asymmetry into the recombination such that recombination occurs mostly in the part of the sequence defined by the primers (the part covered by the primers). Example 4 also demonstrates that specific mutations can be introduced into the recombinant sequences by using a suitable primer sequence or sequences containing the desired mutation(s).

The two heat-stable subtilisin E variant encoding genes (R1 and R2) used in Example 1 were recombined using the defined primer-based recombination method using first primers. Figure 6 shows the four defined internal primers used to generate recombinant progeny genes from the R1 and R2 template genes in this example. Primer P50F contains a mutation (A->T at base 598) that eliminates the HindIII restriction site and simultaneously creates a new, unique NheI site. This primer was used to demonstrate that specific mutations can be introduced into the population of recombinant sequences by specifically designing the defined primer. The R2 gene also contains an A->G mutation at the same site that eliminates the HindIII site. Thus, restriction analysis of random clones from the recombinant library (digested with NheI and HindIII) can be used to demonstrate the efficiency of recombination and the introduction of specific mutations introduced by the mutagenic primer. Sequence analysis of randomly picked (unscreened) clones provides additional information about the recombination and mutagenic events that occur during recombination based on the specific primer.

Materials and methods

Primer-based recombination

A variant of the recombination based on a specific primer, which was also performed using StEP, is shown in Figure 2.

1. Production of genes to be recombined

Approximately 10 μg of plasmid containing the R1 and R2 genes were digested at 37°C for 1 h with Nbel and BamHI [using 30 units of each (50 μΐ once in B buffer (Boehringer Mannheim, Indianapolis, IN, USA)]. Inserts of approximately 1 kb were purified on a 0.8% preparative agarose gel using the QIAEX TT gel extraction kit. The DNA inserts were dissolved in 10 mM Tris-HCl buffer (pH 7.4). DNA concentrations were estimated and the inserts were mixed 1:1 to a final concentration of 50 ng/μΐ.

2. Wandering chain extension PCR and gene assembly

The following conditions were used (in a final volume of 100 μΐ): approximately 100 ng of insert as template, 50 ng of each of the four internal primers, one Taq buffer, 0.2 mM of each of the four dNTPs, 1.5 mM MgCl ₂ and 0.25 units of Tag polymerase. The program was as follows: seven cycles of 30 seconds at 94°C, 15 seconds at 55°C, followed by another 10 cycles of 30 seconds at 94°C, 15 seconds at 55°C, 5 seconds at 72°C (roaming extension), followed by 53 cycles of 30 seconds at 94°C, 15 seconds at 55°C, and one minute at 72°C (gene assembly).

3. DpnI digestion of templates

1 μΐ of the reaction mixture was diluted to 9.5 μΐ with distilled H ₂ 0, then 5 μΐ of DpnI restriction enzyme was added, the DNA template was digested for 45 minutes, and the mixture was incubated at 70 C° for 10 minutes. Then, as a template,

- 44 10 μΐ were used for a PCR lasting 10 cycles.

4. PCR amplification of reassembled products

PCR conditions (final volume 100 μΐ) were as follows: 30 pmol each of the external primers P5N and P3B, 1 time Taq buffer, 0.2 mM of each dNTP, and 2.5 units of Taq polymerase. The following PCR program was used: 10 cycles of 30 seconds at 94°C and 30 seconds at 55°C, followed by 1 minute at 72°C. After running the program, a single band of the correct size appeared. The product was purified and subcloned into the pBE3 transfer vector. This gene library was then amplified in E. coli HB101 cells and then transferred into B. subtilis DB428 competent cells for expression and screening, as previously described (35). The thermal stability of the enzyme variants was determined in a 96-well plate-based array, as previously described (33).

DNA sequencing

Ten E. coli HB101 transformants were selected for sequencing. The genes were purified using the QIAEprep spin plasmid miniprep kit to obtain DNA of suitable quality for sequencing. Sequencing was performed using an ABI 373 DNA sequencing system with the Dye Terminator Cycle Sequencing kit (Perkin-Elmer, Branchburg, NJ, USA).

- 45 Results

1) Restriction analysis:

Forty clones randomly picked from the recombinant library were digested with NheI and BamHI restriction enzymes. In a separate experiment, the same 40 plasmids were also digested with HindIII and BamHI enzymes. These reaction products were analyzed by gel electrophoresis. As shown in Figure 7, 8 of the 40 clones (approximately 20%) contained the newly introduced NheI restriction site, confirming that the mutagenic primer was indeed able to introduce the specific mutation into the population.

2) DNA sequence analysis

The first 10 randomly picked clones were subjected to sequence analysis, the results of which are summarized in Figure 8. At least 6 of the 10 genes recombined. The minimum crossover event (recombination) between the R1 and R2 genes of the 6 genes varies from 1 to 4. All of the visible crossovers occurred within the region defined by the four primers. Mutations outside this region rarely, if ever, recombine, as shown by the fact that no recombination occurred between the two mutations at base positions 484 and 520. These results indicate that the specific primers can introduce asymmetry into the recombination, so that it occurs most frequently in the region of the sequence defined by the primers (within the primers). Mutations that are very close to each other tend to stay together (for example, base substitutions 731 and 745 and 1141 and

(Base substitution 1153 always remains as a pair.) However, the sequence of clone 7 shows that two mutations located 33 bases apart can recombine (base positions 1107 and 1141).

During the procedure, 23 new point mutations were introduced into the ten genes. This error rate (0.23%) corresponds to two to three new point mutations per gene, a rate previously considered optimal for directed enzymatic evolution to generate mutant libraries (15). The types of mutations are shown in Table 4. The mutations are mainly base substitutions and are evenly distributed along the gene.

Table 4

Novel point mutations identified in 10 recombinant genes

Base exchange Frequency Transversion Frequency G - > The 4 The - > T 1 The - + G 4 The - + c 1 C - + T 3 C - + The 1 T - > C 5 C - > G 0 G — > C 1 G — + T 0 T - > The 3 T - > G 0

A total of 9860 bases were sequenced. The mutation rate was 0.23%.

4) Phenotype analysis

Approximately 450 B. subtilis DB428 clones were picked and grown in SG medium supplemented with 20 µg/ml kanamycin in 96-well plates. Approximately 56% of the clones expressed active enzymes. Based on previous experience, this degree of inactivation corresponds to a mutation rate of the order of 2–3 mutations/gene (35). Approximately 5% of the clones showed a phenotype consistent with the double mutant (N181D + N218S) (which is below the expected 25%, which would correspond to random recombination primarily due to point mutagenesis). (DNA sequencing showed that two clones, 7 and 8, out of ten randomly picked clones, contained both the N218S and N181D mutations).

Example 6

Optimization of the ECB deacylase of Actinoplanes utahensis using a random priming-based recombination process

In this example, the method of the invention was used to prepare short DNA fragments from denatured, linear, double-stranded DNA (e.g., restriction fragments purified by gel electrophoresis; 22). The purified DNA, mixed with a polar excess of primer, was denatured by boiling, and then the synthesis was carried out using the Klenow fragment of E. coli DNA polymerase I. This enzyme has no 5'->3' exonuclease activity, so the product produced by random priming is exclusively from the primers starting from the primers.

- 48 is formed by chain elongation and is not degraded by exonuclease. The reaction is carried out at pH 6.6, where the 3' ->5’ exonuclease activity of the enzyme is greatly reduced (36). These conditions are favorable for random initiation of synthesis.

The procedure consists of the following steps:

1. The DNA in question is cleaved with an appropriate restriction endonuclease or endonucleases, and the corresponding DNA fragment is purified by gel electrophoresis using the Wizard PCR Prep Kit (Promega, Madison, WI, USA). For example, the ECB deacylase gene of Actinoplanes utahensis was excised from recombinant plasmid pSHP100 as a 2.4 kb XhoI-Psh AI fragment. It was important to linearize the DNA for the next denaturation step. The fragment was purified by agarose gel electrophoresis using the Wizard PCR Prep Kit (Promega, Madison, WI, USA) (Figure 9, step a). Gel purification is also important to remove the restriction endonuclease buffer from the DNA, as Mg2 ⁺ ions make it difficult to denature the DNA in the next step.

2. 400 ng (approximately 0.51 pmol) of double-stranded DNA was dissolved in H ₂ O and mixed with 2.75 μg (approximately 1.39 nmol) of dp(N) ₆ random primer. After immersing the mixture in boiling water for three minutes, it was immediately placed in an ice-cold ethanol bath.

The size of products produced by random priming is inversely related to the concentration of the primer (33). The presence of high concentrations of primers is likely to

- 49 led to spherical inhibition. Under the reaction conditions described in the example, the products produced by random priming are approximately 200-400 bp long, as determined by alkaline agarose gel electrophoresis (Figure 9, step b).

3. 10 μΐ of 10-fold reaction buffer (10-fold buffer: 900 mM HEPES, pH 6.6, 0.1 M magnesium chloride, 10 mM dithiothreitol, 5 mM dATP, dCTP, dGTP and dTTP) was added to the denatured sample, then the total reaction volume was made up to 95 μΐ with H ₂ 0.

4. 10 units (approximately 5 μΐ) of E. coli DNA polymerase I Klenow fragment were added to the mixture. All components were mixed gently by tapping the outer wall of the tube and then centrifuged at 12,000 g for 1-2 seconds in a microfuge tube to remove all liquid to the bottom of the tube. The reaction was carried out at 22°C for 35 minutes.

