MXPA98009854A - Recombination of polynucleotide sequences using random or defined primers - Google Patents

Abstract

A method for in vitro mutagenesis and recombination of polynucleotide sequences based on polymerase-catalyzed extension of primer oligonucleotides is disclosed. The method involves priming template polynucleotide(s) with random-sequences or defined-sequence primers to generate a pool of short DNA fragments with a low level of point mutations. The DNA fragments are subjected to denaturization followed by annealing and further enzyme-catalyzed DNA polymerization. This procedure is repeated a sufficient number of times to produce full-length genes which comprise mutants of the original template polynucleotides. These genes can be further amplified by the polymerase chain reaction and cloned into a vector for expression of the encoded proteins.

Description

RECOMBINATION OF POLYUCLEOTIDE SEQUENCES USING RANDOMIZED OR DEFINED PRIMERS The government of the United States of America has certain rights in this invention in accordance with the donation No. DE-FG02-93-CH10578 awarded by the Department of energy and the donation No. N00014-96-1-0340 awarded by the Office of Naval Research.

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates in general to two in vitro methods for the utagénesis and recombination of polynucleotide sequences. More particularly, the present invention involves a simple and efficient method for mutagenesis and recombination in sequence of polynucleotide sequences based on extension. catalyzed by polymerase oligonucleotide primers, followed by gene array and optional gene amplification. 2. Description of Related Art The publications and other reference materials referred to herein to describe the background of the invention and to provide additional details with respect to its practice are incorporated herein by reference. For convenience, the reference materials are numerically referenced and grouped in the accompanying bibliography. The proteins are interspersed in order to improve their performance for practical applications. The desirable properties depend on the application of interest and may include stronger links to a receiver, high catalytic activity, high stability, the ability to accept a wider (or narrower) range of substrates, or the ability to function in non-target environments. naturals such as organic solvents. A variety of approaches have been successfully used, including 'rational' design and random mutagenesis methods to optimize protein functions (1). The choice of approximation form for a given optimization problem will depend on the degree of understanding of the relationships between the sequences, structure and function. The rational redesign of a catalytic site of an enzyme, for example, frequently requires extensive knowledge of the structure of the enzyme, the structures of its complexes with various ligands and intermediate reaction analogs and details of the catalytic mechanism. This information is available only for very few well-studied systems: little is known about the vast majority of potentially interesting enzymes. Identifying the amino acids responsible for the functions of an existing protein and those that could give rise to new functions remains a frequently overwhelming challenge. This, together with the growing appreciation that many protein functions are not confined to a small number of amino acids, 'but are affected by residues away from active sites, has prompted an increasing number of groups to turn to random mutagenesis, or 'directed' evolution, to overlap novel proteins (1). Several optimization procedures such as genetic algorithms (2,3) and evolutionary strategies (4,5) have been inspired by natural evolution. These procedures employ mutation, which makes small random changes in members of the population, as well as crossing, which combines properties of different individuals, to ace a specific optimization goal. There are also strong interplay between mutation and crossing, as shown by computer simulations of different optimization problems (6-9). Developing efficient and practical experimental techniques to mimic these key processes is a scientific challenge. The application of these techniques should allow one, for example, to explore and optimize the functions of biological molecules such as proteins and nucleic acids, in vivo or even completely free of the constraints of a living system (10,11). The directed evolution, inspired by the natural evolution, involves the generation and selection or filter of a breeding ground of mutated molecules which have enough diversity for a molecule to encode a protein with altered or increased function that is present in it. It usually begins with the creation of a library of mutated genes. The gene products that show improvement with respect to the desired property or set of properties are identified by the selection or filter. The gene or genes that code for these products can be subjected to other cycles of the process in order to accumulate beneficial mutations. This evolution may involve a few or many generations, depending on how much one wants to progress and the effects of mutations typically observed in each generation. These approaches have been used to create novel functional nucleic acids (12), peptides and other small molecules (12), antibodies (12), as well as enzymes and other proteins (13, 14, 16). The directed evolution requires little specific knowledge about the product itself, only a means to evaluate the function to be optimized. These procedures are still fairly tolerant of inaccuracies and noise in the evaluation of functions (15). The diversity of genes for directed evolution can be created by introducing novel point mutations using a variety of methods, including polymerase chain reaction (15) or combinatorial cassette mutagenesis (16). The ability to recombine genes, however, can add an important dimension to the evolutionary process, as evidenced by its key role in natural evolution. Homologous recombination is an important natural process in which organisms exchange genetic information between related genes, increasing the accessible genetic diversity in a species. Although adaptive competencies and powerful diversification are potentially introduced to their hosts, these trajectories also operate at very low efficiencies, often obtaining insignificant changes in the structure or function of the trajectory, even after tens of generations. Thus, although these mechanisms prove to be beneficial to host organisms / species over an extended geological time, in vivo recombination methods represent voluminous combinatorial processes, if not unusable, of customizing the behavior of enzymes or other non-protein proteins. so strongly linked to the metabolism and survival of the organism's intermediary. Several groups have recognized the utility of gene recombination in directed evolution. Methods for the in vivo recombination of genes are described, for example, in PCT application WO 97/07205 and in U.S. Patent Number: 5,093,257. As explained above, these in vivo methods are bulky and poorly optimized for rapid evolution of function. Stemmer has described a method for the recombination in vi tro of related DNA sequences in which parent sequences are cut into fragments, generally using an enzyme such as DNase I, and rearranged (17,18,19). The fragmentation of the non-random DNA associated with DNase I and other endonucleases, however, introduces bias in the recombination and limits the diversity of the recombination. In addition, this method is limited to the recombination of double-stranded polynucleotides and can not be used in single-chain anneals. In addition, this method does not work well with certain combinations of genes or primers. It is not efficient for the recombination of short sequences (less than 200 nucleotides (nts)), for example. Finally, it is quite laborious, requiring several steps. Alternative, convenient methods are needed to create novel genes by point mutagenesis and in vitro recombination.

SUMMARY OF THE INVENTION The present invention provides a new and significantly improved approach for creating novel polynucleotide sequences by point mutation and in vitro recombination of a set of parent sequences. (the temperate). The novel polynucleotide sequences may be useful per se (eg, for DNA-based computing), or may be expressed in recombinant organisms for the targeted evolution of the gene products. One embodiment of the invention involves priming the gene or genes hardened with oligonucleotides of random sequence to generate a deposit of short DNA fragments. Under appropriate reaction conditions, these short DNA fragments can prime each other based on complementarity and thus can be rearranged to form full length genes by thermocycling in the presence of thermostable DNA polymerase. These rearranged genes, which contain point mutations as well as novel combinations of sequences from different parent genes, can be further expanded by polymerase chain reaction and cloned into a suitable vector for the expression of encoded proteins. The filter or selection of gene products leads to new variants with improved or even novel functions. These variants can be used as they are, or they can serve as new starting points for other cycles of mutagenesis and recombination. A second embodiment of the invention involves priming the gene or the temperate genes with a set of oligonucleotides of defined sequence or defined sequence that exhibits limited randomness to generate a deposit of short fragments of DNA, which rearrange then as described above in genes full length A third embodiment of the invention involves a novel process that we name the process of 'stepped extension', or StEP. Instead of rearranging the deposit of fragments created by the extended primers, the full length genes are arranged directly in the presence of quenched or tempered. StEP consists of repeated cycles of denaturation followed by extremely abbreviated tempering / extension steps. In each cycle the extended fragments can be hardened to different tempers based on complementarity and extended a little more to create "recombinant cassettes". Because of this change in annealing, most polynucleotides contain sequences from different parent genes (ie, they are novel recombinants). This process is repeated until full-length genes are formed. It can be followed by an optional step of gene amplification. The different embodiments of the invention provide features and advantages for different applications. In the most preferred embodiment, one or more defined primers or defined primers exhibiting limited randomness corresponding to or flanking the 5 'and 3' ends of the tempered polynucleotides are used with the StEP to generate gene fragments that grow in the length sequences totally new. This simple method does not require knowing the sequence or tempered sequences. In another preferred embodiment, multiple defined primers or defined primers exhibiting limited randomness are used to generate short fragments of genes which rearrange into full length genes. Using multiple defined primers allows the user to bias the recombination frequency in vi tro. If sequence information is available, the primers can be designed to generate overlapping recombination cassettes which increase the recombination frequency at particular locations. Among other features, this method introduces the flexibility to take advantage of available structural and functional information as well as information accumulated through previous generations of mutagenesis and selection (or filter). In addition to recombination, the different modalities of the primer-based recombination process will generate point mutations. It is desirable to know and be able to control this point mutation regimen, which can be done by manipulating the conditions of DNA synthesis and rearrangement of genes. Using the defined primer approach, specific point mutations can also be targeted to specific positions in the sequence through the use of mutagenic primers. The various primer-based recombination methods according to this invention have been shown to increase the activity of the ECB decylation of Actinoplanes utahensis over a wide range of pH values and in the presence of organic solvent and improve the thermostability of subtilisin E of the Bacill us subtilis. DNA sequencing confirms the role of point mutation and recombination in the generation of novel sequences. It has been found that these protocols are both simple and reliable. The above-explained features and many other concomitant advantages and advantages will be better understood with reference to the following detailed description when taken in conjunction with the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 depicts recombination according to the present invention using primers of random sequence and rearrangement of genes. The steps shown are: (a) Synthesis of single-stranded DNA fragments using mesophilic or ter ophilic polymerase with random sequence oligonucleotides as primers (the primers are not shown); (b) Temperate removal, - (c) Rearrangement with thermophilic DNA polymerase; (d) Amplification with thermostable polymerase (s); (e) cloning and selection (optional); and (f) Repeat the process with the selected gene or genes (optional). Figure 2 depicts recombination according to the present invention using defined primers. The method is illustrated for the recombination of two genes, where x = mutation. The diagrammed steps are: (a) the genes are primed with defined primers in polymerase chain reactions that can be done separately (2 primers per reaction) or combined (multiple primers per reaction); (c) Initial products are formed until the defined primers are depleted. The tempering is removed (optional); (d) The initial fragments are primed and extended in other polymerase chain reaction cycles without addition of external primers. The assembly continues until full length genes are formed; (e) (optional) Full-length genes are amplified in a polymerase chain reaction with external primers; (f) (optional) Repeat the process with the selected gene or genes. Figure 3 depicts recombination according to the present invention using two defined flanking primers and the StEP. Only one primer and two simple two-templated chains are shown here to illustrate the recombination process. The steps outlined are: (a) After denaturation, the temperate genes are primed with a defined primer; (b) Short fragments are produced by primer extension for a short time, - (c) in the next StEP cycle, the fragments are randomly primed and further extended; (d) Denaturation and annealing / extension are repeated until full-length genes are made (visible on an agarose gel), - (e) Full-length genes are purified, or amplified in a polymerase chain reaction with external primers (optional); (f) (optional) The process is repeated with the selected gene or genes. Figure 4 is a diagrammatic representation of the results of the recombination of two genes using two step flanking and extension primers according to the present invention. The DNA sequences of five genes chosen from the recombined library are indicated, where x is a mutation present in the parent 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 Example 3. The temperate genes 2-13 and 5-B12 were harvested using the defined primer approach. The positions of the primers are indicated by arrows, and the positions where the parent sequences differ from each other are indicated by letters x. The new point mutations are indicated by triangles. The mutations identified in these recombined genes are listed (only positions that differ in parent sequences are listed). Both 6E6 and 6H1 are products of the recombination of the temperate genes. Figure 6 shows the positions and sequences of the four defined internal primers used to generate recombined genes from the Rl and R2 hardened genes by intercalated primer-based recombination. Primer P50F contains a mutation (A? T at base position 598) which simultaneously removes a HindIII restriction site and adds a new exclusive Nhel site. The R2 gene also contains an A? G mutation in the same base position, which eliminates the HindIII site. Figure 7 is an electrophoresis gel showing the results of the restriction-digestion analysis of the plasmids of the 40 clones. Figure 8 shows the results of sequencing ten genes from the primer-based recombination library. The lines represent 986 base pairs of the subtilisin E gene that include 45 nucleotides of its prosequence, the entire mature sequence and 113 nucleotides after the stop codon. Crosses indicate mutations positions of the parent Rl and R2 gene, while triangles indicate positions of new point mutations introduced during the recombination procedure. The circles represent the mutation introduced by the mutagenic P50F primer. Figure 9 represents the results of applying the random sequence primer recombination method for the ECB deacylase gene of Actinoplanes utahensis. (a) The 2.4 kb of the ECB deacylase gene is purified from an agarose gel. (b) The size of the random priming products varies from 100 to 500 bases, (c) The shorter fragments of 300 bases were isolated. (d) The purified fragments were used to rearrange the full-length gene with a stained background, (e) A single polymerase chain reaction product of the same size as the. Deacilasa ECB gene was obtained after conventional polymerase chain reaction with the two primers located in the start and stop regions of this gene. (f) After digestion with Xhol and Psh AI, the product of the polymerase chain reaction was cloned into a modified pIJ702 vector to form a mutant library. (g) Introducing this library in Streptomyces li vidans TK23 resulted in approximately 71 percent of clones producing the active ECB deacylase. Figure 10 shows the specific activity of the wild type ECB deacylase and the mutant MI6 obtained according to the present invention. Figure 11 shows activity pH profiles of the wild type ECB deacylase and the M16 mutant obtained according to the present invention. Figure 12 shows the DNA sequence analysis of 10 clones chosen randomly from the library / Klenow. The lines represent 986 base pairs of the subtilisin E gene that include 45 nucleotides of its prosequence, the entire mature sequence and 113 nucleotides after the stop codon. The crosses indicate positions of mutations from R1 to R2, while the triangles indicate positions of new point mutations introduced during the random priming recombination process. Figure 13. Thermostability index profiles of selected clones from five libraries produced using different polymerases: (a) library / Klenow, (b) library / T4, (c) library / Secuenase, (d) library / Stoffel and (e) library / Pfu. The normalized residual activity (Ar / Ai) after incubation at 65 ° C was used as an index of the thermostability of the enzyme. The data was classified and plotted in descending order.