The rate of chain extension depends on the concentration of the template and the concentrations of the four nucleotide precursors. Since the reaction was carried out under conditions that minimized exonucleolytic digestion, the newly synthesized products were not degraded to any detectable extent.

5. After allowing the reaction to proceed for 35 minutes at 22 C°, it was frozen by cooling the sample to 0 C° on ice. 100 μΐ ice-cold H ₂ O was added to the reaction mixture.

6. Randomly primed products were purified by eluting the entire reaction mixture with Centricon-100 (to remove template and proteins) and Centricon-10

- 50 filters (to remove primers and fragments smaller than 50 bases). Centricon filters were purchased from Amicon Inc. (Beverly, MA, USA). The retained fraction (approximately 85 μΐ) was recovered from Centricon 10. This fraction contained the desired random priming products (Figure 9, step c) and was thus used to reassemble the entire gene.

Reassembly of the entire gene was performed by performing the following steps:

1. For gene assembly by PCR, the following were mixed on ice: 5 μΐ randomly primed DNA fragment from Centricon-10, 20 μΐ 2x PCR master mix [5x diluted cloning Pfu buffer, 0.5 mM of each dNTP, 0.1 unit/μΐ cloned Pfu polymerase (Stratagene, La Jolla, CA, USA)], 8 μΐ 30% v/v glycerol, and 7 μΐ H ₂ 0. Since the concentration of randomly primed DNA fragments used for assembly is the most important variable, it is preferable to perform several separate reactions with different concentrations to find the preferred concentration.

2. After incubation at 96°C for 6 minutes, 40 temperature cycles were performed, each cycle program was as follows: 1.5 minutes at 95°C, 1.0 minutes at 55°C, and 1.5 minutes + 5 seconds/cycle at 75°C, with the chain extension step at 72°C for 10 minutes in the last cycle. The cycles were performed on a DNA Engine PTC-200 (MJ

- 51 Research Inc., Watertown, MA, USA) without the addition of mineral oil.

3. At cycles 20, 30, and 40, 3 μΐ aliquots of the reaction mixture were removed and analyzed by agarose gel electrophoresis. At cycle 40, the reassembled PCR product contained the correct size product, along with smaller and larger products (see Figure 9, step ”d).

The properly assembled products of this first PCR were further amplified in a second PCR run using PCR primers complementary to the ends of the template DNA. The amplification procedure was as follows:

1. 2.0 μΐ aliquots of the assembly PCR mixture were used as templates in standard PCR reactions performed in a volume of 100 μΐ, which contained 0.2 μΜ of each of the following primers: xhoF28 (5'-GGTAGAGCGAGTCTCGAGGGGGAGATGC-3'; SEQ ID NO: 13) and pshR22 (5'-AGCCGGCGTGACGTGGGTCAGC-3'; SEQ ID NO: 14), as well as 1.5 mM MgCl ₂ , 10 mM Tris-HCl (pH 9.0), 50 mM potassium chloride, 200 mM each of dNTPs, 6% glycerol, 2.5 units of Taq polymerase (Promega, Madison, WI, USA) and 2.5 units of pfu polymerase (Stratagene, La Jolla, CA, USA) .

2. After incubating the mixture at 96°C for 5 minutes, 15 temperature cycles were performed, the program of which was as follows: 1.5 minutes at 95°C, 1.0 minutes at 55°C and 1.5 minutes at 72°C, followed by another 15 temperature cycles: 1.5 minutes at 95°C, 1.0 minutes at 55°C and 1.5 minutes + 5 seconds.

- 52 min/cycle at 72 C°, while in the last cycle the chain extension step lasted for 10 min at 72 C°. The cycles were performed in a DNA Engine PTC-200 (MJ Research Inc., Watertown, MA, USA) without the addition of mineral oil.

3. The amplification resulted in a large amount of PCR product corresponding to the size of the entire ECB deacylase gene (Figure 9, step e).

Cloning was performed as follows:

1. The PCR product of the ECB deacylase gene was digested with XhoI and PshAI restriction enzymes and then cloned into a modified pIJ702 vector.

2. S. lividans TK23 protoplasts were transformed with the above ligation mixture, creating a mutant library.

In situ screening of ECB deacylase mutants

Each transformant of the S. lividans TK23 library prepared as described above was screened for deacylase activity using ECB as a substrate in an in situ plate assay. Transformed protoplasts were allowed to regenerate on R2YE agar plates by incubation for 24 h at 30°C and then developed by incubation in the presence of thiostrepton for an additional 48-72 h. When colonies reached the appropriate size, 6 ml of 45°C purified agarose (Sigma) (containing 0.5 mg/ml ECB in 0.1 M sodium acetate buffer, pH 5.5) was poured onto the top of each R2YE agar plate and development was continued for an additional 18-24 h at 37°C. The wild-type recombinant plasmid, pSHP150

Colonies surrounded by a clearing zone larger than the control colony containing -53 2 showed more efficient ECB hydrolysis, which was due to an improved enzyme property or increased expression or secretion levels. These colonies were selected as potential positive mutants. The colonies were picked for further preservation and manipulation.

HPLC analysis of ECB deacylase mutants

Each positive transformant was inoculated into 20 ml of fermentation medium containing 5 μg/ml thiostrepton and grown at 30°C for 48 h. At this stage, each culture was subjected to HPLC analysis using ECB as substrate. 100 μΐ of the total reaction mixture was used for each HPLC reaction, which was carried out at 30°C for 30 min in the presence of 0.1 M NaAc (pH 5.5), 10% MeOH and 200 pg/ml ECB substrate. 20 μΐ of each reaction mixture was loaded onto a PolyLC polyhydroxyethylaspartamide column (4.6 x 100 mm) and eluted with an acetonitrile gradient at a flow rate of 2.2 ml/min. The ECB core was detected at 225 nm.

Purification of ECB deacylase mutants

After HPLC analysis, 50 ml of fermentation medium was inoculated with 2.0 ml precultures prepared separately from each possible positive mutant and the cells were grown at 30°C for 96 hours with shaking at 280 rpm. These 50 ml cultures were then centrifuged at 7000 g for 10 minutes. The supernatants were again decanted

- 54 were centrifuged at 16 g for 20 minutes. The supernatants containing the mutant ECB deacylase enzymes were stored at -20 C°.

Supernatants from positive mutants were further concentrated to 1/30 of their original volume using an Amicon filter unit with a filter cut-off of 10 kDa. The resulting enzyme samples were diluted with an equal volume of 50 mM KH ₂ PO ₄ buffer (pH 6.0) and 1.0 ml of this was loaded onto a Hi-Trap ion exchange column. The binding buffer was 50 mM KH ₂ PO ₄ (pH 6.0) and the elution buffer was 50 mM KH ₂ PO ₄ (pH 6.0) and 1 M NaCl. A linear gradient of 8 column volumes of NaCl from 0 M to 1.0 M was applied at a flow rate of 2.7 ml/min. The ECB-deacylase mutant fraction was eluted with 0.3 M NaCl, concentrated, and the buffer was exchanged with 50 mM KH ₂ PO ₄ (pH 6.0) in Amicon Centricon-10 units. The purity of the enzyme was confirmed by SDS-PAGE, and the concentration was determined using the Bio-Rad Protein Assay.

Specific activity assay procedure for ECB deacylase mutants

4 μg of each purified ECB deacylase mutant was used in the activity assay, which was performed at 30°C for 0-60 min in the presence of 0.1 M NaAc (pH 5.5), 10% MeOH (v/v) and 200 μg/ml ECB substrate. 20 μΐ of each reaction mixture was loaded onto a PolyLC-aspartamide column (4.6 x 100 mm) and eluted with an acetonitrile gradient at a flow rate of 2.2 ml/min. The reaction products were monitored at 225 nm and the data were recorded using an IBM PC data acquisition system. The peak of the ECB core

- 55 were numerically integrated to calculate the specific activity of each mutant.

As shown in Figure 10, when this random priming method is applied to the wild-type ECB deacylase gene in just one round, a mutant (M16) from the original 2012 transformant was found to have a 2.4-fold higher specific activity than the wild-type enzyme. Figure 11 shows that the activity of M16 was increased compared to the wild-type enzyme over a wide pH range.

Example 7

Enhancing the thermal stability of Bacillus subtilis subtilisin E by a recombination method based on random sequence primers

This example demonstrates that different DNA polymerases can be used in primer-based recombination. The example also demonstrates the stabilization of subtilisin E by recombination.

In this example, the genes of the two thermostable subtilisin E variants described in Example 1, the R1 and R2 genes, were used as templates for recombination.

(1) Production of target genes

The genes of the thermostable mutant of subtilisin E, the R1 and R2 genes (Figure 11), were subjected to random primer DNA synthesis. The 986 bp fragment, which included the 45 nucleotide subtilisin E prosequence, the complete mature sequence, and the 113 nucleotides after the stop codon, was generated by digesting the pBE3 plasmid with 2x BamHI and NdeI, and then 0.8%

- 56 were purified from agarose gels using the Wizard PCR Prep Kit (Promega, Madison, WI, USA). It was essential to linearize the DNA for the next denaturation step. Gel purification was also essential to remove the restriction endonuclease buffer from the DNA, as MG ²⁺ ions make it difficult to denature the DNA in the next step.

(2) Random-primed DNA synthesis

Short DNA fragments were generated from denatured, linear, double-stranded DNA by random primer DNA synthesis. Purified B. subtilis subtilisin E mutant genes were mixed with a molar excess of primers, denatured by boiling, and then synthesis was performed using one of the following DNA polymerases: Klenow fragment of E. coli DNA polymerase I, T4 bacteriophage DNA polymerase, and T7 sequence version 2.0 DNA polymerase.

Under optimal operating conditions (29), bacteriophage T4 DNA polymerase produces similar synthesis results to the Klenow fragment. When T7 sequence version 2.0 DNA polymerase (31, 32) was used, the length of the synthesized DNA fragments was generally greater. During the synthesis, a small amount of MnCl ₂ can be added to the mixture in order to adjust the length of the synthesized fragments to between 50 and 400 bases.

Short, nascent DNA fragments can also be produced by PCR with Pfu DNA polymerase or the Stoffel fragment of Tag DNA polymerase. It is important to proceed with caution and to determine the conditions under which

- 57 which ensure that short, random primers can hybridize to templates and provide sufficient DNA amplification at higher temperatures. We have found that primers of approximately 12 nucleotides [dp(N) ₁₂ ] can be used to generate fragments in PCR.

2.1 Random-primed DNA synthesis with Klenow fragment

The Klenow fragment of E. coli DNA polymerase I lacks 5’ —> 3’ exonuclease activity, so the random primer product is synthesized exclusively by primer extension and is not degraded by exonuclease. The reaction was performed at pH 6.6, where the 3’ -> 5' exonuclease activity of the enzyme is greatly reduced (36). These conditions favor random initiation of synthesis.

1. 200 ng (approximately 0.7 pmol) of Rl-DNA and an equal amount of R2-DNA were dissolved in H ₂ 0 and mixed with 13.25 pg (6.7 nmol) of dp(N) ₆ random primer. After placing the sample in boiling water for 5 min, the mixture was immediately placed in an ice-cold ethanol bath.

The size of the products produced by random priming is inversely proportional to the concentration of the primer (30). The presence of high concentrations of primers presumably led to spherical inhibition. Under the reaction conditions described in the example, the products produced by random priming are approximately 50-500 bp in length, as determined by agarose gel electrophoresis.

2. 10 μΐ of 10-fold reaction buffer (10-fold buffer: 900 mM HEPES, pH 6.6, 0.1 M magnesium chloride, 20 mM dithiothreitol, 5 mM dATP, dCTP, dGTP and dTTP) was added to the denatured sample, then the total reaction volume was made up to 95 μΐ with H ₂ 0.

3. 10 units (approximately 5 μΐ) of E. coli DNA polymerase I Klenow fragment (Boehringer Mannheim, Indianapolis, IN, USA) were added to the mixture. All components were mixed gently by tapping the outer wall of the tube and then centrifuged at 12,000 g for 1–2 seconds in a microfuge tube to remove all liquid to the bottom of the tube. The reaction was carried out at 22°C for 3 hours.

The rate of chain extension depends on the concentration of the template and the concentrations of the four nucleotide precursors. Since the reaction was carried out under conditions that minimized exonucleolytic digestion, the newly synthesized products were not degraded to any detectable extent.

4. After allowing the reaction to proceed for 3 hours at 22 C°, it was frozen by cooling the sample to 0 C° on ice. 100 μΐ ice-cold H ₂ O was added to the reaction mixture.

5. Random primer products were purified by passing the entire reaction mixture through a Microcon-100 filter (to remove template and proteins) and a Microcon-10 filter (to remove primers and fragments smaller than 40 bases). The Microcon filters were purchased from Amicon Inc. (Beverly, MA, USA). The retained fraction (approximately 65 μΐ) was recovered from the Microcon 10. In this fraction, which is the desired random primer,

- It contained products produced by 59 primers, the buffer was replaced with PCR reaction buffer, with the new Microcon 10, and this fraction was used to reassemble the entire gene.

2.2 Random-primed DNA synthesis with T4 bacteriophage DNA polymerase

Bacteriophage T4 DNA polymerase and the Klenow fragment of E. coli DNA polymerase I are similar in that they have 5' —>3' polymerase activity and 3' -> 5' exonuclease activity. The exonuclease activity of bacteriophage T4 DNA polymerase is more than 200-fold greater than that of the Klenow fragment. Because this enzyme does not remove short oligonucleotide primers from single-stranded DNA templates (23), its mutagenesis efficiency differs from that of the Klenow fragment.

1. 200 ng (approximately 0.7 pmol) of Rl-DNA and an equal amount of R2-DNA were dissolved in H ₂ 0 and mixed with 13.25 μg (6.7 nmol) of dp(N) ₆ random primer. After the sample was placed in boiling water for 5 min, the mixture was immediately placed in an ice-cold ethanol bath. The presence of a high concentration of primer presumably leads to spherical inhibition.

2. 10 μΐ of 10-fold reaction buffer [10-fold buffer: 500 mM Tris-HCl, pH 8.8; 150 mM (NH ₄ ) ₂ SO ₄ , 70 mM magnesium chloride, 100 mM 2-mercaptoethanol, 0.2 mg/ml bovine serum albumin, and 2 mM each of dATP, dCTP, dGTP and dTTP] was added to the denatured sample, then the total reaction volume was made up to 90 μΐ with H ₂ O.

3. 10 units (approximately 10 μΐ) of T4 DNA polymerase I (Boehringer Mannheim, Indianapolis, IN, USA) were added to the mixture. All components were mixed gently by tapping the outer wall of the tube and then centrifuged at 12,000 g for 1-2 seconds in a microfuge tube to remove all liquid to the bottom of the tube. The reaction was carried out at 37°C for 30 minutes. Under the reaction conditions described in the example, the size of the products generated by random priming is between approximately 50 and 500 bp.

4. After allowing the reaction to proceed for 30 minutes at 37 C°, it was frozen by cooling the sample to 0 C° on ice. 100 μΐ ice-cold H ₂ O was added to the reaction mixture.

5. The random primer products were purified by passing the entire reaction mixture through a Microcon-100 (to remove template and proteins) and a Microcon-10 filter (to remove primers and fragments smaller than 40 bases). The retained fraction (approximately 65 μΐ) was recovered from the Microcon 10. In this fraction, which contained the desired random primer products, the buffer was replaced with PCR reaction buffer, with fresh Microcon 10, and this fraction was used to reassemble the entire gene.

2.3 Random-primed DNA synthesis with T7-Sequenase v2.0 DNA polymerase

Because T7 Sequenase v2.0 DNA polymerase lacks exonuclease activity and is a fast-moving enzyme, the average length of the synthesized DNA is greater than that of DNA synthesized by Klenow fragment or T4 DNA polymerase.

- 61 However, by adding a sufficient amount of McC1 ₂ to the reaction mixture, the size of the synthesized fragments can be controlled and reduced to below 400 bp.

1. 200 ng (approximately 0.7 pmol) of Rl-DNA and an equal amount of R2-DNA were dissolved in H ₂ O and mixed with 13.25 μg (6.7 nmol) of dp(N) ₆ random primer. After placing the sample in boiling water for 5 min, the mixture was immediately placed in an ice-cold ethanol bath. The presence of a high concentration of primer presumably leads to spherical inhibition.

2. 10 μΐ of 10-fold reaction buffer (the composition of the 10-fold buffer: 400 mM Tris-HCl, pH 7.5; 200 mM magnesium chloride, 500 mM NaCl, 3 mM MnCl ₂ and 3 mM each of dATP, dCTP, dGTP and dTTP) was added to the denatured sample, then the total volume of the reaction mixture was made up to 99.2 μΐ with H ₂ 0.

3. 10 units (approximately 0.8 μΐ) of T7-sequenase v2.0 (Amersham Life Science, Cleveland, Ohio, USA) were added to the mixture. All components were mixed by gently tapping the outside of the tube, and the sample was then centrifuged at 12,000 g for 1–2 seconds in a microfuge tube to remove all liquid to the bottom of the tube. The reaction was performed at 22°C for 15 minutes. Under the reaction conditions described here, the size of the random primer products is approximately 50–400 bp.

4. After allowing the reaction to proceed for 15 minutes at 22 C°, it was frozen by cooling the sample to 0 C° on ice. 100 μΐ ice-cold H ₂ O was added to the reaction mixture.

5. The random priming products were purified by passing the entire reaction mixture through a Microcon-100 filter (to remove template and proteins) and a Microcon-10 filter (to remove primers and fragments smaller than 40 bases). The retained fraction (approximately 65 μΐ) was recovered from the Microcon 10. In this fraction, which contained the desired random priming products, the buffer was replaced with PCR reaction buffer, with fresh Microcon 10, and this fraction was used to reassemble the entire gene.

2.4 Random-primed DNA synthesis by PCR using the Stoffer fragment of Taq DNA polymerase

Like the Klenow fragment of E. coli DNA polymerase I, the Stoffel fragment of Taq DNA polymerase lacks 5’-3’ exonuclease activity. It is also more thermostable than Taq DNA polymerase. The Stoffel fragment has a slow propagation speed, extending a primer by an average of only 5-10 nucleotides before dissociating. Due to its slower propagation, it copies with greater accuracy.

1. 50 ng (0.175 pmol) of Rl-DNA and an equal amount of R2-DNA were dissolved in H ₂ 0 and mixed with 6.13 μg (approximately 1.7 nmol) of dp(N) _i2 random primer.

2. 10 μΐ of a 10-fold concentration premix (composition: 100 mM Tris-HCl, pH 8.3; 30 mM magnesium chloride, 100 mM potassium chloride, 2 mM each of dATP, dCTP, dGTP and dTTP) was added to the reaction mixture, then the total volume of the mixture was made up to 99 μΐ with H ₂ 0.