DETAILED DESCRIPTION OF THE INVENTION In a preferred embodiment of the present invention, a set of primers with all possible nucleotide sequence combinations (dp (N) L: where L = primer length) was used for primer-based recombination. For years it has been known that oligodeoxynucleotides of different lengths can serve as primers for the initiation of DNA synthesis in single-chain anneals by the Klenow fragment of E. coli polymerase I (21). Although they are smaller than the size of a normal polymerase chain reaction primer (ie less than 13 bases), oligomers as short as hexanucleotides can adequately prime the reaction and are often used to label reactions (22). The use of random primers to create a deposit of gene fragments followed by a rearrangement of genes according to the invention is shown in Figure 1. The steps include generation of various "parenting blocks" from the polynucleotide annealing Through random priming, the rearrangement of full-length DNA from short nascent DNA fragments by tertnocyclycation in the presence of DNA polymerase and nucleotides, and the amplification of the desired genes from rearranged products by conventional polymerase reaction for cloning and subsequent selection. This procedure introduces new mutations mainly in the priming step but also during other steps. These new mutations and the mutations already present in the hardened sequences are recombined during the rearrangement to create a library of novel DNA sequences. The process can be repeated in the selected sequences, if desired. In order to carry out the random priming procedure, the quenching (or quenching) can be single or double stranded denatured linear or closed circular polynucleotide (or polynucleotides). Tempering (or tempering) can be mixed in equimolar quantities, or in weighted amounts, for example, by its functional attributes. Since, at least in some cases, the temperate genes are cloned into vectors in which no additional mutations should be introduced, they are usually first dissociated with restriction endonuclease (or endonucleases) and purified from the vectors. The resulting linear DNA molecules are denatured by boiling, quenched to random sequence oligodeoxynucleotides and incubated with DNA polymerase in the presence of an adequate amount of dNTPs. Hexanucleotide primers are preferred, although longer random primers (up to 24 bases) may also be used, depending on the DNA polymerase and conditioning used during the random priming synthesis. Thus the oligonucleotides prime the DNA of interest at various positions throughout the entire target region and extend to generate short DNA fragments complementary to each strand of the tempered DNA. Due to cases such as poor base incorporations and poor priming, these short DNA fragments also contain point mutations. Under routine reaction conditions, short DNA fragments can prime each other based on homology and rearrange to full length genes by repeated thermocycling in the presence of thermostable DNA polymerase. The resulting full-length genes will have various sequences, most of which, however, still resemble that of the original temperate DNA. These sequences can be further amplified by conventional polymerase chain reaction and cloned into a vector for expression. The filtering or selection of mutants expressed in a vector for expression should lead to variants with improved specific functions or even new ones. These variants can be used immediately as partial solutions to a practical problem, or they can serve as new starting points for other cycles of directed evolution. Compared with other techniques used for protein optimization, such as the combinatorial cassette and oligonucleotide-directed mutagenesis (24, 25, 26), error-prone polymerase chain reaction (27, 28), or redistribution of / DNA (17). , 18, 19), some of the advantages of the random primer-based procedure for the evolution of protein in vi tro are summarized as follows: 1. The annealing (or temperate) used for the random priming synthesis can either be polynucleotides single or double chain. In contrast, the error-prone polymerase chain reaction and the method of redistribution of DNA for recombination (17, 18, 19) necessarily employ only double-stranded polynucleotides. Using the technique described herein, mutations and / or crossings can be introduced at the level of / DNA using different DNA-dependent DNA polymerases, or even directly from the mRNA using different RNA-dependent DNA polymerases. Recombination can be carried out using single chain DNA anneals. 2. In contrast to the DNA redistribution procedure, which requires the fragmentation of double-stranded DNA tuning (generally done with DNAse I) to generate random fragments, the technique described here employs random priming synthesis to obtain DNA fragments from size controllable as "parenting blocks" for later rearrangement (Figure 1). An immediate advantage is that two sources of activity (DNase I and 5'-3 'exonuclease) are eliminated, and this allows for easier control over the size of the final rearrangement and amplification gene fragments. 3. Since the random primers are a population of synthetic oligonucleotides that contain all four bases in all positions, they are uniform in length and lack a sequence bias. The sequence heterogeneity allows them to form hybrids with the strings of DNA tuned in many positions, so that any nucleotide of the temperate (except, perhaps, those of the extreme 5 'term) should be copied at a similar frequency in products. In this way, both mutations and crosses can happen more randomly than, for example, with polymerase chain reaction with error propensity or redistribution of DNA. 4. The synthesis of randomly primed DNA is based on the hybridization of a mixture of hexanucleotides to the DNA tempers, and the complementary strands are synthesized from the 3'-OH terms to the random hexanucleotide primer using polymerase and the four triphosphates of deoxynucleotide. Thus the reaction is independent of the length of the DNA tempering. DNA fragments of 200 bases in length can be primed equally well as linearized plasmid or DNA? (29) This is particularly useful, for example, for overlapping peptides. 5. Since DNase I is an endonuclease that hydrolyzes double-stranded DNA at sites adjacent to pyrimidine nucleotides, its use in DNA redistribution may be the result of a bias (particularly for genes with high G + C content or high content). of A + T) in the passage of digestion and temperate gene. The effects of this potential bias on the overall mutation regimen and the frequency of recombination using the random priming approach can be avoided. The bias in the random priming due to preferential hybridization to the GC rich regions of the tempered DNA could be overcome by increasing the content of A and T in the library of random oligonucleotides. An important part of practicing the present invention is to control the average size of the single stranded, nascent DNA synthesized during the random priming process. This step has been studied in detail by others. Hodgson and Fisk (30) found that the average size of the single-stranded DNA synthesized is an inverse function of the primer concentration: Length = k / (lnPc), where Pe is the primer concentration. The inverse relationship between the concentration of primer and the size of the DNA fragment produced may be due to steric hindrance. Based on this line, suitable conditions for random priming synthesis can be easily set for individual genes of different lengths. Since dozens of polymerases are currently available, the synthesis of nascent, short DNA fragments can be achieved in a variety of ways. For example, the DNA polymerase of bacteriophage T4 (23) or the DNA polymerase version 2.0 of T7 sequence (31, 32) can be used for the random priming synthesis. For tempered single-stranded polynucleotides (particularly for RNA tempers), a reverse transcriptase is preferred for the random priming synthesis. Since this enzyme lacks 3 '? 5' exonuclease activity, it is quite prone to error. In the presence of high concentrations of dNTPs and Mn, approximately 1 base in each 500 is poorly incorporated (29). By modifying the reaction conditions, the polymerase chain reaction can be adjusted for the random priming synthesis using thermostable polymerase for the nascent, short DNA fragments. An important consideration is to identify by routine experimentation the reaction conditions which ensure that the short random primers can anneal to the hardened ones and give sufficient amplification of DNA at high temperatures. We have found that random primers as short as dp (N) 12 can be used with polymerase chain reaction to generate the extended primers. Adapting the polymerase chain reaction to a random priming synthesis provides a convenient method for making nascent, short DNA fragments, and makes this random priming recombination technique very robust. In many evolution scenarios, recombination should be conducted between oligonucleotide sequences for which sequence information is available during at least some of the annealing sequences. In these scenarios, it is often possible to define and synthesize a series of primers that are interspersed between the different mutations. When defined primers are used, they can be 6 to 100 bases long. In accordance with the present invention, it was discovered that by leaving these defined primers to initiate a series of primer extension overlap reactions (which can be facilitated by thermocycling), it is possible to generate recombination cassettes each containing one or more of the accumulated mutations , allelic or isotypic differences between temperate. Using the defined primers in a manner that produces overlap extension products in the DNA polymerization reactions, the depletion of the available primers leads to the progressive cross-hybridization of the extended primer products until the complete gene products are generated. . The repeated rounds of tempering, extension and denaturation ensure the recombination of each overlap cassette with each of the others. A preferred embodiment of the present invention involves methods in which a set of defined oligonucleotide primers is used to prime DNA synthesis. Figure 2 illustrates an exemplary version of the present invention in which defined primers are used. The design and careful placement of the oligonucleotide primers facilitates the generation of non-random extended recombination primers and is used to determine the most important recombination events (co-segregation) along the length of homologous templates. Another embodiment of the present invention is an alternative approach to the assembly and recombination of genes based on primers in the presence of tempers. Thus, as illustrated in Figure 3, the present invention includes recombination in which the polymerization of DNA catalyzed by enzyme is allowed to continue only briefly (limiting the time and lowering the temperature of the extension step) before denaturation . The denaturation is followed by the random tempering of the extended fragments to temper sequences and continue the partial extension. This process is repeated multiple times, depending on the concentration of primer and quenched, until the entire length sequences are made. This process is called a stepped extension, or StEP. Although random primers can also be used for the stepped extension process, gene synthesis is not as efficient as with defined primers. Thus, the defined primers are preferred. In this method, a short tempering / extension step (steps) is used to generate the partially extended primer. A typical tempering / extension step is made under conditions that allow tempering of high-fidelity primer primed greater than Tm "25), but limits polymerization / extension to no more than a few seconds (or an average extension to less). of 300 nucleotides.) Minimal extensions are preferably in the order of 20-50 nucleotides. Thermostable DNA polymerases have been shown to typically exhibit maximum polymerization rates of 100-150 nucleotides / second / enzyme molecule at optimal temperatures, but follow the approximate Arrhenius kinetics at temperatures approaching the optimum temperature (Topt) .Thus, at a temperature of 55 ° C, a thermostatic polymerase presents only 20-25 percent of the steady-state polymerization regime that presents 72 C (Topt), or 24 nucleotides / second (40) At 37 ° C and 22 ° C, it is reported that the Tag polymerase has extension activities of 1.5 and 0.25 nucleotides / second, r Specifically (24) Both time and temperature can be routinely altered based on the desired recombination events and knowledge of the basic kinetics and biochemistry of the polymerase. The progress of the stepped extension process is monitored by removing aliquots from the reaction tube at various time points in the primer extension and separating the DNA fragments by agarose gel electrophoresis. The evidence of the effective primer extension is seen from the appearance of a "spot" of low molecular weight initially in the process that increases in molecular weight with an increasing number of cycles. Unlike the gene amplification process (which generates new DNA exponentially), the stepped extension process generates new DNA fragments in an additive manner in their initial cycles containing DNA segments corresponding to the different temperate genes. Under non-amplification conditions, 20 cycles of stepped extension process generates a maximum DNA molar yield of approximately 40 times the initial annealing concentration. In comparison, the idealized polymerase chain process for the amplification of genes is of multiplicative yield, giving a maximum mole yield of approximately 1 x 10 times through the same number of steps. In practice, the difference between the two or processes can be observed by polymerase chain reaction giving a clear 'band' after only a few cycles (less than 10) when starting with tempering at concentrations less than 1 ng / ul and primers at 10-500 fold of excess (against 10 times the typical excess of gene amplification). Under similar reaction conditions, the stepped extension process would be expected to give a less visible 'stain', which increases in molecular weight with increasing number of cycles. When significant numbers of DNA molecules extended from the primer begin to reach sizes greater than 1/2 the length of the total length gene, a rapid jump in molecular weight occurs, according to the chains extending halfway forward and Reverse begin to cross hybridize to generate fragments almost 2 times the size of those who are at that point in the process. At this point, the consolidation of the spot in a discrete band of the appropriate molecular weight can be presented quickly either by proceeding to subject the DNA to the step of stepped extension, or alternating the thermocycle to allow the full extension of the primed DNA to drive the amplification exponential of genes.

After gene arrangement (and, if necessary, conversion to the double-stranded form) the recombined genes are amplified (optional), digested with convenient restriction enzymes and ligated into expression vectors to select expressed gene products . The process can be repeated if desired, in order to accumulate changes of sequences that lead to the evolution of desired functions. The stepped extension process and homologous gene arrangement process (StEP) represents a powerful, flexible method for recombining similar genes in a random or deviant manner. The process can be used to concentrate the recombination within or outside specific regions of a known series of sequences by controlling the placement of primers and the time allowed for the annealing / extension steps. It can also be used to recombine specific cassettes of homologous genetic information generated separately or within a single reaction. The method is also applicable to recombinant genes for which sequence information is not available but for which the 5 'and 3d functional amplification primers can be prepared. Unlike other recombination methods, the step extension process can be performed in a single tube using conventional procedures without complex separation or purification steps. Some of the advantages of the defined primer modalities of the present invention are summarized as follows: 1. The StEP method does not require the separation of parent molecules from the assembled products. 2. Defined primers can be used to skew the location of recombination events. 3. The stepped extension process allows the recombination frequency to be adjusted by varying extension times. 4. The recombination process can be carried out in a single tube. 5. The process can be carried out in single chain or double chain polynucleotides. 6. The process avoids the bias introduced by DNase I or other endonucleases. 7. Universal primers can be used. 8. Defined primers that exhibit limited randomness can be used to increase the frequency of mutation in selected areas of the gene. As will be appreciated by skilled artisans, various embodiments of the present invention are possible. Exemplary embodiments include: 1. Recombination and point mutation of related genes using only defined flanking primers and stepped extension. 2. Recombination and mutation of related genes using flanking primers and a series of internal primers at sufficiently low concentration that depletion of the primers will occur during the course of thermocycling, forcing the overlapping gene fragments to cross-hybridization and extending until they are form the recombined synthetic genes. 3. The recombination and mutation of genes using primers of random sequence at high concentration to generate a deposit of short fragments of DNA that rearrange to form new genes. 4. Recombination and mutation of genes using a set of defined primers to generate a deposit of DNA fragments that rearrange to form new genes. 5. Recombination and mutation of single-stranded polynucleotides using one or more defined primers and stepped extension to form new genes. 6. Recombination using defined primers with randomness defined to more than 30 percent or more than 60 percent of nucleotide positions within the primer. Examples of the practice showing the use of the primer-based recombination method are the following.

EXAMPLE 1 Use of defined flanking primers and stepped extension to recombine and improve the thermostability of subtilisin E This example shows how the recombination method of defined primers can be used to improve the thermostability of subtilisin E by the recombination of two known genes. encoding subtilisin E variants with thermostabilities that exceed that type of subtilisin E of the wild type. This example demonstrates the general method delineated in Figure 3 which uses only two primers corresponding to the 5 'and 3' ends of the annealed ones. As outlined in Figure 3, the extended recombination primers are first generated by the stepped extension process (StEP), which consists of repeated cycles of denaturation followed by the short step (or steps) of tempering / extension. The extended fragments rearrange into full-length genes by assembling homologous genes assisted by thermocycling in the presence of DNA polymerase, followed by an optional step of gene amplification. Rl and R2 mutants of thermostable subtilisin E were used to test the recombination technique based on defined primers using stepped extension. The positions in which these two genes differ from each other are shown in Table 1. Among the ten nucleotide positions that differ in Rl and R2, only those mutations that lead to amino acid substitutions Asn 181-Asp (N181D) and Asn 218 -Ser (N218S) confer thermostability. The remaining mutations are neutral with respect to their effects on thermostability (33). The half-life at 65 ° C of the simple variants NI8ID and N218S are approximately 3 times and 2 times higher than that of subtilisin E of the wild type, respectively, and their melting temperatures, Tm, are 3.7 ° C and 3.2 ° C greater than those of the wild-type enzyme, respectively. Random recombination events that produce sequences that contain both of these functional mutations will result in enzymes whose half lives at 65 ° C are approximately 8 times greater than subtilisin E of the wild type, provided they do not enter these genes during the recombination process. In addition, the global point mutagenesis regime associated with the recombination process can be estimated from the catalytic activity profile of a small sample of the recombinant variant library. If the mutagenesis regime is zero, 25 percent of the population should exhibit activity similar to the natural one, 25 percent of the population should have activity similar to (N181D + N218S) double mutant and the remaining 50 percent should have activity similar to (N181D or N218S) mutant. Finite point mutagenesis increases the fraction of the library that encodes enzymes with activity similar to the natural (or minor) type. This fraction can be used to estimate the point mutagenesis regime. TABLE 1 Substitutions of DNA and amino acids in the Rl and R2 mutants of subtilisin E thermostable The mutations listed are related to subtilisin E of the wild type with base substitution in 780 in common.

Materials and methods Procedure for, primer-based recombination \ defined using two flanking primers. Two defined primers, P5N (5'-CCGAG CGTTG CATAT GTGGA AG-3 '(SEQ ID NO: 1), the underlined sequence is the restriction site Ndel) and P3B (5'-CGACT CTAGA GGATC CGATT C-3' ( SEQ ID NO: 2), the underlined sequence is the restriction site BamHI), which corresponds to the flanked 5 'and 3' primers, respectively, were used for recombination. The conditions (100 ul final volume): 0.15 pmol plasmid DNA containing the Rl and R2 genes (mixed at 1: 1) were used as quenched, 15 pmol of each flanking primer, 1 time regulatory tag, 0.2 mM of each dNTP , 1.5 mM MgCl2 and 0.25 U of Tag polymerase. Program: 5 minutes at 95 ° C, 80 cycles of 30 seconds at 94 ° C, 5 seconds at 55 ° C. The correct size product (approximately 1 kb) was cut from a 0.8 percent agarose gel after electrophoresis and purified using the QIAEX II gel extraction gel. This purified product was digested with Ndel and BamHI and subcloned into the shuttle vector pBE3. This gene library was amplified in E. coli HB101 and transferred to competent DB428 cells of B. subtilis for its expression and selection, as described elsewhere (35). DNA sequencing The genes were purified using the QIAprep spin plasmid miniprep kit to obtain DNA of sequencing quality. Sequencing was done in an ABI DNA Sequencing System 373 using the Dye Finisher Cycle Sequencing set (Perkin-Elmer, Branchburg, NJ). Results The progress of the step extension was monitored by removing aliquots (10 ul) from the reaction tube at various time points in the primer extension process and separating DNA fragments by agarose gel electrophoresis. Electrophoresis of the primer extension reactions revealed that the 5-second quenching / extension reactions at 55 ° C resulted in the occurrence of a stain reaction reaching 100 base pairs (after 20 cycles), 400 base pairs (after 40 cycles), 800 base pairs (after 60 cycles) and finally a band * of 1 kb within this spot. This band (mixture of rearranged products) was purified with gel, digested with restriction enzyme BainHI and Ndel, and ligated with vector generated by BamHI digestion -Ndel shuttle vector pBE3 E. coli / B. subtilis. This gene library was amplified in E. coli HBlOl and transferred into competent DB428 cells of B. subtilis for its expression and selection (35). The thermostability of enzyme variants was determined in the 96-well plate format described above (33). Approximately 200 clones were analyzed, and approximately 25% retained subtilisin activity. Among these active clones, the frequency of the double mutant-like phenotype (high thermostability) was approximately 23 percent, the simple mutant-like phenotype was approximately 42 percent, and the wild type-like phenotype was approximately 34 percent. hundred. This distribution is very close to the expected values when the two thermostable mutations N218S and Ni8ID can recombine with each other completely freely. Twenty clones were randomly chosen from the HBlOl gene library of E. coli. Their plasmid DNAs were isolated and digested with Ndel and BamHl. Nine out of 20 (45 percent) had inserts of the correct size (approximately 1 kb). Thus, approximately 55 percent of the previous library had no activity due to the lack of the correct subtilisin E gene. These clones are not members of the subtilisin library and should be removed from our calculations. Taking this factor into account, we found that 55 percent of the library (25 percent of active clones / 45 percent of clones with correct size insert) retained subtilisin activity. This activity profile indicates a point mutagenesis regimen of less than 2 mutations per gene (36). Five clones with inserts of correct size were sequenced. The results are summarized in Figure 4. All five genes are recombination products with minimal crosses that vary from 1 to 4. Only one new point mutation was found in these genes. EXAMPLE 2 Use of defined flanking primers and stepped extension to recombine pNB esterase mutants The two primer recombination method used here for the pNB esterase is analogous to that described in Example 1 for subtilisin E. Two mutant genes were used. esterase pNB tempered that differ in 14 bases. Both hardened (61C7 and 4G4) are used in the form of a plasmid. Both target genes are present in the extension reaction at a concentration of 1 ng / ul. The flanking primers (RM1A and RM2A, Table 2) are added to a final concentration of 2 ng / ul (approximately 200 times molar excess over annealed). TABLE 2 Primers used in the recombination of the pNB esterase genes Primer Sequence RM1A GAG CAC ATC AGA TCT ATT AAC (SEQ ID NO: 3) RM2A GGA GTG GCT CAC AGT CGG TGG (SEQ ID NO: 4) Clone 61C7 was isolated based on its activity in organic solvent and contained 13 DNA mutations against the wild-type sequence. Clone 4G4 was isolated for thermostability and contains 17 DNA mutations when \ compares with the natural type. Eight mutations are shared among them, due to the common ancestor. The product of the 4G4 gene is significantly more thermostable than the product of the 61C7 gene. Thus, a measure of the recombination between the genes is the cosegregation of high solvent activity and high thermostability or the loss of both properties in the recombined genes. In addition, the frequency of recombination and the mutagenic regime can be checked by sequencing random clones. For the pNB esterase gene, the extension of the primer proceeds through 90 rounds of extension with a thermocycle consisting of 30 seconds at 94 ° C followed by 15 seconds at 55 ° C. Aliquots (10 μl) are removed after cycle 20, 40, 60, 70, 80 and 90. Agarose gel electrophoresis reveals the formation of a low molecular weight 'blot' by cycle 20, which increases in size average and global intensity at each successive sample point. In cycle 90, a pronounced spot extending from 0.5 kb to 4 kb is evident, and exhibits maximum intensity signal even size of approximately 2 kb (the length of the full-length genes). The jump of genes from half-length to full-length appears to occur between cycles 60 and 70. The intense blot is amplified through 6 cycles of the polymerase chain reaction to more clearly define the population of recombined genes, in length complete A less primer control is also amplified with flanking primers to determine the bottom due to residual quenching in the reaction mixture. The band intensity of the extended gene population of the primer exceeds that of the control by more than 10 times, indicating that the amplified, non-recombined annealed comprises only a small fraction of the population of amplified genes. The amplified recsmbired gene pool is digested with restriction enzymes Xbal and BamHI and ligated into the expression vector pNB106R described by Zock et al. (35). The transformation of ligated DNA into the TG1 strain of E. coli is done using the well-characterized calcium chloride transformation process. Transformed colonies were selected on LB / agar plates containing 20 μg of tetracycline. The mutagenic regime of the process is determined by measuring the percent of clones expressing an active esterase (20). In addition, random colonies are chosen and sequenced and used to define the mutagenic frequency of the method and the efficiency of the recombination.

EXAMPLE 3 Recombination of pNB esterase genes using defined internal intercalated primers and stepped extension This example demonstrates that the intercalated defined primer recombination technique can produce novel sequences through point mutagenesis and recombination of mutations present in the parent sequences. Experimental design and background information Two genes of enterasa pNB (2-13 and 5-B12) were recombined using the recombination technique of defined primers. The products of the genes of both 2-13 and 5-B12 were measurably more thermostable than the wild type. The 2-13 gene contained 9 mutations not originally present in the wild type sequence, while the 5-B12 gene contained 14. The positions in which these two genes differ from each other are shown in Figure '5. Table 3 shows the sequences of the eight primers used in this example. The place (at the 5 'end of the temperate gene) of oligo tempering to the temperate genes is indicated in the table, as its primer orientation (the F indicates a forward primer, R indicates reverse). These primers are shown as arrows along the 2-13 gene in Figure 5.

TABLE 3 Sequences of primers used in this example name positioning sequence sequence RM1A F -76 GAGCACATCAGATCTATTAAC (SEQ ID NO: 3) RM2A R +454 GGAGTGGCTCACAGTCGGTGG (SEQ ID NO: 4) S2 F 400 TTGAACTATCGGCTGGGGCGG (SEQ ID NO: 5) S5 F 1000 TTACTAGGGAAGCCGCTGGCA (SEQ ID NO: 6) S7 F 1400 TCAGAGATTACGATCGAAAAC (SEQ ID NO: 7) S8 R 1280 GGATTGTATCGTGTGAGAAAG (SEQ ID NO: 8) S10 R 880 AATGCCGGAAGCAGCCCCTTC (SEQ ID NO: 9) S13 'R 280 CACGACAGGAAGATTTTGACT (SEQ ID NO: 10) Materials and Methods Recombination based on defined primers 1. Preparation of genes to be recombined. Plasmids containing the genes to be recombined were purified from TGl cells using the Qiaprep game (Qiagen, Chateworth, CA). The plasmids were quantified by ultraviolet absorption and mixed 1: 1 for a final concentration of 50 ng / ul. 2. Stepped extension, chain reaction of polymers and rearrangement. 4 μl of the plasmid mixture was used as a template in a standard reaction of 100 μl (1.5 mM MgCl 2, 50 mM KCl, 10 mM Tris-HCl pH 9.0, 0.1 percent Triton X-100, 0.2 mM dNTPs, 0.25 U of Tag polymerase (Promega, Madison, Wl) which also contained 12.5 ng of each of the 8 primers. A control reaction that did not contain primers was also assembled. The reactions were thermocycled through 100 cycles of 94 ° C, 30 seconds, - AH248 55 ° C, 15 seconds. Verifying an aliquot of the reaction on an agarose gel at this point showed that the product was a large blot (no visible product in the control without primer). 3. Dp.nl digestion of tempers. 1 μl of the array reactions was then digested with Dpnl to remove the hardened plasmid. The lOμl of the Dpnl digest contained 1 x NEBuffer 4 and 5 U Dpnl (both obtained from New England Biolabs, Beverly, MA) and incubated at 37 ° C for 45 minutes, then AH-248 by incubation at 70 ° C for 10 minutes to kill the enzyme with heat. 4. Amplification of polymerase chain reaction of rearranged products. The 10 μl digest was then added to 90 μl of standard polymerase chain reaction (as described in step 2) containing 0.4 μM * + of primers 5b (ACTTAATCTAGAGGGTATTA) (SEQ ID NO: 11) and 3b (AGCCTCGCGGGATCCCCGGG) (SEQ ID NO: 12) specific for the ends of the gene. After 20 cycles of standard polymerase reaction (94 ° C, 30 seconds, 48 ° C, 30 seconds, 72 ° C, 1 minute) a strong band of the correct size (2kb) was visible when the reaction was verified on a gel of agarose, while only a very blurred band was visible in the control lane without primer. The product band was purified and re-cloned into the expression plasmid pNB106R and transformed by electroincorporation into TG1 cells. Results Four 96 well trays of colonies resulting from this transformation were tested to see the initial activity of esterase pNB and thermostability. Approximately 60 percent of the clones exhibited initial activity and thermostability within 20 percent of the parent gene values. Very few (10 percent) of the clones were inactive (less than 10 percent of the father's initial activity values). These results suggest a low mutagenesis regimen. Four mutants with the highest thermostability values were sequenced. Two clones (6E6'and 6H1) were the result of recombination between the parent genes (Figure 5). One of the remaining clones contained a new point mutation, and one showed no difference from the 5B12 parent. The combination of the T99C and C204T mutations in the 6E6 mutant is evidence of a recombination event at these two sites. In addition, the 6H1 mutant shows the loss of the A1072G mutation (but the retention of C1038T mutations and \ T1310C) which is evidence of two recombination events (one between sites 1028 and 1072, and another between 1072 and 1310).