3. After incubation at 96°C for 5 min, 2.5 units (approximately 1.0 μΐ) of Taq DNA polymerase Stoffelfragmens (Perkin-Elmer Corp., Norwalk, CT) were added. 35 temperature cycles were performed, each with the following program: 60 s at 95°C, 60 s at 55°C, and 50 s at 72°C, this time without chain extension in the final step. The reaction was performed in a DNA Engine PTC-200 (MJ Research, Inc., Watertown, MA, USA). Under the reaction conditions described here, the length of the priming products was approximately 50-500 bp.

4. The reaction was stopped by cooling the sample on ice to 0 C°. 100 μΐ ice-cold H ₂ O was added to the reaction mixture.

5. The random primer products were purified by passing the entire reaction mixture through a Microcon-100 (to remove template and proteins) and a Microcon-10 filter (to remove primers and fragments smaller than 40 bases). The retained fraction (approximately 65 μΐ) was recovered from the Microcon 10. In this fraction, which contained the desired random primer products, the buffer was replaced with PCR reaction buffer, with fresh Microcon 10, and this fraction was used to reassemble the entire gene.

2.5 Random-primed DNA synthesis by PCR using PfuDNA polymerase

Pfu DNA polymerase is particularly thermostable, and the enzyme has inherent 3’ —>5’ exonuclease activity, but no

- 64 5'->3' exonuclease activity. Its base substitution accuracy is estimated at 2xl0 ⁶ .

1. 50 ng (0.175 pmol) of Rl-DNA and an equal amount of R2-DNA were dissolved in H ₂ 0 and mixed with 6.13 μg (approximately 1.7 nmol) of dp(N) _i2 random primer.

2. 50 μΐ of a 2-fold concentration premix [composition: 5-fold diluted cloned-Pfu-buffer (Stratagene, La Jolla, CA, USA), 2 mM each of dATP, dCTP, dGTP and dTTP] was added to the reaction mixture, then the total volume of the mixture was made up to 99 μΐ with H ₂ 0.

3. After incubation at 96°C for 5 min, 2.5 units (approximately 1.0 μΐ) of Pfu DNA polymerase (Stratagene, La Jolla, CA, USA) were added. 35 temperature cycles were performed, each with the following program: 60 s at 95°C, 60 s at 55°C, and 50 s at 72°C, this time without extension in the final step. The reaction was performed in a DNA Engine PTC200 (MJ Research, Inc., Watertown, MA, USA). Under the reaction conditions described here, the length of the priming products was approximately 50-500 bp.

4. The reaction was stopped by cooling the sample on ice to ⁰ C°. 100 μΐ ice-cold H ₂ O was added to the reaction mixture.

5. Randomly primed products were purified by passing the entire reaction mixture through a Microcon-100 filter (to remove template and proteins) and a Microcon-10 filter (to remove primers and fragments smaller than 40 bases). The retained fraction

- 65 (about 65 μΐ) was recovered from Microcon 10. In this fraction, which contained the desired random priming products, the buffer was replaced with PCR reaction buffer, with new Microcon 10, and this fraction was used to reassemble the entire gene.

(3) Reassembly of the entire gene

1. For PCR assembly, 10 μΐ DNA fragment from Microcon10, 20 μΐ twice-concentrated PCR premix [5-fold diluted cloned Pfu buffer, 0.5 mM dNTPs each, 0.1 unit/μΐ cloned Pfu polymerase (Stratagene, La Jolla, USA)], and 15 μΐ H ₂ O were mixed on ice.

2. After incubation at 96°C for three minutes, 40 temperature cycles were performed with the following program: 1.0 min at 95°C, 1.0 min at 55°C, and 1.0 min +5 sec/cycle at 72°C, with the chain extension step of the last cycle at 72°C for 10 minutes in a DNA Engine PTC-200 (MJ Research, Inc., Watertown, MA, USA) without the addition of mineral oil.

3. At cycles 20, 30 and 40, 3 μΐ aliquots were removed from the reaction mixture and analyzed by agarose gel electrophoresis. The reassembled PCR product after 40 cycles contained the correct size product in a band smeared in the direction of the lower and higher lifetime ranges.

(4) Amplification

The correctly assembled product of this first PCR was further amplified in a second PCR run, during which the

- PCR primers complementary to the ends of 66 template DNA were used.

1. 2.0 μΐ aliquots of the gene assembly PCR mixture were used as templates in 100 μΐ standard PCR reactions containing 0.3 mM each of the P1 primer (5'CCGAGCGTTGC ATATGTGGAAG3'; SEQ ID NO: 15) and the P2 primer (5'CGACTCTAGAGGATCCGATTC3'; SEQ ID NO: 16), 1.5 mM MgCl ₂ , 10 mM Tris-HCl (pH 9.0), 50 mM potassium chloride, 200 mM each of four dNTPs, 2.5 units of Taq polymerase (Promega, Madison, WI, USA), and 2.5 units of Pfu polymerase (Stratagene, La Jolla, CA, USA).

2. After incubation for three minutes at 96°C, 15 temperature cycles were performed with the following program: 60 seconds at 95°C, 60 seconds at 55°C, and 50 seconds at 72°C, followed by another 15 temperature cycles with the following program: 60 seconds at 95°C, 60 seconds at 55°C, and 50 seconds (+5 seconds/cycle) at 72°C, with the chain extension step in the last cycle lasting 10 minutes at 72°C. The reaction was performed in a DNA Engine PTC-200 (MJ Research, Inc., Watertown, MA, USA) without the addition of mineral oil.

3. The amplification resulted in a large amount of PCR product, corresponding in size to the entire subtilisin E gene.

(5) Cloning

Since the short DNA fragments were produced with five different DNA polymerases, the final amplified, assembled

- Five different mixtures of 67 assembled PCR gene products were available. Each DNA mixture was used to create the corresponding mutant subtilisin E library.

1. The PCR-amplified assembled product was purified using the Wizard DNA-CleanUp kit (Promega, Madison, WI, USA), digested with BamHI and NdeI, and electrophoresed in a 0.8% agarose gel. The 986 bp product was excised from the gel and purified using the Wizard PCR Prep kit (Promega, Madison, WI, USA). The products were ligated with the vector obtained from the pBE3 transfer vector by digestion with BamHI and NdeI.

2. E. coli HB101 competent cells were transformed with the above ligation mixture to generate a mutant library. Approximately 400 transformants from the library were pooled and a recombinant plasmid mixture was isolated from the pooled sample.

3. B. subtilis DB428 competent cells were transformed with the above isolated plasmid mixture, thus creating another library of subtilisin E variants.

4. The five libraries prepared from short nascent DNA fragments were named as follows, according to the DNA polymerase used for random priming: library/Klenow, library/T4, library/sequenase, library/Stoffel, and library/Pfu. Approximately 400 transformants were randomly picked from each library and tested for thermal stability [see step (7)].

(6) Sequencing of random clones

Ten random clones were selected from the B. subtilis DB428 library/Klenow for DNA sequence analysis. Recombinant plasmids were individually purified from B. subtilis DB428 using the QIAprep spin plasmid miniprep kit (QIAGEN) with the modification that 2 mg/ml lysozyme was added to the PI buffer and the cells were incubated for 5 min at 37°C, then transformed back into competent E. coli HB101 cells and repurified using the QIAprep spin plasmid miniprep kit to obtain DNA of sufficient quality for sequencing. Sequencing was performed on an ABI 373 DNA sequencing system using the Dye Terminator Cycle Sequencing reagent kit (Perkin-Elmer Corp., Norwalk, CT, USA).

(7) Screening for thermal stability

Approximately 400 transformants were screened from each of the five libraries described in step (4). Screening was performed using the previously described assay procedure (33, 35) using succinyl-Ala-Ala-Pro-Phe-p-nitroanilide (SEQ ID NO: 25) as the substrate. B. subtilis DB428 containing the plasmid library was grown on LB plates containing 20 pg/ml kanamycin at 37°C. After 18 hours, one colony per well was inoculated into a 96-well plate containing 100 μΐ kanamycin in SG medium. These colonies were shaken and incubated at 37°C for 24 hours to allow the cells to grow to saturation. The cells were then centrifuged and a sample of the supernatant was taken for thermal stability analysis.

- For 69 assays. Each culture plate was replicated in triplicate, i.e. 96-well replica assay plates were prepared, each well containing 10 ml of supernatant. Subtilisin activities were measured by adding 100 μl of activity assay solution [0.2 mM succinyl-Ala-Ala-Pro-Phe-p-nitroanilide (SEQ ID NO: 25, 100 mM Tris-HCl, 10 mM CaCl ₂ , pH 8.0, 37°C]. The reaction rate was monitored at 405 nm for one minute using a ThermoMax microplate reader (Molecular Devices, Sunnyvale, CA, USA). The activity measured at room temperature was used to calculate the fraction of active clones (clones with less than 10% activity compared to wild type were considered inactive). The initial activity (Ai) was measured after incubating the assay plate at 65°C for 10 minutes by immediately adding 100 μl of pre-warmed (37°C) assay solution. [0.2 mM succinylAla-Ala-Pro-Phe-p-nitroanilide (SEQ ID NO: 25, 100 mM Tris-HCl, 10 mM CaCl ₂ , pH 8.0} to each well. The residual activity (Ar) was measured after 40 min of incubation.