A total of five New point mutations were found in the four sequenced genes. EXAMPLE 4 Recombination of two thermostable subtilisin E variants using internal defined primers and stepped extension This example demonstrates that the defined primer recombination technique can produce novel sequences containing new combinations of mutations present in the parent sequences. It also demonstrates the utility of the defined primer recombination technique to obtain further improvements in the functioning of the enzyme (here, thermostability). This example further shows that the defined primers can bias recombination so that recombination appears more frequently in the portion of the sequence defined by the primers (inside the primers). In addition, this example shows that specific mutations can be introduced into the recombined sequences using the sequence or sequences of suitable defined primers that contain the desired mutation or mutations. The genes encoding two variants of thermostable subtilisin E of Example 1 (R1 and R2) were recombined using \ the primer recombination procedure defined with internal primers. Figure 6 shows the four defined internal primers used to generate recombinant progeny genes from hardened genes Rl and R2 in this example. Primer P50F contains a mutation (A? T at base position 598) which removes a HindIII restriction site and simultaneously adds a new single Nhel site. This primer is used to demonstrate that specific mutations can also be introduced into the population of recombined sequences by the specific design of the defined primer. The R2 gene also contains an A? G mutation in the same base position, which eliminates the Hind III site. Thus the restriction analysis (cutting by Nhel and HindIII) of random clones sampled from the recombined library will indicate the efficiency of recombination and the introduction of a specific mutation via the mutagenic primer. Sequence analysis of randomly selected clones (not selected) provides additional information about the recombination and mutagenesis events that occur during recombination based on defined primers. Materials and methods Recombination based on defined primers A version of the defined primer-based recombination illustrated in Figure 2 is carried out with the addition of the stepped extension process. 1. The preparation of the genes that are going to recombine. Approximately 10 ug of plasmids containing Rl and R2 were digested at 37 ° C for 1 hour with Ndel and BamHI (30 U each) in 50 μl of buffer B (Boehringer Mannheim, Indianapolis, I). Inserts of approximately 1 kb were purified from 0.8 percent preparative agarose gels using the QIAEX II gel extraction set. The inserts of AD? were dissolved in 10 mM Tris-HCl (pH 7.4). The concentrations of AD? were estimated, and the inserts were mixed 1: 1 at a concentration of 50 ng / ul. 2. Stepwise extension, polymerase chain reaction and rearrangement. Conditions (100 ul final volume): approximately 100 ng inserts were used as quenched, 50 ng each of 4 internal primers, lx Taq regulator, 0.2 mM of each TP, 1.5 mM MgCl2 and .25 U polymerase Tag. Program: 7 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 (stepped extension), followed by 53 cycles of 30 seconds at 94 ° C, 15 seconds at 55 ° C, 1 minute at 72 ° C (gene assembly). 3. Dpnl digestion of tempers. 1 μl of this reaction was diluted to 9.5 μl with dH20 and 0.5 μl of restriction enzyme Dpnl was added to digest the tempering of AD? for 45 minutes, followed by incubation at 70 ° C for 10 minutes and then these 10 ul were used as a template in a 10-cycle polymerase chain reaction. 4. Amplification by polymerase chain reaction of rearranged products. Polymerase chain reaction conditions (100μl final volume): 30 pmol of each P5N and P3B external primer, Taq regulator lx, 0.2 mM of each dNTP and 2.5 U of Tag polymerase. Polymerase chain reaction program: 10 cycles of 30 seconds at 94 ° C, 30 seconds at 55 ° C, 1 minute at 72 ° C. This program gave a single band at the correct size. The product was purified and subcloned in shuttle vector pBE3. This gene library was amplified in E. coli HBlOl and transferred to competent DB428 cells of B. subtil is for its expression and selection, as described elsewhere (35). The thermostability of the enzyme variants was determined in a 96-well tray format described above (33). DNA sequencing Ten HBlOl transformants of E. coli were chosen for sequencing. The genes were purified using the QIAprep spin plasmid miniprep set to obtain DNA of sequencing quality. The sequencing was done in an ABI 373 DNA Sequencing system using the Dye Finisher Cycle Sequencing game (Perkin-Elmer, Branchburg, NJ). Results 1) restriction analysis: Forty clones randomly chosen from the recombined library were digested with Nhel and BamEI restriction enzymes. In a separate experiment the same forty plasmids were digested with Hindl I and JBamHI. These products of the reaction were analyzed by gel electrophoresis. As shown in Figure 7, eight out of 40 clones (approximately 20 percent) contain the newly introduced Nhel restriction site, demonstrating that the mutagenic primer has certainly been able to introduce the specified mutation into the population. 2) DNA sequence analysis The first ten clones chosen at random were subjected to sequence analysis, and the results are summarized in Figure 8. A minimum of 6 out of 10 genes have undergone recombination. Among these 6 genes, the minimal crossing events (recombination) between the Rl and R2 genes vary from 1 to 4. All visible crosses occurred within the region defined by the four primers. Mutations outside of region rarely, if they are, recombine, as shown by the fact that there is no recombination between the two mutations at base positions 484 and 520. These results show that the defined primers can bias recombination so that it appears more frequently in the portion of the sequence defined by the primers (inside the primers). Very close mutations also tend to remain together (for example, base substitutions 731 and 745 and the base substitutions 1141 and 1153 always remain as a pair). However, the sequence of clone 7 shows that two mutations as close as they are separated by 33 bases can recombine (base position 1107 and 1141). Twenty-three new point mutations were introduced into the ten genes during the process. This error rate of 0.23 percent corresponds to 2-3 new point mutations per gene, which is a regime that has been determined optimal to generate mutant libraries for the evolution of directed enzyme (15). The types of mutation are listed in Table 4. Mutations are mainly transitions and are distributed evenly throughout the gene.

TABLE 4 New point mutations identified in ten recombined genes Transition Frequency Transversion Frequency G? A 4 A? T 1 A? G 4 A? C 1 C? T 3 C? A 1 T? C 5 C? G 0 G? C 1 G? T 0 T? A 3 T? G 0 A total of 9860 bases were sequenced. The mutation regimen was 0.23 percent. 4) Phenotypic analysis Approximately 450 DB428 clones of B. Subtili s were chosen and cultured in SG medium supplemented with 20 ug / ml kanamycin in 96-well trays. Approximately 56 percent of the clones expressed activity enzymes. From previous experience, we know that this level of inactivation indicates a mutation regimen of the order of 2-3 mutations per gene (35). Approximately 5 percent of clones showed similar phenotypes (N181D + N218S) to double mutants (which is lower than the expected value of 25 percent for random recombination alone mainly due to point mutagenesis). (DNA sequencing showed that two clones, 7 and 8, of the 10 randomly chosen ones contained both N218S and N181D mutations). EXAMPLE 6 Optimization of the ECB decysate of Actinoplan.es utafiensis by the random priming recombination method In this example, the method is used to generate short DNA fragments from denatured DNA, linear, double-stranded DNA (e.g. restriction fragments purified by gel electrophoresis; 22). The purified DNA, mixed with a molar excess of primers, is denatured by boiling, and the synthesis is then carried out using the Klenow fragment of E. coli DNA polymerase I. This enzyme lacks 5 '? 3' exonuclease activity, so that the random priming product is synthesized exclusively by primer extension and is not degraded by exonuclease. The reaction is carried out at a pH of 6.6, where the 3 '? 5' exonuclease activity of the enzyme is greatly reduced (36). These conditions favor the random initiation of the synthesis The procedure involves the following steps: 1. Dissociate the DNA of interest with the appropriate restriction endonuclease (s) and purify the DNA fragment of interest by electrophoresis using the Wizard PCR Prep Kit game (Promega , Madison, Wl). As an example the ECB deacetylase gene of Actinoplanes utahensis was dissociated as an Xho I-Psh AI fragment of 2.4 kb in length from the recombinant plasmid pSHPlOO. It was essential to linearize the DNA for the subsequent denaturing step. The fragment was purified by agarose gel electrophoresis using the Wizard PCR Prep Kit (Promega, Madison, Wl) (Figure 9, step (a)). Purification was also essential in order to remove the restriction endonuclease regulator from DNA, since Mg ions make it difficult to denature the DNA in the next step. 2. 400 ng (approximately 0.51 pmol) of the double-stranded DNA dissolved in H20 was mixed with 2.75 μg. (approximately 1.39 nmol) of dp (N) 6 random primers.

After immersion in boiling water for 3 minutes, the mixture was immediately placed in an ice / ethanol bath. The size of the random priming products is an inverse function of the primer concentration (33). It is thought that the presence of high concentrations of primer leads to steric hindrance. Under the reaction conditions described here the random priming products are approximately 200-400 base pairs, as determined by electrophoresis through an alkaline agarose gel (Figure 9, step b). 3. Ten μl of 10 x reaction regulator [10X regulator: 900 mM HEPES, pH 6.6, 0.1 M magnesium chloride, 10 mM 'dithiothreitol, and 5' mM each of dATP, dCTP, dGTP and dTTP) added to the denatured sample, and the total volume of the reaction mixture was brought to 95 μl with H0. 4. Ten units (approximately 5 μl) of the Klenow fragment of polymerase I of E. coli DNA were added. All components were mixed by gently tapping the outside of the tube and centrifuged at 12 ° C., 000 g for 1-2 seconds in a microfuge to move all the liquid to the bottom. The reaction was carried out at 22 ° C for 35 minutes. The rate of extension depends on the concentrations of the annealing and the four nucleotide precursors. Because the reaction was carried out under conditions that minimize exonucleolytic digestion, the newly synthesized products did not degrade to a detectable degree. 5. After 35 minutes at 22 ° C, the reaction was terminated by cooling the sample to 0 ° C on ice. 100 μl ice H20 was added to the reaction mixture. 6. The random primed products were purified by passing the entire reaction mixture through Centricon-100 filters (to remove tempering and proteins) and Centricon-10 filters (to remove primers and fragments less than 50 bases), successively. Centricon filters are available from Amicon Inc (Berverly, MA). The retentate fraction (approximately 85 μl in volume) was recovered from Centricon-10. This fraction contained the desired random priming products (Figure 9, step c) and was used for the rearrangement of the total gene. The rearrangement of the total gene was carried out by the following steps: 1. To rearrange by polymerase chain reaction, 5 μl of the randomly primed DNA fragments of Centricon-10, 20 μl of 2x pre-mixed chain reaction of polymerase (Pfu regulator cloned 5 times diluted, 0.5 mM each of dNTP, 0. lU / μl cloned Pfu polymerase (Stratagene, La Jolla, CA)), 8 μl of 30 percent (volume / volume) glycerol and 7 μl of H20 were mixed on ice. Since the concentration of randomly primed DNA fragments used for rearrangement is the most important variable, it is useful to establish several reactions separately at different concentrations to establish the preferred concentration. 2. After incubation at 96 ° C for 6 minutes, 40 thermocycles were made, each with 1.5 minutes at 95 ° C, 1.0 minute at 55 ° C and 1.5 minutes + 5 seconds / cycle at 72 ° C, with the extension step of the last cycle proceeding at 72 ° C for 10 minutes, in a DNA Engine PTC-200 apparatus (MJ Research Inc., Watertown, MA) without adding mineral oil. 3. Aliquots of 3 μl in cycles 20, 30 and 40 were removed from the reaction mixture and analyzed by agarose gel electrophoresis. The product of the rearranged polymerase chain reaction at 40 cycles contained the correct size product in a larger and smaller size spot (see Figure 9, step d). The correctly rearranged product of this first polymerase chain reaction was further amplified in a second polymerase chain reaction containing the polymerase chain reaction primers complementary to the ends of the quenched DNA. The amplification procedure was as follows: 1. 2.0 μl polymerase chain reaction rearrangement aliquots were used as an annealing in the standard 100-μl polymerase chain reactions, which contained 0.2 mM each of the primers of xhoF28 (5 'GGTAGAGCGAGTCTCGAGGGGGAGATGC3') (SEQ ID NO: 13) and pshR22 (5 'AGCCGGCGTGACGTGGGTCAGC 3') (SEQ ID NO: 14), 1.5 mM MgCl 2, mM Tris-HCl [pH 9.0], 50 mM KCl, 200 μM each of the four dNTPs, 6 percent (volume / volume) of glycerol, 2.5 U polymerase Tag (Promega, Madison, Wl) and 2.5 U polymerase PJGU (Stratagene, La Jolla, CA). 2. After incubation at 96 ° C for 5 minutes, se. carried out 15 thermocycles, each with 1.5 minutes at 95 ° C, 40 minutes at 55 ° C and 1.5 minutes at 72 ° C, followed by 15 additional thermocycles of 1.5 minutes at 95 ° C, 1.0 minute at 55 ° C and 1.5 minutes + 5 seconds / cycle at 72 ° C with the extension step of the last cycle proceeding at 72 ° C for 10 minutes, in a DNA Engine PTC-200 apparatus (MJ Research Inc., Watertown, MA) without adding mineral oil . 3. Amplification resulted in a large amount of polymerase chain reaction product with the correct size of the total ECC deacylase gene (Figure 9, step e). The cloning was carried out as follows: 1. The product of the polymerase chain reaction of the deacylase ECB gene was digested with the restriction enzymes Xhol and Psh AI, and cloned into a modified pIJ702 vector. 2. Protoplasts TK23 of S. lividans were transformed with the above ligation mixture to form a mutant library. ECB deacylase mutant selection In each transformant with the TK23 library of S. lividans obtained as described above was analyzed to see deacylase activity with a plaque assay method in itself using ECB as a substrate. The transformed protoplasts were allowed to regenerate on R2YE agar plates by incubation at 30 ° C for 24 hours and developed in the presence of thiostrepton for another 48-72 hours. When the colonies grew to the appropriate size, 6 ml of solution (Sigma) of purified agarose at 45 ° C containing 0.5 mg / ml of ECB in 0.1 M sodium acetate buffer (pH 5.5) was poured on top of each plate. R2YE agar and allowed to develop further for 18-24 hours at 37 ° C. Colonies surrounded by a zone of free space greater than that of a control colony containing recombinant plasmid pSHP150-2 of the wild type were indicative of a more efficient ECB hydrolysis resulting from improved enzyme properties or improved enzyme expression and level of secretion, and were chosen as potential positive mutants. These colonies were chosen for later preservation and manipulation. High performance liquid chromatography assay of ECC deacylase mutants Single positive transformants were inoculated in a 20 ml fermentation medium containing 5 μg / ml thiostreptory and allowed to grow at 30 ° C for 48 hours. In this step, all cultures were subjected to high performance liquid chromatography assay using ECB as substrate. 100 μl of whole broth was used for a high performance liquid chromatography reaction at 30 ° C for 30 minutes in the presence of 0.1 M NaAc (pH 5.5), 10 percent (volume / volume) of MeOH and 200 μg / ml of ECB substrate. 20 μl of each reaction mixture were loaded onto a polyhydroxyethylaspartamide (4.6 × 100 mm) poly (poly) column and eluted by acetonitrile gradient at a flow rate of 2.2 milliliters / minute. The ECB core was detected at 225 nm. Purification of ECC deacylase mutants After the HPLC assay, 2.0 milliliters of precultures of all potential positive mutants were then used to inoculate 50 ml of fermentation medium and allowed to grow at 30 ° C, 280 rpm for 96 hours. These 50 milliliter cultures were then centrifuged at 7,000 g for 10 minutes. The supernatants were recentrifuged at 16,000 g for 20 minutes. Supernatants containing deacylase ECB mutant enzymes were stored at -20 ° C. The supernatants of the positive mutants were further concentrated to 1/30 of their original volume with an Amicon filtration unit with 10 kD molecular weight disruption. The resulting enzyme samples were diluted with an equal volume of 50 mM regulator KH2P04 (pH 6.0) and 1.0 ml was applied to a Hi-Trap ion exchange column. The binding buffer was 50 mM KH2P04 (pH 6.0), and the elution buffer was 50 mM KH2P0 (pH 6.0) and 1.0 NaCl. A linear gradient of 0 to 1.0 M NaCl was applied in 8 column volumes with a flow rate of 2.7 ml / min. The mutant fraction of ECB deacylase was eluted at 0.3 M NaCl and concentrated and exchanged by regulator in 50 mM KH2P04 (pH 6.0) in Amicon Centricon-10 units. The purity of the enzyme was verified by SDS-PAGE, and the concentration was determined using the Bio-Rad protein assay. Specific activity assay of the ECB deacylase mutants 4.0 μg of each purified ECB deacylase mutant was used for the activity assay at 30 ° C for 0-60 minutes in the presence of 0.1 M NaAc (pH 5.5), 10 percent (volume / volume) of MeOH and 200 μg / ml of ECB substrate. 20μl of each reaction mixture was loaded onto a polyhydroxyethylaspartamide (4.6 x 100 millimeters) poly (poly) column and eluted with a gradient of acetonitrile at a flow rate of 2.2 milliliters / minute. The products of the reaction were monitored at 225 nm and recorded in an IBM PC data acquisition system. The core peak of ECB was numerically integrated and used to calculate the specific activity of each mutant. As shown in Figure 10, after only one round of applying this technique based on random priming on the wild type ECB deacylase gene, a mutant (M16) of 2.012 original transformants was found to possess 2.4 times the specific activity of the enzyme of natural type. Figure 11 shows that the activity of M16 has been increased relative to that of the wild type enzyme over a wide pH range. EXAMPLE 7 Improving the thermostability of subtilisin E from Bacillus subtilis using the random sequence primer recombination method This example demonstrates the use of several DNA polymerases for primer-based recombination. It also demonstrates the stabilization of subtilisin E by recombination. The Rl and R2 genes encoding two thermostable subtilisin E variants described in Example 1 were chosen as the hardened ones for recombination. (1) Preparation of the Obj ective Gene The heat-stable Rl and R2 mutant genes of subtilisin E (Figure 11) were subjected to random barley DNA synthesis. The fragment of 986 base pairs including 45 nucleotides of prosecuencia of subtilisina E, the whole mature sequence and 113 nucleotides after the stop codon were obtained by means of the double digestion of the plasmid pBE3 with Bam Hl and Nde I and were purified from a gel of agarose at 0.8 percent using the Wizard PCR Prep Kit game (Promega, Madison, Wl). It was essential to linearize the DNA for the subsequent denaturing step. Gel purification was also essential in order to remove the restriction endonuclease regulator from DNA, since Mg ions make it difficult to denature the DNA in the next step. (2) Randomized barley DNA synthesis The synthesis of random barley DNA used to generate short DNA fragments from linear, denatured double-stranded DNA. The mutant genes of subtilisin E purified from B. subtilis, mixed with a molar excess of primers, were denatured by boiling, and the synthesis was carried out using one of the following DNA polymerases: the Klenow fragment of E. coli DNA polymerase I, bacteriophage T4 DNA polymerase and DNA polymerase version 2.0 sequencing of T7. Under its conditions of optimal performance (29), the DNA polymerase of bacteriophage T4 gives synthesis results similar to those of the Klenow fragment. When using DNA polymerase version 2.0 of T7 sequence (31, 32), the lengths of the DNA fragments synthesized are usually larger. Some amount of MnCl2 has to be included during the synthesis in order to control the lengths of the synthesized fragments within 50-400 bases. Short, nascent DNA fragments with polymerase chain reaction can also be generated using the Stoffel fragment for Tag DNA polymerase or pfu DNA polymerase. An important consideration is to identify by routine experimentation the reaction conditions which ensure that short random primers can anneal to the hardened ones and give sufficient amplification of DNA at higher temperatures. We have found that random primers as short as dp (N) J2 can be used with polymerase chain reaction to generate fragments. 2.1 Synthesis of random barley DNA with the Klenow fragment. The Klenow fragment of DNA polymerase I E. coli lacks 5 '? 3' exonuclease activity, so that the random priming product is synthesized exclusively by primer extension, and is not degraded by the exonuclease. The reaction was carried out at pH 6.6, where the 3 '? 5' exonuclease activity of the enzyme was greatly reduced (36). These conditions favor the random initiation of the synthesis. 1. 200 ng (approximately 0.7 pmol) of RI DNA and an equal amount of R2 DNA dissolved in H20 were mixed with 13.25 μg (approximately 6.7 nmol) of random primers dp (N) g. After immersion in boiling water for 5 minutes, the mixture was immediately placed in an ice / ethanol bath. The size of the random priming products is an inverse function of the primer concentration (30). The presence of high concentrations of primer is thought to lead to spherical hindrance. Under the reaction conditions described here the random priming products are about 50-500 base pairs, as determined by agarose gel electrophoresis. 2. Ten μl of 10 x reaction regulator [10X regulator: 900 mM HEPES, pH 6.6, 0.1 M magnesium chloride, 20 mM dithiothreitol, and 5 mM each of dATP, dCTP, dGTP and dTTP) were added to the denatured sample, and the total volume of the mixture of the reaction was carried to 95 μl with H20. 3. Ten units (approximately 5 μl) of the Klenow fragment of polymerase I (Boehringer Mannheim, Indianapolis, IN) DNA from E. coli were added. All components were mixed by gently tapping the outside of the tube and centrifuged at 12,000 g for 1-2 seconds in a microfuge to move all the liquid to the bottom. The reaction was carried out at 22 ° C for 3 hours. The rate of extension depends on the concentrations of the annealing and the four nucleotide precursors. Because the reaction was carried out under conditions that minimize exonucleolytic digestion, the newly synthesized products did not degrade to a detectable degree. 4. After 3 hours at 22 ° C, the reaction was terminated by cooling the sample to 0 ° C on ice. 100 μl of ice-cold H20 was added to the reaction mixture. 5. Random primed products were purified by passing the entire reaction mixture through Microcon-100 (Amicon, Beverly MA) filters (to remove tempering and proteins) and Microcon-10 filters (to remove primers and fragments) under 40 bases), successively. The retentate fraction (approximately 65 μl in volume) was recovered from Microcon-10. This fraction containing the desired random priming products was exchanged by regulator against polymerase chain reaction regulator with the additional use of the new Microcon-10 for the rearrangement of the total gene. 2.2 Synthesis of random primed DNA with DNA polymerase of bacteriophage T4 The polymerase of bacteriophage T4 DNA and the Klenow fragment of polymerase I of E. coli DNA are similar in that each has a 5'-3 'polymerase activity and a 3'-5 exonuclease activity The exonucleases activity of the bacteriophage T4 DNA polymerase is more than 200 times that of the Klenow fragment. Since it does not displace the short oligonucleotide primers from the single-stranded DNA templates (23), the mutagenesis efficiency is different from the Klenow fragment. 1. 200 ng (approximately 0.7 pmol) of RI DNA and an equal amount of R2 DNA dissolved in H20 were mixed with 13.25 μg (approximately 6.7 nmol) of random primers dp (N) g. After immersion in boiling water for 5 minutes, the mixture was immediately placed in an ice / ethanol bath. The presence of high primer concentrations is thought to lead to steric hindrance. 2. Ten μl of 10 x reaction regulator [10X regulator: 500 mM Tris-HCl, pH 8.8; 150 mM (NH4) 2S04; 70 M of magnesium chloride, 100 mM of 2-mercaptoethanol, 0.2 mg / ml of bovine serum albumin and 2 mM of each of dATP, dCTP, dGTP and dTTP) were added to the denatured sample, and the total volume of the reaction mixture was brought to 90 μl with H20. 3. Ten units (approximately 10 μl) of polymerase I (Boehringer Mannheim, Indianapolis, IN) DNA were added. All components were mixed by gently tapping the outside of the tube and centrifuged at 12,000 g for 1-2 seconds in a microfuge to move all the liquid to the bottom. The reaction was carried out at 37 ° C for 30 minutes. Under the reaction conditions described here the random priming products are about 50-500 base pairs. 