(8) Sequence analysis

After screening, one clone that showed the highest thermal stability among the 400 transformants from the library/Klenow was inoculated onto a kanamycin-containing LB agar plate, and individual colonies grown on this plate were used to establish liquid cultures in tubes for the generation of glycerol stocks and plasmid preparation. The recombinant plasmid was prepared using a QIAprep spin plasmid miniprep

- 70 reagent kit (QUIAGEN) was used to purify the plasmid, modifying the procedure by adding 2 mg/ml lysozyme to PI buffer and incubating the cells for 5 min at 37°C, then transforming the plasmid into competent E. coli HB101 cells and then purifying it again with the QIAprep spin plasmid miniprep kit to obtain DNA of suitable quality for sequencing. Sequencing was performed on an ABI373 DNA sequencing system using the Dye Terminator Cycle Sequencing reagent kit (Perkin-Elmer Corp., Norwalk, CT, USA).

Results

1. Typical recombination frequency and efficiency for random sequence-based recombination

The random priming procedure was performed as described above. The procedure is illustrated in Figure 1. Ten clones were randomly selected from the mutant library/Klenow and sequenced. As summarized in Figure 12 and Table 5, each clone differed from the original gene. The frequency of a single point mutation in the recombinant genes relative to the original R1 or R2 genes varied between 40% and 70%, fluctuating around the expected value of 50%. This indicates that the two original genes recombined almost randomly when the random priming procedure was used. Figure 12 also shows that all 10 mutations could recombine or be excised, even though they were only 12 base pairs apart.

- 71 The rate of heat inactivation of subtilisin at 65°C was then estimated by analyzing 400 random clones collected from each of the five libraries prepared in step (5). The thermostability values obtained in a 96-well plate are shown in Figure 13, in descending order. Approximately 21% of the clones resulted in thermostability comparable to that of the mutant containing both the N181D and N218S mutations. This indicates that the N181D mutation from RC2 and the N218S mutation from RC1 recombined randomly. Sequence analysis of the clone with the highest thermostability among the 400 transformants screened from the library/Klenow indicated that the clone contained both the N181D and N218S mutations.

2. Frequency of newly introduced mutations during the random priming procedure

Approximately 400 transformants were picked from each of the five B. subtilis DB428 libraries [see step (5)] and cultured in 96-well plates in SG medium supplemented with 20 µg/ml kanamycin. These cultures were screened for subtilisin E activity. Approximately 77-84% of the clones expressed active enzyme, while 16-23% of the transformants were inactive, presumably due to newly introduced mutations. From previous experience, this inactivation rate is indicative of a mutation rate of the order of 1-2 mutations/gene (35).

As shown in Figure 12, 18 new point mutations were introduced during the procedure. This error rate, 0.18%, corresponds to 1-2 new point mutations per gene, which is in line with the

- Determined from 72 inactivation curves. Mutations are distributed almost randomly along the gene.

Table 5

Nucleotide and amino acid substitutions in 10 random clones from the library/Klenow

Clone Positive Base substitution Substitution type Amino acid substitution Substitution type C#1 839 The —> c transversion Gly -> Gly synonym C#2 722 The —> G transition Ser -> Cheese synonym C#2 902 T —> C transition With -> With synonym C#2 1117 C —> G transversion Ser —> Cheese synonym C#4 809 T -> C transition Asn —> Asn synonym C#4 1098 G —> C transversion Gly —> Under non-synonymous C#4 1102 T —> C transition Under —> Under synonym C#6 653 C — The transversion His -> Hey non-synonymous C#6 654 The —> T transversion His —Hey synonym of gender C#6 657 T —> C transition With —> Under non-synonymous C#6 658 The —> C transversion With —> Under non-synonymous C#6 1144 The —> G transition Under —> Under synonym C#6 1147 The —> G transition Under —> Under synonym C#7 478 T — C transition Hey —Hey synonym C#9 731 The G transition Under —> Under synonym C#9 994 The -> G transition With —> With synonym C#10 1111 The —> G transition Gly —Gly synonym C#10 1112 The —> T transversion Thr —> Cheese non-synonymous

- 73 The types of mutations are listed in Table 5. The direction of mutation is clearly not random. For example, A changes to G more often than to either T or C. All transitions, especially T-C and A-G, occur more frequently than transversions. Some nucleotides mutate more readily than others. One G-C, one C-G, and one C-A transversion were found in the ten clones sequenced. These mutations are very rare. During error-prone PCR mutagenesis of subtilisin (37) . The random priming procedure may allow for a wider range of amino acid substitutions than PCR-based point mutagenesis.

It is interesting to note that a short sequence fragment at positions 646-667 of the original R1 and R2 genes (5' C GGT ACG CAT GTA GCC GGT ACG 3', SEQ ID NO: 16) changed to the following sequence: 5'-C GGT ACG ATT GCC GCC GGT ACG 3', SEQ ID NO: 17) in the C#6 random clone. Since this sequence fragment contains a short segment repeated at both ends, the newly introduced mutation may be the result of a slipped-strand mispairing process, rather than a mere point mutation process. Since no phase shift occurs, this type of slippage may be useful during domain conversion.

3. Comparison of the copying accuracy of different DNA polymerases during the random priming process

During random primer recombination, homologous DNA sequences recombine almost randomly, and

- 74 in addition, new point mutations are generated. Although these point mutations may provide useful diversity in some in vitro evolutionary applications, they are problematic when recombining previously identified beneficial mutations, especially when the mutation rate is so high. Controlling the error rate in the random priming procedure is therefore particularly important if this method is to be successfully applied to solve in vitro evolutionary problems. By choosing different DNA polymerases and modifying the reaction conditions, the random priming molecular propagation method can be adapted to generate mutant libraries with different error rates.

The Klenow fragment of E. coli DNA polymerase I, the DNA polymerase of bacteriophage C4, the DNA polymerase of T7 sequence version 2.0, the Stoffel fragment of Taq polymerase, and the Pfu polymerase were tested in nascent DNA fragment synthesis. The activity profiles of the five resulting populations [see step (5)] are shown in Figure 13. To generate these profiles, the activities of the individual clones, as measured in the 96-well plate screening assay, were plotted in descending order. The library/Stoffel and the library/Klenow contained a higher percentage of wild-type or inactive subtilisin E clones than the library/Pfu. The percentage of wild-type and inactive clones in all five populations was 17-30%.

Example 8

Use of specific flanking primers and wandering primers for single-stranded DNA recombination

This example demonstrates the use of specific primer recombination supplemented with wandering primers during single-stranded DNA recombination.

Description of the procedure

Single-stranded DNA can be produced by several methods, most easily by using a helper phage, from a plasmid. Many vectors currently in use are derived from filamentous phages, such as the M13mp derivative. After transformation into cells, these vectors can either form a new double-stranded ring or a single-stranded ring derived from one of the two strands of the vector. The single-stranded rings are packaged into phage particles, which are secreted from the cells and can be easily purified from the culture supernatant.

Two specific primers (i.e., those that hybridize to the 5’ and 3’ ends of the template) were used in this example to recombine single-stranded genes. Only one of the primers is needed before final PCR amplification. The extended-strand recombination primers are first prepared in the wandering priming procedure (StEP), which consists of repeated cycles of denaturation followed by extremely shortened hybridization/extension step(s). The extended fragments are then assembled into full-length genes by homologous gene assembly using temperature cycling.

- 76 in the presence of DNA polymerase, followed by a gene amplification step.

The progress of the wandering primer process is monitored by removing aliquots (10 μΐ) from the reaction tube (100 μΐ starting volume) at different times during primer extension and separating the DNA fragments by agarose gel electrophoresis. Evidence of efficient primer extension is the appearance of a low molecular weight smear early in the process, which shifts toward the higher molecular weight range with increasing number of cycles. Initial reaction conditions are set to allow template denaturation (e.g., denaturation at 94°C for 30 seconds), followed by a very short hybridization/extension step (or steps) (e.g., 55°C for 1-15 seconds). This is continued for 5-20 cycles before the reaction is sampled. Typically, 20-200 cycles of wandering chain elongation are required to create single-stranded DNA smears that are larger than the length of the entire gene.

The experimental design was the same as in Example 1. Two thermostable mutant subtilisin E genes, the R1 and R2 genes, were subcloned into the M13mpl8 vector by digestion with EcoRI and BamHI restriction enzymes. Single-stranded DNA was prepared as described previously (39).

- 77 Recombination based on two flanking primers Two specific primers, P5N (5'-CCGAG CGTTG CATAT GTGGA AG-3', sequence given in ID number 18, the underlined section is the Mdel restriction site) and P3B (5'-CGACT CTAGA GGATC CGATT C-3', sequence given in ID number 19, the underlined section is the BamHI restriction site) were used during the recombination, which correspond to the 5'-end and 3'-end flanking primers, respectively. The conditions were as follows for a final volume of 100 μΐ: 0.15 pmol of single-stranded DNA containing the R1 or R2 gene (in a 1:1 ratio) was used as template, 15 pmol of one of the flanking primers (P5N or P3B), once Taq buffer, 0.2 mM each of the dNTPs, 1.5 mM MgCl ₂ and 0.25 units of Taq polymerase. The following program was used: 5 min at 95°C, 80-200 cycles of 30 s at 94°C, followed by 5 s at 55°C. The single-stranded DNA product of the appropriate size (approximately 1 kb) was excised from a 0.8% agarose gel after electrophoresis and purified using the QIAEX II Gel Extraction Kit. This purified product was amplified by conventional PCR. The conditions (in a final volume of 100 μΐ) were as follows: 1-10 ng of template, 30 pmol of each flanking primer, 1x Taq buffer, 0.2 mM of each dNTP, 1.5 mM MgCl ₂ and 0.25 units of Taq polymerase. The following program was used: 5 min at 95°C, 20 cycles of 30 s at 94°C, 30 s at 55°C, and 1 min at 72°C. The PCR product was purified,

- 78 was then digested with NdeI and BamHI and subcloned into the pBE3 transfer vector. This gene library was amplified in E. coli HB101 cells and transferred into B. subtilis DB428 competent cells for expression and screening as described elsewhere (35). The thermal stability of the enzyme variants was determined in a 96-well plate array as previously described (33).