4. After 30 minutes at 37 ° C, the reaction was terminated by cooling the sample to 0 ° C on ice. 100 μl of ice-cold H20 was added to the reaction mixture. 5. Random primed products were purified by passing the entire reaction mixture through Microcon-100 filters (to remove tempering and proteins) and Microcon-10 filters (to remove primers and fragments less than 40 bases), successively. The retentate fraction (approximately 65 μl in volume) was recovered from Microcon-10. This fraction containing the desired random priming products was exchanged by regulator against polymerase chain reaction regulator with the additional use of the new Microcon-10 for the rearrangement of the total gene. 2.3 Synthesis of random primed DNA with DNA polymerase version 2.0 of T7 sequence. Since DNA polymerase version 2.0 of T7 sequence has no exonuclease activity and is highly processive, the average length of the DNA synthesized is greater than that of the DNA synthesized by the Klenoy fragment or T4 DNA polymerase. But in the presence of an adequate amount of MnCl 2 in the reaction, the size of the synthesized fragments can be controlled to less than 400 base pairs. 1. 200 ng (approximately 0.7 pmol) of RI DNA and an equal amount of R2 DNA dissolved in H20 were mixed with 13.25 μg (approximately 6.7 nmol) of random primers dp (N) 6. After immersion in boiling water for 5 minutes, the mixture was immediately placed in an ice / ethanol bath. The presence of high primer concentrations is thought to lead to steric hindrance. 2. Ten μl of 10 x reaction regulator [10X regulator: 400 mM Tris-HCl, pH 7.5; 200 mM magnesium chloride, 500 mM NaCl, 3 mM MnCl2, and 3 mM each of dATP, dCTP, dGTP and dTTP) were added to the denatured sample, and the total volume of the reaction mixture was led to 99.2 μl with H20. 3. Ten units (approximately 0.8 μl) of the sequencing version 2.0 of T7 (Amersham Life Science, Cleveland, Ohio) were added. All components were mixed by gently tapping the outside of the tube and centrifuged at 12,000 g for 1-2 seconds in a microfuge to move all the liquid to the bottom. The reaction was carried out at 22 ° C for 15 minutes. Under the reaction conditions described here the random priming products are about 50-400 base pairs. 4. After 15 minutes at 22 ° C, the reaction was terminated by cooling the sample to 0 ° C on ice. 100 μl of ice-cold H20 was added to the reaction mixture. 5. Random primed products were purified by passing the entire reaction mixture through Microcon-100 filters (to remove tempering and proteins) and Microcon-10 filters (to remove primers and fragments less than 40 bases), successively. The retentate fraction (approximately 65 μl in volume) was recovered from Microcon-10. This fraction containing the desired random priming products was exchanged by regulator against polymerase chain reaction regulator with the subsequent use of the new Microcon-10 for the rearrangement of the total gene. 2. 4 Synthesis of barley DNA with polymerase chain reaction using the Stoffel fragment of Tag DNA polymerase Similar to the Klenow fragment of polymerase I DNA. coli, the Stoffel fragment of Tag DNA polymerase lacks 5 'to 3' exonuclease activity. It is also more thermostable than Tag DNA polymerase. The Stoffel fragment has low processivity, with a primer extending an average of only 5-10 nucleotides before dissociating. As a result of its low processivity, it can also have improved fidelity. 1. 50 ng (approximately 0.175 pmol) of RI DNA and an equal amount of R2 DNA dissolved in H20 were mixed with 6.13 μg (approximately 1.7 nmol) of random primers dp (N) 12. 2. Ten μl of 10 x premix of the reaction [10X premix of the reaction: 100 mM Tris-HCl, pH 8.3; 30 mM magnesium chloride, 100 mM KCl, and 2 mM each of dATP, dCTP, dGTP and dTTP) were added to the denatured sample, and the total volume of the reaction mixture was brought to 99.0 μl with H20 3. After incubation at 96 ° C for 5 minutes, 2.5 units (approximately 1.0 μl) of the Stoffel fragment of Tag DNA polymerase (Perkin-Elmer Corp., Norwalk, CT) were added. Thirty-five thermocycles were made, each with 60 seconds at 95 ° C, 60 seconds at 55 ° C and 50 seconds at 72 ° C, without the extension step of the last cycle, in a DNA Engine PTC-200 apparatus (MJ Research Inc., Watertown, MA). B the reaction conditions described here the random priming products are approximately 50-500 base pairs. 4. The reaction was terminated by cooling the sample to 0 ° C on ice. 100 μl of ice H0 was added to the reaction mixture. 5. Random primed products were purified by passing the entire reaction mixture through Microcon-100 filters (to remove tempering and proteins) and Microcon-10 filters (to remove primers and fragments less than 40 bases), successively. The retentate fraction (approximately 65 μl in volume) was recovered from Microcon-10. This fraction containing the desired random priming products was exchanged by regulator against polymerase chain reaction regulator with the subsequent use of the new Microcon-10 for the rearrangement of the total gene. 2.5 Synthesis of random barley DNA with polymerase chain reaction using Pfu DNA polymerase Pfu DNA polymerase is extremely thermostable, and the enzyme possesses an inherent exonuclease activity of 3 'to 5' but does not possess an exonuclease activity. '?3' . Its base substitution fidelity has been estimated to be 2 x 10. 1. 50 ng (approximately 0.175 pmol) of RI DNA and an equal amount of R2 DNA dissolved in H20 were mixed with 6.13 μg (approximately 1.7 nmol) of random primers dp (N) 12. 2. Fifty μl of 2 x premix of the reaction [2X premix of the reaction: cloned Pfu regulator (Stratagene, La Jolla, CA), 0.4 mM of each dNTP], were added, to the denatured sample, and the total volume of the reaction mixture was brought to 99.0 μl with H20. 3. After incubation at 96 ° C for 5 minutes, 2.5 units (approximately 1.0 μl) of the Pfu DNA polymerase (Stratagene, La Jolla, CA) were added. Thirty-five thermocycles were made, each with 60 seconds at 95 ° C, 60 seconds at 55 ° C and 50 seconds at 72 ° C, without the extension step of the last cycle, in a DNA Engine PTC-200 apparatus (MJ Research Inc., Watertown, MA). Under the reaction conditions described here the random priming products are about 50-500 base pairs. 4. The reaction was terminated by cooling the sample to 0 ° C on ice. 100 μl of ice-cold H20 was added to the reaction mixture. 5. Random primed products were purified by passing all the reaction mixture through Microcon-100 filters (to remove tempering and proteins) and Microcon-10 filters (to remove primers and fragments less than 40 bases), successively. The retentate fraction (approximately 65 μl in volume) was recovered from Microcon-10. This fraction containing the desired random priming products was exchanged by regulator against polymerase chain reaction regulator with the subsequent use of the new Microcon-10 for the rearrangement of the total gene. (3) Rearrangement of the complete gene 1. For rearrangement by polymerase chain reaction 10 μl of random primed DNA fragments (from Microcon-10, 20 μl of 2X polymerase chain reaction premix (diluted Pfu regulator diluted 5 times, 0.5 mM of each dNTP, 0. lU / μl cloned Pfu polymerase (Stratagene, La Jolla, CA)), 15 μl of H20 was mixed on ice 2. After incubation at 96 ° C for 3 minutes, 40 Thermocycles were performed, each with 1.0 minute at 95 ° C, 1.0 minute at 55 ° C and 1.0 minute + 5 seconds / cycle at 72 ° C, with the extension step of the last cycle proceeding at 72 ° C for 10 minutes, in a DNA Engine PTC-200 apparatus (MJ Research Inc., Watertown, MA) without adding mineral oil 3. Aliquots of 3 μl in cycles 20, 30 and 40 were removed from the reaction mixture and analyzed by electrophoresis The product of the polymerase chain reaction rearranged at 40 cycles contained the product of the correct size in a larger and smaller size spot. (4) Amplification The correctly rearranged product of this first polymerase chain reaction was further amplified in a second polymerase chain reaction containing the polymerase chain reaction primers complementary to the ends of the quenched DNA. 1. 2.0 μl of polymerase chain reaction rearrangement aliquots were used as an annealing in the standard 100-μl polymerase chain reactions, which contained 0.3 mM each of the primers of Pl (5 'CCGAGCGTTGC ATATGTGGAAG 3' ) (SEQ ID NO: 15) and P2 (5 'CGACTCTAGAGGATCCGATTC 3') (SEQ ID NO: 16), 1.5 mM MgCl2, 10 mM Tris-HCl [pH 9.0], 50 M KCl, 200 mM of each of the four dNTPs, 2.5 U of Tag polymerase (Promega, Madison, Wl, USA)) and 2.5 U of Pfu polymerase (Stratagene, La Jolla, CA). . 2. After incubation at 96 ° C for 3 minutes, 15 thermocycles were carried out, each with 60 seconds at 95 ° C, 60 seconds at 55 ° C and 50 seconds at 72 ° C, followed by 15 additional thermocycles. from 60 seconds at 95 ° C, 60 seconds at 55 ° C and 50 seconds (+ 5 seconds / cycle) at 72 ° C with the extension step of the last cycle proceeding at 72 ° C for 10 minutes, on a DNA Engine apparatus PTC-200 (MJ Research Inc., Watertown, MA) without adding mineral oil. 3. The amplification resulted in a large amount of polymerase chain reaction product with the correct size of the whole subtilisin E gene. (5) Cloning Since the short DNA fragments were generated with five different DNA polymerases, there was five depots of amplified end products of polymerase chain reaction. Each of the DNA deposits was used to construct the corresponding subtilisin E mutant library. 1. The amplified rearrangement product of the polymerase chain reaction was purified by the Wizard DNA-CleanUp kit (Promega, Madison, Wl), digested with Bam Hl and Nde I, electrophoresed on 0.8 percent agarose gel. The 986 base pair product was cut out of the gel and purified by the Wizard PCR Prep kit (Promega, Madison, Wl). The products were ligated with vector generated by the BamHI digestion -Ndel of shuttle vector pBE3. 2. Competent HBlOl cells of E. coli were transformed with the above ligation mixture to form a mutant library. Approximately 4,000 transformants of this library were deposited, and the recombinant plasmid mixture was isolated from this deposit. 3. The competent cells DB428 of B. Sutbilis were transformed with the mixture of previous isolated plasmids to form another library of the variants of subtilisin E. 4. Based on AD polymerase? used to randomly prime the nascent fragments, short, AD ?, the five libraries built were named: library / Klenow, library / T4, library / Secuenasa, library / Stoffel and library / Pfu. Approximately 400 transformants from each library were randomly chosen and subjected to selection to see the thermostability [see Step (7)]. (6) Sequencing of random clones Ten random clones of B were chosen. subtilis DB428 library / Klenow for sequence analysis of AD ?. The recombinant plasmids were purified individually from B. subtilis DB428 using a miniprep kit of plasmid spinning QIAprep (QIAGE?) with the modification that 2 mg / ml of lysozyme was added to the regulator Pl and the cells were incubated for 5 minutes at 37 ° C, retransformed in E. competent HB 101 coli and then purified again using the miniprep set of plasmid spinning QIAprep to obtain DNA of sequencing quality. Sequencing was done on an ABI 373 DNA Sequencing System using the Dye Finisher Cycle Sequencing set (Perkin-Elmer Corp., Norwalk, CT). (7) Selection to see thermostability Approximately 400 transformants for each of the five libraries described in Step (4) were subjected to selection. The selection was based on the previously described assay (33, 35), using succinyl-Ala-Ala-Pro-Phe-p-nitroanilide (SEQ ID NO: 25) as a substrate. B subtilis DB428 containing the plasmid library was plated on LB / kanamycin plates (20 μg / ml). After 18 hours at 37 ° C single colonies were chosen in 96-well trays containing 100 μl of SG / kanamycin medium per well. These trays were shaken and incubated at 37 ° C for 24 hours to allow the cells to grow to saturation. The cells were centrifuged, and the supernatants were sampled for the thermostability test. Three replicates of 96-well assay trays were duplicated for each culture tray, each well containing 10 milliliters of supernatant. The activities of subtilisin were then measured by adding 100 milliliters of activity assay solution (0.2 mm succinyl-Ala-Ala-Pro-Phe-p-nitroanilide (SEQ ID NO: 25), 100 mM Tris-HCl, 10 mM CaCl2, pH 8.0, 37 ° C). The reaction rates were measured at 405 nm over 1.0 minute in a ThermoMax microplate reader (Molecular Devices, Sunnyvale CA). The average activity at room temperature was used to calculate the fraction of active clones (clones with activity less than 10 percent of that of the wild type were classified as inactive). The initial activity (Ai) was measured after incubating a test plate at 65 ° C for 10 minutes by immediately adding 100 μl of preheated assay solution (37 ° C (0.2 mM succinyl-Ala-Ala-Pro-Phe-p- nitroanilide (SEQ ID NO: 25), 100 mM Tris HCl, pH 8.0, 10 mM CaCl 2) in each well Residual activity (Ar) was measured after 40 minutes of incubation. selection, a clone that showed the highest thermostability within 400 transformants of the library / Klenow was again scratched on the LB / kanamycin agar plate, and simple colonies derived from this plate were inoculated into cultures in tubes, for the preparation of glycerol broth and plasmid preparation The recombinant plasmid was purified using a miniprep set of plasmid spinning QIAprep (QIAGEN) with the modification that 2 mg / ml of lysozyme was added to the Pl regulator and the cells were incubated for 5 minutes at 37 minutes. ° C, retransformed in competent E. coli HB 101 and then purified again using the miniprep set of plasmid spinning QIA prep. to obtain DNA of sequencing quality. Sequencing was done in an ABI 373 DNA Sequencing System using the Dye Finisher Cycle Sequencing set (Perkin-Elmer Corp., Norwalk, CT). Results 1. The frequency of recombination and the efficiency associated with random sequence recombination. The random priming process was carried out as described above. The process is illustrated in Figure 1. Ten clones of the library / Klenow of mutants were randomly selected and sequenced. As summarized in Figure 12 and Table 5, all clones were different from the parent genes. The frequency of occurrence of a particular point mutation of the father Rl or R2 in the recombined genes varied from 40 percent to 70 percent, fluctuating around the expected value of 50 percent. This indicates that the two parent genes have already almost been randomly recombined with the random primer technique. Figure 12 also shows that all ten mutations can be recombined or dissected, even those that are only 12 base pairs apart. We then estimated the rates of thermoinactivation of subtilisin at 65 ° C by analyzing the 400 random clones from each of the five libraries built at Step (5). The thermostabilities obtained from a 96-well tray are shown in Figure 13, plotted in descending order. Approximately 21 percent of the clones exhibited thermostability comparable to the mutant with the NI8ID and N218S double mutations. This indicates that the N181D mutation of RC2 and the N218S mutation of RCl have been randomly recombined. Sequence analysis of the clone exhibiting the highest thermostability among the 400 transformants selected from the library / Klenow showed that the mutation NI8ID and N218S did exist. 2. Frequency of mutations introduced again during the random priming process. Approximately 400 transformants were chosen for each of the five B subtili DB428 libraries [see Step (5)], were grown in SG medium supplemented with 20 ug / ml kanamycin in 96-well trays and subjected to selection by subtilisin E activity. Approximately 77-84 percent of the clones expressed active enzymes, while 16-23 percent of the transformants were inactive, presumably as a result of newly introduced mutations. From previous experience, we know that this inactivation regime indicates a mutation regimen of the order of 1 to 2 mutations per gene (35). - As shown in Figure 12, 18 new point mutations were introduced in the process. This error rate of 0.18 percent corresponds to 1-2 point mutations per gene, which is a regimen that has been determined from the "inactivation" curve.The mutations are distributed almost randomly throughout the gene.