This requirement leads to the generation of new sequences containing new combinations of mutations and new point mutations from the original sequences. Screening allows the identification of enzyme variants that are more thermostable than the original enzymes, as described in Example 1.

As is evident from the above examples, primer-based recombination can be used to explore the vast field of potential catalysts, achieving their optimal performance in a wide range of applications, and to develop new enzymes for fundamental structure-function studies.

Although the description describes the use of DNA-dependent DNA polymerases and single-stranded DNA as templates, other provisions can be made for the use of single-stranded RNA as templates. Using the mRNA of a given protein as a template and an RNA-dependent DNA polymerase (reverse transcriptase) as a catalyst, the methods described herein can be modified to introduce mutations and crossovers into cDNA clones, and to achieve molecular diversity directly from the mRNA level to optimize protein function. This greatly reduces the

- 79 simplifies the ETS procedure (expression-tagged strategy), which can be used to identify new catalysts.

In addition to the above, the solution according to the invention is suitable for the study of obligate intracellular pathogens or other systems where the cells in question cannot proliferate (38).

Having thus described exemplary embodiments of the invention, it should be noted that it will be apparent to those skilled in the art that the teachings are not limited to the examples set forth, and that various other alternatives, adaptations, and modifications may be made within the scope of the invention. Accordingly, the scope of our invention is not limited to the specific embodiments set forth, and is defined solely by the following claims.

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18. Stemmer, W.P.C. 1994b. DNA shuffling by random fragmentation and reassembly - in vitro recombination for molecular evolution. Proc. Natl. Acad. Sci., USA, 91:10747-10751.

19. U.S. Patent No. 5,605,793.

20. Moore, J.C., H.-M. Jin, O. Kuchner and F.H. Arnold. 1997. Strategies for the in vitro Evolution of Protein Function: Enzyme Evolution by Random Combination of Improved Sequences, J. Molecular Biology, in press.

21. Klenow, H. and I. Henningsen. 1970. Selective elimination of the exonuclease activity of the deoxyribonucleic acid polymerase from Escherichis coli B by limited proteolysis. Proc. Natl. Acad. Sci. 65:168

22. Feinberg, A. P. and Vogdstein, B. 1983. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal. Biochem. 132: 6-13.

23. Nossal, N.G. 1974. DNA synthesis on a double-stranded DNA template by the T4 bacteriophage DNA polymerase and the T4 gene 32 DNA unwinding protein. J. Biol. Chern. 249: 5668-5676.

24. Ehrlich, H.A., PCR Technology, Stockton Press (1989).

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26. Hermes, J.D., Blacklow, S.C., and Knowles, J.R. 1990. Searching sequence space by definably random mutagenesis - improving the catalytic potency of an enzyme. Proc. Natl. Acad. Sci. USA 87: 696-700.

27. Leung, D. W., Chen, E., and Goeddel, D. V. 1989. A method for random mutagenesis of a defined DNA segment using a modified polymerase chain reaction. BioTechnique 11-15.

28. Chen, K. and Arnold, F.H. 1993. Tuning the activity of an enzyme for unusual environments: sequential random mutagenesis of subtilisin E for catalysis in dimethylformamide. Proc. Natl. Acad. Sci. USA 90:56815622.

29. Sambrook, J., Fritsch, E.F. and Maniatis, T. 1989. Molecular cloning: A Laboratory Manual. 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.

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32. Tabor, S. and Richardson, C. C. 1989. Selective inactivation of the exonuclease activity of bacteriophage-T7 DNA polymerase by in vitro mutagenesis. J. Biol. Chem. 264:6447-6458.

33. Zhao, H. and Arnold, F.H. 1997. Functional and non-functional mutations distinguished by random recombination of homologous genes. Proc. Natl. Acad. Sci. USA 94:7997-8000.

34. Zock, J., Cantwell, C., Swarding, J., Hodges, R., Pohl, T., Sutton, K., Rosteck Jr., P., McGilvray, D. & Queener, S. 1994. The Bacillus subtilis pnbA gene encoding p-nitrobenzyl esterase - cloning, sequence and high-level expression in Escherichia coli Gene, 151, 37-43.

35. Zhao, H. and Arnold, F.H. 1997. Optimization of DNA shuffling for high fidelity recombination. Nucleic Acids Research, 25:1307-1308.

36. Lehman, I.R. and Richardson, C.C. 1964. The deoxyribonucleases of Escherichia coli. ARC. An exonuclease activity present in purified preparations of deoxyribonucleic acid polymerase. J. BioL Chem. 239:233.

37. Shafikhani, S., Siegel, R. A., Ferrari, E. & Schellenberger, V. 1997. Generation of large libraries of random mutants in Bacillus subtilis by PCR-based plasmid multimerization. Biotechniques, in press.

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- 83 SEQUENTIALIST

DATA FOR SEQUENCE IDENTIFICATION NUMBER 1:

SEQUENCE CHARACTERISTICS:

LENGTH: 22 nucleotides

TYPE: nucleotide

TOPOLOGY: linear

MOLECULE TYPE: oligonucleotide

DESCRIPTION OF SEQUENCE IDENTIFICATION NUMBER 1:

CCG AGC GTT GCA TAT GTG GAA G

DATA OF SEQUENCE IDENTIFICATION NUMBER 2:

SEQUENCE CHARACTERISTICS:

LENGTH: 21 nucleotides

TYPE: nucleotide

TOPOLOGY: linear

MOLECULE TYPE: oligonucleotide

DESCRIPTION OF SEQUENCE IDENTIFICATION NUMBER 2:

CGA CTC MEMBER AGG ATC CGA TTC

DATA OF SEQUENCE IDENTIFICATION NUMBER 3:

SEQUENCE CHARACTERISTICS:

LENGTH: 21 nucleotides

TYPE: nucleotide

TOPOLOGY: linear

MOLECULE TYPE: oligonucleotide

DESCRIPTION OF SEQUENCE IDENTIFICATION NUMBER 3:

GAG CAC ATC AGA TCT ATT AAC

- 84 DATA OF SEQUENCE IDENTIFICATION NUMBER 4:

SEQUENCE CHARACTERISTICS:

LENGTH: 21 nucleotides

TYPE: nucleotide

TOPOLOGY: linear

MOLECULE TYPE: oligonucleotide

DESCRIPTION OF SEQUENCE IDENTIFICATION NUMBER 4:

GGA GTG GCT CAC AGT CGG TGG

DATA FOR SEQUENCING IDENTIFICATION NUMBER 5:

SEQUENCE CHARACTERISTICS:

LENGTH: 21 nucleotides

TYPE: nucleotide

TOPOLOGY: linear

MOLECULE TYPE: oligonucleotide

DESCRIPTION OF SEQUENCE IDENTIFICATION NUMBER 5:

TTG AAC TAT CGG CTG GGG CGG

DATA OF SEQUENCE IDENTIFICATION NUMBER 6:

SEQUENCE CHARACTERISTICS:

LENGTH: 21 nucleotides

TYPE: nucleotide

TOPOLOGY: linear

MOLECULE TYPE: oligonucleotide

DESCRIPTION OF SEQUENCE IDENTIFICATION NUMBER 6:

TTA CTA GGG AAG CCG CTG GCA

- 85 DATA OF SEQUENCE IDENTIFICATION NUMBER 7:

SEQUENCE CHARACTERISTICS:

LENGTH: 21 nucleotides

TYPE: nucleotide

TOPOLOGY: linear

MOLECULE TYPE: oligonucleotide

DESCRIPTION OF SEQUENCE IDENTIFICATION NUMBER 7:

TCA GAG ATT ACG ATC GAA AAC

DATA OF SEQUENCE IDENTIFICATION NUMBER 8:

SEQUENCE CHARACTERISTICS:

LENGTH: 21 nucleotides

TYPE: nucleotide

TOPOLOGY: linear

MOLECULE TYPE: oligonucleotide

DESCRIPTION OF SEQUENCE IDENTIFICATION NUMBER 8:

GGA TTG TAT CGT GTG AGA AAG

DATA FOR SEQUENCING IDENTIFICATION NUMBER 9:

SEQUENCE CHARACTERISTICS:

LENGTH: 21 nucleotides

TYPE: nucleotide

TOPOLOGY: linear

MOLECULE TYPE: oligonucleotide

DESCRIPTION OF SEQUENCE IDENTIFICATION NUMBER 9:

AAT GCC GGA AGC AGC CCC TTC

- 86 DATA OF SEQUENCE IDENTIFICATION NUMBER 10:

SEQUENCE CHARACTERISTICS:

LENGTH: 21 nucleotides

TYPE: nucleotide

TOPOLOGY: linear

MOLECULE TYPE: oligonucleotide

DESCRIPTION OF SEQUENCE IDENTIFICATION NUMBER 10:

CAC GAC AGG AAG ATT TTG ACT 21

DATA OF SEQUENCE ID NO. 11: SEQUENCE CHARACTERISTICS: LENGTH: 20 nucleotides TYPE: nucleotide TOPOLOGY: linear MOLECULE TYPE: oligonucleotide

DESCRIPTION OF SEQUENCE IDENTIFICATION NO. 11: ACT TAA TCT AGA GGG TAT TA

DATA OF SEQUENCE ID NO. 12: SEQUENCE CHARACTERISTICS: LENGTH: 20 nucleotides TYPE: nucleotide TOPOLOGY: linear MOLECULE TYPE: oligonucleotide

DESCRIPTION OF SEQUENCE IDENTIFIER 12: AGC CTC GCG GGA TCC CCG GG

- 87 DATA OF SEQUENCE IDENTIFICATION NUMBER 13:

SEQUENCE CHARACTERISTICS:

LENGTH: 28 nucleotides

TYPE: nucleotide

TOPOLOGY: linear

MOLECULE TYPE: oligonucleotide

DESCRIPTION OF SEQUENCE IDENTIFICATION NUMBER 13:

GGT AGA GCG AGT CTC GAG GGG GAG ATG C

DATA OF SEQ ID NO: 14:

SEQUENCE CHARACTERISTICS:

LENGTH: 22 nucleotides

TYPE: nucleotide

TOPOLOGY: linear

MOLECULE TYPE: oligonucleotide

DESCRIPTION OF SEQUENCE IDENTIFICATION NUMBER 14:

AGC CGG CGT GAC GTG GGT CAG C

DATA OF SEQ ID NO: 15:

SEQUENCE CHARACTERISTICS:

LENGTH: 22 nucleotides

TYPE: nucleotide

TOPOLOGY: linear

MOLECULE TYPE: oligonucleotide

DESCRIPTION OF SEQUENCE IDENTIFICATION NUMBER 15:

CCG AGC GTT GCA TAT GTG GAA G

- 88 DATA OF SEQUENCE IDENTIFICATION NUMBER 16:

SEQUENCE CHARACTERISTICS:

LENGTH: 21 nucleotides

TYPE: nucleotide

TOPOLOGY: linear

MOLECULE TYPE: oligonucleotide

DESCRIPTION OF SEQUENCE IDENTIFICATION NUMBER 16:

CGA CTC MEMBER AGG ATC CGA TTC

CONSCIOUSNESS OF SEQUENCE IDENTIFICATION NUMBER 17:

SEQUENCE CHARACTERISTICS:

LENGTH: 22 nucleotides

TYPE: nucleotide

TOPOLOGY: linear

MOLECULE TYPE: oligonucleotide

DESCRIPTION OF SEQUENCE IDENTIFICATION NUMBER 17:

CGG TAC GCA TGT AGC CGG TAC G

DATA OF SEQ ID NO: 18:

SEQUENCE CHARACTERISTICS:

LENGTH: 22 nucleotides

TYPE: nucleotide

TOPOLOGY: linear

MOLECULE TYPE: oligonucleotide

DESCRIPTION OF SEQUENCE IDENTIFICATION NUMBER 18:

CGG TAC GAT TGC CGC CGG TAC G

- 89 DATA OF SEQUENCE IDENTIFICATION NUMBER 19:

SEQUENCE CHARACTERISTICS:

LENGTH: 22 nucleotides

TYPE: nucleotide

TOPOLOGY: linear

MOLECULE TYPE: oligonucleotide

DESCRIPTION OF SEQUENCE IDENTIFICATION NUMBER 19:

CCG AGC GTT GCA TAT GTG GAA G

DATA FOR SEQUENCING IDENTIFICATION NUMBER 20:

SEQUENCE CHARACTERISTICS:

LENGTH: 21 nucleotides

TYPE: nucleotide

TOPOLOGY: linear

MOLECULE TYPE: oligonucleotide

DESCRIPTION OF SEQUENCE IDENTIFICATION NUMBER 20:

CGA CTC MEMBER AGG ATC CGA TTC

DATA OF SEQ ID NO: 21:

SEQUENCE CHARACTERISTICS:

LENGTH: 18 nucleotides

TYPE: nucleotide

TOPOLOGY: linear

MOLECULE TYPE: oligonucleotide

DESCRIPTION OF SEQUENCE IDENTIFICATION NUMBER 21:

GGC GGA GCT AGC TTC GTA

- 90 DATA OF SEQUENCE IDENTIFICATION NUMBER 22:

SEQUENCE CHARACTERISTICS:

LENGTH: 18 nucleotides

TYPE: nucleotide

TOPOLOGY: linear

MOLECULE TYPE: oligonucleotide

DESCRIPTION OF SEQUENCE ID NO: 22: GAT GTG ATG GCT CCT GGC

DATA OF SEQ ID NO: 23:

SEQUENCE CHARACTERISTICS:

LENGTH: 18 nucleotides

TYPE: nucleotide TOPOLOGY: linear

MOLECULE TYPE: oligonucleotide

DESCRIPTION OF SEQUENCE ID NO: 23: CAG AAC ACC GAT TGA GTT

DATA OF SEQ ID NO: 24:

SEQUENCE CHARACTERISTICS:

LENGTH: 18 nucleotides

TYPE: nucleotide

TOPOLOGY: linear

MOLECULE TYPE: oligonucleotide

DESCRIPTION OF SEQUENCE IDENTIFICATION NUMBER 24:

AGT GCT TTC TAA ACG ATC

- 91 DATA OF SEQUENCE IDENTIFICATION NUMBER 25:

SEQUENCE CHARACTERISTICS:

LENGTH: 4 amino acids

TYPE: peptide

TOPOLOGY: linear

MOLECULE TYPE: peptide

DESCRIPTION OF SEQUENCE IDENTIFICATION NUMBER 25:

Ala Ala Pro Phe

Claims (40)