TABLE 5 Substitutions of DNA and amino acid residues in the random clones of the Library / Klenow The types of mutation are listed in TABLE 5. The direction of the mutation is clearly non-random. For example, A changes more frequently to G than either T or C. all transitions, and in particular T-C and A-G. They occur more frequently than transversion. Some nucleotides are more mutable than others. A transversion G? C, a C? G and a C? A were found within the 10 sequenced clones. These mutations were generated very rarely during the mutagenesis of polymerase chain reaction prone to subtilisin error (37). The random priming process can allow access to a greater range of amino acid substitutions than point mutagenesis based on polymerase chain reaction. It is interesting to note that a short stretch of 5 'C GGT ACG CAT GTA GCC GGT ACG 3' (SEQ ID NO: 16) at position 646-667 in parents Rl and R2 was mutated to 5 'C GGT ACG ATT GCC GCC GGT ACG 3 '(SEQ ID NO: 17) in the random clone C # 6. Since the stretch contained two short repetitions on both ends the newly introduced mutations may be the result of a sliding chain disparity process instead of just the process of point mutation. Since there is no frame change, this kind of slip can be useful for domain conversion. 3. Comparison of different fidelity of DNA polymerases in the random priming process During the random priming recombination, the homologous DNA sequences are almost randomly recombined and new point mutations are also introduced. Although these point mutations may provide useful diversity for some amplifications of in vi tro evolution, there is problematic recombination of beneficial mutations already identified previously, especially when the mutation regimen is that high. It is particularly important to control the error regime during the random priming process to successfully apply this technique to solve problems of in vi tro evolution. By choosing different DNA polymerase and modifying the reaction conditions, the molecular priming technique of random priming can be adjusted to generate mutant libraries with different error rates. The Klenow fragment of polymerase I of E. coli DNA, the DNA polymerase of bacteriophage T4, the DNA polymerase of Secuenase version 2.0 of T7, the Stoffel fragment of Tag polymerase and the Pfu polymerase have been tested for the synthesis of the fragment of nascent DNA. 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 measured in the 96-well tray selection test are plotted in order falling. The Library / Stoffel and the Library / Klenow contain higher percentage of subtilisin E clones of the wild type or inactive than that of the Library / Pfu. In the five populations, the percentage of naturally occurring and inactive clones varies from 17 to 30 percent. EXAMPLE 8 Use of defined flanking primers and stepped extension to recombine single-stranded DNA This example demonstrates the use of recombination of defined primers with stepped extension in the recombination of single-stranded DNA. Description of the method Single-stranded DNA can be prepared by a variety of methods, most easily from plasmids using an auxiliary phage. Many vectors of current use are derived from filamentous phages, such as those derived from M13mp. After transformation into cells, these vectors give rise to both new double-stranded circles and single-stranded circles from one of the two strands of the vector. The single-stranded circles are packed into phage particles, secreted from the cells and can be easily purified from the culture supernatant. Two defined primers (for example, hybridizing to the 5 'and 3' ends of the annealed ones) are used here to recombine single chain genes. Only one of the primers is needed before the final amplification of the polymerase chain reaction. The extended recombination primers are first generated by the stepped extension process (StEP)), which consists of repeating denaturation cycles followed by step or extremely abbreviated steps of tempering / extension. The extended fragments are then rearranged into full length genes by arrangement of homologous genes aided by thermocycling in the presence of a DNA polymerase, followed by a step of gene amplification. The progress of the stepped extension process is monitored by removing aliquots (10 ul) from the reaction tube (100 ul initial volume) at several time points in the extension of the primer and separating DNA fragments by agarose gel electrophoresis. Evidence of effective primer extension 5 is seen as the appearance of a low molecular weight 'blot' at the beginning in the process which increases in molecular weight by increasing the number of the cycle. The conditions of the initial reaction are set to allow denaturation of the annealing (for example, denaturation 0 to 94 ° C-30 seconds) followed by very short step (or steps) of annealing / extension (for example 55 ° C-1 a 15 seconds) repeated through increments of 5-20 cycles before sampling the reaction. Typically, 20 to 200 cycles of stepped extension are required to generate single-chain DNA 'spots' corresponding to sizes greater than the length of the entire gene. . The experimental design is as in Example 1. Two mutant Rl and R2 genes of thermostable subtilisin E are subcloned into the M13mpl8 vector by restriction digestion with EcoRI and Ba HI. Single-stranded DNA is prepared as described (39). Recombination based on two flanking primers Two defined primers, P5N (5'-CCGAG CGTTG CATAT GTGGA AG-3 ') (SEQ ID NO: 18), the underlined sequence is the restriction site Ndel) and P3B (5'-CGACT CTAGA GGATC CGATT C-31 (SEQ ID NO: 19), the underlined sequence is the restriction site BamHI), corresponding to the 5 'and 3' flanking primers, respectively, are used for recombination. Conditions (100 ul final volume): 0.15 pmol single-stranded DNA containing Rl and R2 genes (mixed at 1: 1) 15 pmol of a flanking primer (either P5N or P3B), lx Tag regulator, 0.2 mM of each dNTP, 1.5 mM MgCl2 and 0.25 U polymerase Tag are used as quenched. Program: 5 minutes of 95 ° C, 80-200 cycles of 30 seconds at 94 ° C, 5 seconds at 55 ° C. The single-stranded DNA products of correct size (approximately 1 kb) are cut out of 0.8% agarose gel after electrophoresis and purified using QIAEX II gel extraction set. This purified product is amplified by a conventional polymerase chain reaction. Condition (100 ul final volume): 1-10 ng of annealing, 30 pmol of each flanking primer, lx Taq regulator, 0.2 mM of each dNTP, 1.5 mM MgCl2 and 0.25 U polymerase Tag. Program: 5 minutes of 95 ° C, 20 cycles of 30 seconds at 94 ° C, 30 seconds at 55 ° C, i minute at 72 ° C. The product of the polymerase chain reaction is purified, digested with. Ndel and BamHI and subcloned in the shuttle vector pBE3. This gene library is amplified in E. coli HBlOl and transferred to the competent DB428 cells of B. subtilis for its expression and selection, as described elsewhere (35). The thermostability of the enzyme variants is determined in the 96-well tray format described above (33). This protocol results in the generation of novel sequences containing novel combinations of mutations of the parent sequences as well as novel point mutations. The selection allows the identification of enzyme variants that are more thermostable than the parent enzymes, as in Example 1. As apparent from the previous examples, primer-based recombination can be used to explore the vast space of potentially useful catalysts for its optimal performance in a wide range of applications as well as to develop or evolve new enzymes for studies of basic structure functions. Although the present specification describes using DNA-dependent DNA polymerase and single-stranded DNA as annealed, alternative protocols for using single-stranded RNA as annealed are also feasible. Using mRNA-specific protein such as RNA-dependent DNA (RNA) -dependent polymerase (reverse transcriptase) as the catalyst, the methods described herein can be modified to introduce mutations and crosses into cDNA clones and to create molecular diversity directly from the mRNA level to achieve the goal of optimizing protein functions. This would greatly simplify the marketed expression strategy) for the discovery of novel catalysts. In addition to the above, the present invention is also useful for obligate intracellular pathogen probe proteins or other systems where the cells of interest can not be propagated (38). Having thus described exemplary embodiments of the present invention, it should be noted by those skilled in the art that the disclosures are exemplary only and that various other alternatives, adaptations, and modifications may be made within the scope of the present invention. In accordance with the foregoing, the present invention is not limited to the specific embodiments as illustrated herein, but is limited only by the following claims.

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Zhao, H. and Arnold, F.H. 1997. Functional and non-functional mutations distinguished by random recombination of homologous genes. Proc. Nati Acad. Sci. USA 94: 7997-8000. Zock, J., Cantwell, C, Swartling, 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. Zhao, H. and Arnold, F.H. 1997. Optimization of DNA shuffling for high fidelity recombination. Nucleic Acids Research, 25: 1307-1308. . Lehman, I.R. and Richardson, C.C. 1964. The deoxyribonucleases of Escherichia coli IV. An exonuclease activity present in purified preparations of deoxyribonucleic acid polymerase. J. Biol. Chem. 239: 233. 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. Ebel, T., Middleton, J. F. S., Frish, A., and Lipp, J. 1997. Characterization of a secretory type Theileria parva glutaredoxin homologue identified by novel screening procedure. J. Biol. Chem. 272 (5): 3042-3048. Messing, J. 1983. Methods Enzymology 101: 20-78. Innis, M. A. et al., 1988. Proc. Nati Acad. Sci. 85: 9436-9440.

LIST OF SEQUENCES (1) GENERAL INFORMATION (i) APPLICANTS: French H. Arnold Zhixin Shao Joseph A. Affholter Huimin Zhao Lori Giver (ii) TITLE OF THE INVENTION: Recombination of Polynucleotide Sequences Using Sequences of Random or Defined Primers (iii) NUMBER OF SEQUENCES : 25 (iv) ADDRESS FOR CORRESPONDENCE: (A) RECIPIENT: Oppenheimer Po s Smith (B) STREET: 2029 Century Park East, Suite 3800 (C) CITY: Los Angeles (D) STATUS: CA (E) COUNTRY: USA ( F) CODE: 90067 (v) COMPUTER LEGIBLE FORM: (A) MIDDLE TYPE: Soft disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: Windows (D) SOFTWARE: Microsoft Word 6.0 (vi) APPLICATION DATA CURRENT: (A NUMBER OF APPLICATION: (B DATE OF SUBMISSION: (C CLASSIFICATION: (ii) PREVIOUS APPLICATION DATA: (A NUMBER OF APPLICATION: 60 / 041,666 (B DATE OF SUBMISSION: March 25, 1997 (C NUMBER OF APPLICATION: 60 / 045,211 (D DATE OF SUBMISSION: April 30, 1997 (E NUMBER OF APPLICATION : 60 / 046,256 (F: DATE OF SUBMISSION: May 12, 1997 (viii) ATTORNEY / AGENT INFORMATION: (A: NAME: Oldenkamp, David J. (B REGISTRATION NUMBER: 29,421 (c: REFERENCE NUMBER / FILE: 330187-84 (ix) TELECOMMUNICATION INFORMATION: (TO TELEPHONE: (310) 788-5000 (B TELEFAX: (310) 277-1297 (2) INFORMATION FOR SEQ ID NO: 1: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 22 nucleotides (B) TYPE: retinoid (C) TOPOLOGY: linear (ii) TYPE OF MOLECULE: oligonucleotide (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 1: CCG AGC GTT GCA TAT GTG GAA G. 22 (2) INFORMATION FOR SEQ ID NO: 2: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 21 nucleotides (B) TYPE: nucleotide (C) TOPOLOGY: linear (ii) TYPE OF MOLECULE: oligonucleotide (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 2: CGA CTC TAG AGG ATC CGA TTC "21 (2) INFORMATION FOR SEQ ID NO: 3: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 21 nucleotides (B) TYPE: nucleotide (C) TOPOLOGY: linear (ii) TYPE OF MOLECULE: oligonucleotide (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 3: GAG CAC ATC AGA TCT ATT AAC 21 (2) INFORMATION FOR SEQ ID NO: 4: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 21 nucleotides (B) TYPE: nucleotide (C) TOPOLOGY: linear (ii) TYPE OF MOLECULE: oligonucleotide (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 4: GGA GTG GCT CAC AGT CGG TGG 21 (2) 'INFORMATION FOR SEQ ID NO: 5: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 21 nucleotides (B) TYPE: nucleotide (C) TOPOLOGY: linear (ii) TYPE OF MOLECULE: oligonucleotide (xi) ) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 5: TTG AAC TAT CGG CTG GGG CGG 21 (2) INFORMATION FOR SEQ ID NO: 6: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 21 nucleotides (B) TYPE: nucleotide (C) TOPOLOGY: linear (ii) TYPE OF MOLECULE: oligonucleotide (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 6: TTA CTA GGG AAG CCG CTG GCA 21 (2) INFORMATION FOR SEQ ID NO: 7: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 21 nucleotides (B) TYPE: nucleotide (C) TOPOLOGY: linear (ii) TYPE OF MOLECULE: oligonucleotide (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 7: TCA GAG ATT ACG ATC GAA AAC 21 (2) INFORMATION FOR SEQ ID NO: 8 (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 21 nucleotides (B) TYPE: nucleotide (C) TOPOLOGY: linear (ii) TYPE OF MOLECULE: oligonucleotide (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 8: GGA TTG TAT CGT GTG AGA AAG 21 (2) INFORMATION FOR SEQ ID NO: 9: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 21 nucleotides (B) TYPE: nucleotide (C) TOPOLOGY: linear (ii) TYPE OF MOLECULE: oligonucleotide (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 9: AAT GCC GGA AGC AGC CCC TTC 21 (2) INFORMATION FOR SEQ ID NO: 10: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 21 nucleotides (B) TYPE: nucleotide (C) TOPOLOGY: linear (ii) TYPE OF MOLECULE: oligonucleotide (xi) DESCRIPTION OF THE SEQUENCE: SEQ TD NO: 10: CAC GAC AGG AAG ATT TTG ACT 21 (2) INFORMATION FOR SEQ ID NO: 11: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 20 nucleotides (B) TYPE: nucleotide (C) TOPOLOGY: linear (ii) TYPE OF MOLECULE: oligonucleotide (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 11: ACT TAA TCT AGA GGG TAT TA 20 (2) INFORMATION FOR SEQ ID NO: 12: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 20 nucleotides (B) TYPE: nucleotide (C) TOPOLOGY: linear (ii) TYPE OF MOLECULE: oligonucleotide (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 12: AGC CTC GCG GGA TCC CCG GG 20 (2) INFORMATION FOR SEQ ID NO: 13:. • (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 28 nucleotides (B) TYPE: nucleotide (C) TOPOLOGY: linear (ii) TYPE OF MOLECULE: oligonucleotide (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 13: GT AGA GCG AGT CTC GAG GGG GAG ATG C 28 (2) INFORMATION FOR SEQ ID NO: 14: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 22 nucleotides (B) TYPE: nucleotide (C) TOPOLOGY: linear (ii) TYPE OF MOLECULE: oligonucleotide (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 14: GC CGG CGT GAC GTG GGT CAG C 22 (2) INFORMATION FOR SEQ ID NO: 15: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 22 nucleotides (B) TYPE: nucleotide (C) TOPOLOGY: linear (ii) TYPE OF MOLECULE: oligonucleotide (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 15: CCG AGC GTT GCA TAT GTG GAA G. 22 (2) INFORMATION FOR SEQ ID NO: 16: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 21 nucleotides (B) TYPE: nucleotide (C) "TOPOLOGY: linear (ii) TYPE OF MOLECULE: oligonucleotide • ( xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 16: CGA CTC TAG AGG ATC CGA TTC 21 (2) INFORMATION FOR SEQ ID NO: 17: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 22 nucleotides (B) TYPE: nucleotide (C) TOPOLOGY: linear (ii) TYPE OF MOLECULE: oligonucleotide (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 17: CGG TAC GCA TGT AGC CGG TAC G 22 (2) INFORMATION FOR SEQ ID NO: 18: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 22 nucleotides (B) TYPE: nucleotide (C) TOPOLOGY: linear (ii) TYPE OF MOLECULE: oligonucleotide (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 18: CGG TAC GAT TGC CGC CGG TAC G 22 (2) INFORMATION FOR SEQ ID NO: 19: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 22 nucleotides (B) TYPE: nucleotide (C) TOPOLOGY: linear (ii) TYPE OF MOLECULE: oligonucleotide (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 19: CCG AGC GTT GCA TAT GTG GAA G 22 (2) INFORMATION FOR SEQ ID NO: 20: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 21 nucleotides (B) TYPE: nucleotide (C) TOPOLOGY: linear (ii) TYPE OF MOLECULE: oligonucleotide (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 20: CGA CTC TAG AGG ATC CGA TTC 21 (2) INFORMATION FOR SEQ ID NO: 21: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 18 nucleotides (B) TYPE: nucleotide (C) TOPOLOGY: linear (ii) TYPE OF MOLECULE: oligonucleotide (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 21: GGC GGA GCT AGC TTC GTA 18 (2) INFORMATION FOR SEQ ID NO: 22: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 18 nucleotides (B) TYPE: nucleotide (C) TOPOLOGY: linear (ii) TYPE OF MOLECULE: oligonucleotide (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 22: GAT GTG ATG GCT CCT GGC 18 (2) INFORMATION FOR SEQ ID NO: 23: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 18 nucleotides (B) TYPE: nucleotide (C) TOPOLOGY: linear (ii) TYPE OF MOLECULE: oligonucleotide (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 23: CAG AAC ACC GAT TGA GTT 18 (2) INFORMATION FOR SEQ ID NO: 24: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 18 nucleotides (B) TYPE: nucleotide (C) TOPOLOGY: linear (ii) TYPE OF MOLECULE: oligonucleotide (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 24: AGT GCT TTC TAA ACG ATC (2) INFORMATION FOR SEQ ID NO: 25: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 4 amino acids (B) TYPE: peptide (C) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 25 Ala Ala Pro Phe

Claims (34)

1. CLAIMS 1. A method for making double stranded mutagenized polynucleotides from at least one polynucleotide wherein the mutagenized polynucleotides have at least one nucleotide that is different from the nucleotide at the same position in said quenched polynucleotide, said method comprising: a) driving the synthesis of DNA polymerization catalyzed by enzyme from primers of random sequence or defined sequence in the presence of the tempered polynucleotide to form a DNA deposit comprising short fragments of polynucleotide and the tempered polynucleotide (s), - b) denaturing the deposit of DNA from a deposit for single chain fragments; c) allowing the single chain fragments to be quenched, under re-warming conditions, to form a deposit of tempered fragments; d) incubating the deposit of tempered fragments with polymerase under conditions that result in the extension of the double-stranded fragments to form a deposit of fragments comprising extended single-stranded fragments: e) repeating steps from (b) to (d) ) until the fragment deposit contains the mutagenized polynucleotides.
2. 2. A method for making double chain mutagenized polynucleotides according to claim 1 wherein the single chain fragments have areas of complementarity wherein the step of incubating the deposit of hardened fragments is carried out under conditions in which the chain of short polynucleotide or the extended short polynucleotide chains of each of the tempered fragments primes each of the others to form the fragment deposit.
3. 3. A method for making double-stranded mutagenized polynucleotides according to claim 1 wherein the step of incubating the deposit of hardened fragments is conducted in the presence of the tempered polynucleotide (s) to provide randomized re-priming of the single-stranded polynucleotides and the tempered polynucleotide (s).
4. 4. A method for making double stranded mutagenized polynucleotides according to claim 1 wherein at least one of the primers is a defined sequence primer.
5. 5. A method for making double-stranded mutagenized polynucleotides according to claim 2 wherein at least one of the primers is a defined sequence primer.
6. 6. A method for making double-stranded mutagenized polynucleotides according to claim 3 wherein at least one of the primers is a defined sequence primer.
7. 7. A method for making double chain mutagenized polynucleotides according to claim 4 wherein the primer comprises from 6 to 100 nucleotides.
8. 8. A method for making double stranded mutagenized polynucleotides according to claim 5 wherein the primer comprises from 6 to 100 nucleotides.
9. 9. A method for making mutagenized double-stranded polynucleotides according to claim 6 wherein the primer comprises from 6 to 100 nucleotides.
10. 10. A method for making mutagenized double-stranded polynucleotides according to claim 4 wherein at least one defined terminal primer is used.
11. 11. A method for making mutagenized double-stranded polynucleotides according to claim 5 wherein at least one defined terminal primer is used.
12. 12. A method for making mutagenized double-stranded polynucleotides according to claim 6 wherein at least one defined terminal primer is used.
13. A method for making mutagenized double-stranded polynucleotides according to claim 1 wherein the primers are defined sequence primers that exhibit limited randomness at one or more nucleotide positions within the primer.
14. A method for making mutagenized double-stranded polynucleotides according to claim 13 wherein the primers comprise from 6 to 100 nucleotides.
15. 15. A method for making mutagenized double-stranded polynucleotides according to claim 13 wherein two or more defined primers are used specific for any region of the annealing.
16. 16. A method for making mutagenized double-stranded polynucleotides according to claim 1 wherein the primers are defined sequence primers that exhibit limited randomness in more than 30 percent of the- nucleotide positions within the primer.
17. 17. A method for making double chain mutagenized polynucleotides according to claim 16 wherein the primers comprise from 6 to 100 nucleotides.
18. 18. A method for making mutagenized double-stranded polynucleotides according to claim 16 wherein two or more defined primers are used specific for any region of the annealing.
19. 19. A method for making mutagenized double-stranded polynucleotides according to claim 1 wherein the primers are defined sequence primers that exhibit limited randomness in more than 60 percent of the nucleotide positions within the primer.
20. 20. A method for making double chain mutagenized polynucleotides according to claim 19 wherein the primers comprise from 6 to 100 nucleotides.
21. 21. A method for making mutagenized double-stranded polynucleotides according to claim 19 wherein two or more defined primers are used that are specific to any region of the annealing (or quenched).
22. 22. A method for making mutagenized double-stranded polynucleotides according to claim 1 wherein the primers are random sequence primers.
23. 23. A method for making mutagenized double-stranded polynucleotides according to claim 22 wherein the lengths of the primers are from 6 to 24 nucleotides long.
24. 24. A method for making mutagenized double-stranded polynucleotides according to claim 22 wherein the temperate polynucleotide (s) is removed from the DNA pool after generation of the short polynucleotide fragments.
25. 25. A method for making mutagenized double-stranded polynucleotides according to claim 1 which includes the additional steps of isolating the mutagenized double-stranded polynucleotides from the DNA deposit and amplifying the mutagenized double-stranded polynucleotides.
26. 26. A method for making mutagenized double-stranded polynucleotides according to claim 25 wherein the mutagenized double-stranded polynucleotides are amplified by the polymerase chain reaction.
27. 27. A method for producing enzymes comprising the steps of: (a) inserting into a vector a double-stranded mutagenized polynucleotide made in accordance with claim 1 to form an expression vector, said mutagenized polynucleotide encoding an enzyme; (b) transforming a host cell with the expression vector; and (c) expressing the enzyme encoded by the mutagenized polynucleotide.
28. 28. A process for preparing mutagenized double-stranded polynucleotides from at least one quenched polynucleotide, the mutagenized polynucleotides having at least one nucleotide that is different from the nucleotide at the corresponding position in the quenched polynucleotide wherein the process comprises: (a) performing polymerization of enzyme-catalyzed DNA from primers of random sequence or sequence defined in the presence of the tempered polynucleotide (s) to form a DNA reservoir containing short fragments of polynucleotides and tempered polynucleotides; (b) denaturing the deposit of DNA in a deposit of both single-stranded fragment polynucleotides and single-stranded, tempered polynucleotides; (c) allowing the single stranded polynucleotides from the deposit to be quenched, under annealing conditions, to form a deposit of double stranded, polynucleotides; (d) incubating the deposit of tempered polynucleotides with DNA polymerase under conditions that result in the extension of the double-stranded polynucleotides to form a deposit of DNA containing double-stranded polynucleotides; and (e) repeating steps of (b) through (d) until the DNA reservoir containing the extended double-stranded polynucleotides contains the mutagenized polynucleotides.
29. 29. The process according to claim 28, wherein the deposit for polynucleotides of single chain fragments and single stranded chain polynucleotides contain single stranded fragment polynucleotides having regions complementary to the regions of other single chain fragment polynucleotides in the deposit so that these fragment polynucleotides are annealed to each other in step (c), and are primed together in step (d).
30. 30. The process according to claim 28, wherein the single-stranded chain polynucleotide (s) is quenched to at least some single chain-fragment polynucleotide, in step (c), so as to provide randomized re-grading of the polynucleotides of single chain fragments in step (d).
31. 31. A process for preparing mutagenized polynucleotides from at least two tempered polynucleotides, the tempered polynucleotides include a first tempered polynucleotide and a second tempered polynucleotide that differ from each other, the mutagenized polynucleotides have at least one nucleotide that is different from the nucleotide in the corresponding position in the first tempered polynucleotide and at least one other nucleotide that is different from that of the corresponding position in the second tempered polynucleotide, wherein the process comprises: (a) performing DNA polymerization catalyzed by either enzyme from a set of primers of random sequence or from at least one defined sequence primer, over hardened polynucleotides under standard DNA polymerization conditions or under conditions that result only in partial extension, to form a DNA combination containing fragments of poly inucleotides and tempered polynucleotides; (b) denaturing the deposit of DNA in a deposit of both single-stranded fragment polynucleotides and single-stranded, tempered polynucleotides; (c) allowing the single chain polynucleotides in the deposit to be quenched, under annealing conditions, to form a deposit of hardened double-stranded polynucleotides; (d) incubating the polynucleotide deposit with DNA polymerase under conditions which result in the full or partial extension of the double-stranded polynucleotides to form a DNA deposit containing double-stranded polynucleotides, and (e) repeating the steps from (b) to (d) until the DNA combination containing extended double-stranded polynucleotides contains the mutagenized polynucleotides; provided that, when (1) the polymerization conditions are used in step (b) or (2) the full extent is the result in step (d), if at least one defined sequence primer is used, when minus one of those primers must be a nonterminal primer.
32. 32. The process according to claim 31, wherein the first tempered polynucleotide differs from the second tempered polynucleotide in at least two base pairs.
33. 33. The process according to claim 32 wherein the two base pairs are separated from each other.
34. 34. The process according to claim 33 wherein the two base pairs are separated from each other by at least 15 base pairs.