PATENT CLAIMS 1. A method for producing a recombinant DNA molecule from one or more single-stranded or double-stranded template polynucleotides, comprising: (I) generating a set of DNA fragments by (A) preparing a reaction mixture comprising: (1) At least one copy of each template polynucleotide, (2) multiple copies of at least one specified or constrainedly randomly specified primer, or (N) multiple copies of at least one set of random primers of the general formula _L , where N is dAMP, dGMP, dCMP, or dTMP without restriction, and L is the length of each primer in the set in number of nucleotides, (3) water, and (4) optionally a DNA polymerase enzyme, dATP, dGTP, dTTP, dCTP, and DNA polymerization reaction medium; (B) denaturing any double-stranded polynucleotides present in the reaction mixture under denaturing conditions, thereby forming a mixture comprising single-stranded template polynucleotides and single-stranded primers; (C) hybridizing the single-stranded primers to the single-stranded template polynucleotide or polynucleotides under hybridization conditions; (D) optionally adding DNA polymerase enzyme, dATP, dGTP, dTTP, dCTP and a DNA polymerization reaction medium to the reaction mixture; (E) initiating an enzyme-catalyzed DNA polymerization synthesis reaction in the reaction mixture under polymerization conditions; (F) continuing the DNA polymerization synthesis reaction until at least partial chain elongation occurs, thereby generating DNA fragments; (G) optionally repeating steps (I.B)-(I.F) until the reaction mixture contains a mixture of DNA fragments in which the average DNA fragment size(s) is/are shorter than the length of the template polynucleotide(s) and the DNA fragment size range is sufficient to allow at least some of the DNA fragments to hybridize to each other, hybridizing them, and then performing additional denaturing hybridization and chain extension step(s) to produce a DNA molecule greater than or equal to the length of one of the template polynucleotides; II. assembling the DNA fragments into at least one recombinant full-length molecule by (A) denaturing any of the double-stranded polynucleotides present in the reaction mixture under denaturing conditions to form a mixture containing single-stranded DNA fragments; (B) hybridizing the single-stranded DNA fragments to each other under hybridization conditions; (C) initiating an enzyme-catalyzed DNA polymerization synthesis reaction in the reaction mixture under polymerization conditions; (D) continuing the DNA polymerization synthesis reaction until at least partial chain elongation of the hybridized DNA fragments occurs, (E) optionally repeating steps (II.A)-(II.D) until at least one DNA molecule is formed that is at least as long as at least one of the template polynucleotides, thereby producing at least one full-length DNA molecule, at least one of which is a full-length DNA molecule whose DNA sequence is a recombination representation of the nucleotide sequence or sequences of at least one of the template polynucleotides, thereby producing a recombinant full-length DNA molecule; (III), wherein at least one of the recombinant full-length DNA molecules (A) encodes a gene product with at least one altered or improved property compared to the properties of the gene product(s) encoded by the template polynucleotide(s) or (B) has at least one altered or improved property compared to the properties of the template polynucleotide(s), or (C) both conditions (III.A) and (III.B) are met; IV. provided that (A) if the reaction mixture contains only internal primers in step (I.A.II.), then the polymerase - 95 PCR reactions are performed using PCR primers on the mixture of DNA fragments prepared in step (I.G) until at least one recombinant full-length DNA molecule is obtained, at least one of which meets the requirements of step (III); (B) if complete chain extension can occur under the conditions used in steps (I.E)-(I.F), then in step (I.A.2) the reaction mixture contains multiple copies of at least one internal primer in addition to terminal or flanking primers; (C) the concentration of primer bound to a template is below a level that would result in spherical inhibition to such an extent that primer extension would be inhibited, thereby not achieving a DNA fragment size range in step (I.G) that allows at least some of the DNA fragments to hybridize to each other and, upon further denaturation, hybridization, and extension step(s), to form a DNA molecule that is at least as long as at least one of the template polynucleotides; and (D) step (I.A.4) or step (I.D) or both step (I.A.4) and step (I.D) are performed in the process. 2. A method for producing a recombinant DNA molecule from one or more single-stranded or double-stranded template polynucleotides, characterized in that (I) at least one recombinant DNA fragment is generated by (A) preparing a reaction mixture comprising: (1) At least one of each template polynucleotide/ (2) at least one copy of a defined terminal or limited random defined terminal primer, (3) water, and (4) optionally a DNA polymerase enzyme, dATP, dGTP, dTTP, dCTP, and DNA polymerization reaction medium; (B) denaturing any double-stranded polynucleotides present in the reaction mixture under denaturing conditions, thereby forming a mixture comprising single-stranded template polynucleotides and single-stranded primers; (C) hybridizing the single-stranded primers to the single-stranded template polynucleotide or polynucleotides under hybridization conditions; (D) optionally adding DNA polymerase enzyme, dATP, dGTP, dTTP, dCTP and a DNA polymerization reaction medium to the reaction mixture; (E) initiating an enzyme-catalyzed DNA polymerization synthesis reaction in the reaction mixture under polymerization conditions; (F) continuing the DNA polymerization synthesis reaction until at least partial chain elongation occurs, thereby generating DNA fragments; (G) repeating steps (I.B)-(I.F) while re-initiating and extending the DNA fragment(s) until the reaction mixture contains a mixture of DNA fragments in which the average DNA fragment size(s) is/are shorter than the length of the template polynucleotide(s) and the DNA sequence of at least one DNA fragment represents a recombination of the nucleotide sequence of at least one of the template polynucleotide(s), thereby generating at least one recombinant DNA fragment; II. forming at least one recombinant full-length DNA molecule from the recombinant DNA fragment(s) by (A) denaturing any of the double-stranded polynucleotides present in the reaction mixture under denaturing conditions, thereby creating a mixture comprising single-stranded template polynucleotide(s) and single-stranded DNA fragment(s); (B) hybridizing the single-stranded DNA fragments to the single-stranded template polynucleotide or polynucleotides under hybridization conditions; (C) initiating an enzyme-catalyzed DNA polymerization synthesis reaction in the reaction mixture under polymerization conditions; (D) continuing the DNA polymerization synthesis reaction until at least partial chain elongation of the hybridized DNA fragment(s) occurs, (E) repeating steps (II.A)-(II.D) if desired until at least one DNA molecule is formed - 98 hands, which is at least as long as at least one of the template polynucleotides, thereby producing at least one full-length DNA molecule, at least one of which is a full-length DNA molecule whose DNA sequence represents recombinations of the nucleotide sequence or sequences of at least one of the template polynucleotides, thereby producing a recombinant full-length DNA molecule; (III), wherein at least one of the recombinant full-length DNA molecules (A) encodes a gene product with at least one altered or improved property compared to the properties of the gene product(s) encoded by the template polynucleotide(s) or (B) has at least one altered or improved property compared to the properties of the template polynucleotide(s), or (C) both conditions (III.A) and (III.B) are met; IV. provided that (A) the concentration of primer bound to a template is below a level that would result in spherical inhibition to such an extent that primer elongation would be inhibited, thereby not achieving at least one DNA fragment in step (I.G) whose DNA sequence represents recombination of the nucleotide sequence of at least one of the template polynucleotide(s), and (B) step (I.A.4) or step (I.D) or both step (I.A.4) and step (I.D) are performed in the process. 3. The method of claim 1 or 2, characterized in that in step (II.E) DNA fragment(s) of an average size approximately equal to the length of the template DNA sequence(s) are generated. 4. The method of claim 1, wherein in step (I.F) the DNA polymerization synthesis reaction is continued until the chain initiated by each hybridized primer reaches full length. 5. The method of claim 1 or 2, wherein in step (I.F) the DNA polymerization synthesis reaction is continued until at least about 24 nucleotides are added to each hybridized primer. 6. The method of claim 5, wherein the synthesis reaction is continued until at least about 50 nucleotides are added to each hybridized primer. 7. The method of claim 6, wherein the synthesis reaction is continued until at least about 500 nucleotides are added to each hybridized primer. - 100 - 8. The method according to claim 1 or 2, characterized in that in step (I.A.2) a reaction mixture is prepared containing a mutagenic primer as one of the primers. 9. The method of claim 1, wherein the template polynucleotide(s) is/are removed from the reaction mixture between steps (I.G) and (II.). 10. The method according to claim 1 or 2, characterized in that RNA is used as the template polynucleotide(s) and RNA-dependent DNA polymerase is used as the DNA polymerase enzyme. 11. The method according to claim 1 or 2, characterized in that DNA is used as the template polynucleotide(s) and DNA-dependent DNA polymerase is used as the DNA polymerase enzyme. 12. The process according to claim 1 or 2, characterized in that the conditions used in step (II.D) allow complete chain extension to occur. 13. The method of claim 1 or 2, wherein in step (I.A.1) a reaction mixture comprising at least two different template polynucleotides, including a first template polynucleotide and a second template polynucleotide, is formed. - 101 - 14. The method according to claim 13, characterized in that the first template polynucleotide is a template polynucleotide that differs from the second template polynucleotide by at least two nucleotides. 15. The method of claim 14, characterized in that a template polynucleotide containing the different nucleotides separately from each other is used. 16. The method of claim 15, wherein a template polynucleotide is used that contains the different nucleotides spaced at least about 15 nucleotides apart. 17. The method according to claim 1 or 2, characterized in that DNA produced as a reverse transcript of an RNA molecule is used as the template polynucleotide. 18. The method according to claim 1 or 2, characterized in that the primer(s) used are primer(s) of at least six nucleotides in length. 19. The method according to claim 18, characterized in that the primer(s) used are primer(s) with a length of 6-100 nucleotides. - 102 - 20. The method according to claim 19, characterized in that the primer(s) used are primer(s) with a length of 6-24 nucleotides. 21. The method of claim 1 or 2, characterized in that the full-length DNA molecule(s) is/are amplified by polymerase chain reaction (PCR). 22. The method of claim 21, characterized in that the full-length DNA molecule(s) is/are first isolated from the reaction mixture and then amplified by PCR. 23. The method of claim 2, wherein in step (I.A.2) multiple copies of the terminal primer(s) are used. 24. The method according to claim 1 or 2, characterized in that the specific primer(s) with limited randomness are primer(s) showing randomness at one or more nucleotide positions within the primer(s). 25. The method according to claim 24, characterized in that the limited randomness specific primer(s) is a primer(s) showing randomness in more than 30% of the nucleotide positions within the primer(s). - 103 - 26. The method according to claim 25, characterized in that the limited randomness specific primer(s) is a primer(s) showing randomness in more than 60% of the nucleotide positions within the primer(s). 27. The method of claim 1 or 2, wherein in step (I.A.2) multiple copies of at least two primers are used. 28. The method of claim 1, wherein random primers are used as primers. 29. The process of claim 28, wherein the conditions used in step (I.F) allow complete chain extension to occur. 30. The method of claim 29, wherein steps (I.B)-(I.F) are not repeated in step (I.G). 31. The method according to claim 1 or 2, characterized in that step (I.A.4) is performed only once during the method and step (I.D) is not performed at all. - 104 - 32. The method according to claim 1 or 2, characterized in that step (I.D) is performed only once during the method and step (I.A.4) is not performed at all. 33. The method of claim 13, wherein at least one of the full-length DNA molecules produced in step (II.E) is produced whose DNA sequence represents the recombination of at least two template polynucleotide sequences. 34. The method of claim 13, wherein in step (I.G), or in step (II), or in both steps (I.G) and (II), the DNA fragments are subjected to random chain initiation of the template polynucleotides. 35. The method of claim 1 or 2, wherein at least one of the template polynucleotides is a recombinant, full-length DNA molecule produced by the method of claim 1 or 2. 36. A recombinant, full-length DNA molecule produced by the method of claim 1 or 2, which (a) encodes a gene product with at least one altered or improved property compared to the properties of the gene product(s) encoded by the template polynucleotide(s), or - 105 - (b) it has at least one altered or improved property compared to the properties of the template polynucleotide(s), or (c) both conditions (III.A) and (III.B) are met. 37. A method for producing a gene product, comprising: (a) inserting a full-length DNA molecule encoding a recombinant gene product produced by the method of claim 1 or 2 into a vector, thereby creating an expression vector, (b) transforming the host cell with the expression vector, and (c) expressing the gene product encoded by the recombinant full-length DNA molecule; wherein the recombinant, full-length DNA molecule (1) encodes a gene product with at least one altered or improved property compared to the properties of the gene product(s) encoded by the template polynucleotide(s) of the DNA molecule or (2) has at least one altered or improved property compared to the properties of the template polynucleotide(s), or (3) both conditions (III.A) and (III.B) are met. 38. The method of claim 37, characterized in that a protein is expressed as the gene product. - 106 - 39. The method of claim 38, characterized in that an enzyme is expressed as a protein. 40. A gene product produced by the method of claim 37. The authorized person: DANUBE Patent and Trademark Office Ltd.