WO1993012257A1 - Mutagenese produisant une banque par amplification enzymatique inverse - Google Patents

Mutagenese produisant une banque par amplification enzymatique inverse Download PDF

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Publication number
WO1993012257A1
WO1993012257A1 PCT/US1992/010647 US9210647W WO9312257A1 WO 1993012257 A1 WO1993012257 A1 WO 1993012257A1 US 9210647 W US9210647 W US 9210647W WO 9312257 A1 WO9312257 A1 WO 9312257A1
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Prior art keywords
nucleic acid
sequence
double
primers
primer
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PCT/US1992/010647
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English (en)
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Willem P. C. Stemmer
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Hybritech Incorporated
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Publication of WO1993012257A1 publication Critical patent/WO1993012257A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/102—Mutagenizing nucleic acids

Definitions

  • PCR polymerase chain reaction
  • PCR is based on the enzymatic amplification of a DNA sequence that is flanked by two oligonucleotide primers which hybridize to opposite strands of the target sequence.
  • the primers are oriented in opposite directions with their 3 ' ends pointing towards each other. Repeated cycles of heat denaturation of the template, annealing of the primers to their complementary sequences and extension of the annealed primers with a DNA polymerase result in the amplification of the segment defined by the 5* ends of the PCR primers. Since the extension product of each primer can serve as a template for the other primer, each cycle results in the exponential accumulation, of the specific target fragment, up to several million fold in a few hours.
  • the method can be used with a complex template such as genomic DNA and can amplify a single- copy gene contained therein. It is also capable of amplifying a single molecule of target DNA in a complex mixture of RNAs or DNAs and can, under some conditions, produce fragments to ten kb long.
  • the PCR technology is the subject matter o United States Patent Nos. 4,683,195, 4,800,159, 4,754,065, an 4,683,202 all of which are incorporated herein by reference
  • this method has also been used to generate site specific mutations in known sequences. Mutations are create by introducing mismatches into the oligonucleotide primer used in the PCR amplification.
  • the oligonucleotides are then incorporated at both ends o the linear PCR product.
  • the primers often contain restriction enzym recognition sequences which are used for subcloning th mutated linear DNAs into vectors in place of the wild typ sequences.
  • restriction enzym recognition sequences which are used for subcloning th mutated linear DNAs into vectors in place of the wild typ sequences.
  • this procedure is relatively simple t perform, its applications are limited because appropriat restriction sequences are not always conveniently located fo substituting the mutant sequence with the wild-type sequence.
  • Restriction sequences can be incorporated into the wild-typ sequences for subcloning. However, such extraneous sequence can cause detrimental effects to the function of the ' gene o resulting gene product.
  • PCR products typicall contain heterogeneous termini resulting from the addition o extra nucleotides and/or incomplete extension of the pri er templates. Such termini are extremely difficult to ligate and therefore result in a low subcloning efficiency.
  • IPCR inverse PCR
  • the resultant PCR product a linear DNA molecule identical in length to the starting plasmid contains any mutations which were designed into the primers
  • the product is then enzymatically prepared for ligation b blunting and phosphorylating the termini.
  • Enzymatic treatmen of the termini is a necessary step for ligation due t heterogeneous termini associated with PCR products. Thes treatments are likely to be incomplete and cause unwante mutations as well as result in a low ligation an transformation efficiency due to the additional require steps.
  • Recombinant circle PCR (RCPCR) , Jones and Howard BioTechniques 8:178-183 (1990), and recombination PCR (RPCR) , Jones and Howard, BioTechniques 10:62-65 (1991), on the othe hand, are two methods similar to IPCR which do not require an enzymatic treatment.
  • RCPCR two separate PCR reactions, requiring a total of four primers, are needed to generate th mutated product.
  • the separate amplification reactions ar primed at different locations on the same template to generat products that when combined, denatured and cross-annealed, form double-stranded DNA with complementary single-stran ends.
  • the complementary ends anneal to form DNA circle suitable for transformation into E. coli.
  • RPCR is a technique that uses PCR primers having a twelv base exact match at their 5' ends, resulting in a PCR produc with homologous double-stranded termini. Transformation o the linear product into recombination-positive (recA-positive) cells produces a circular plasmid through in viv recombination. Although this method reduces the number o steps and primers used compared to RCPCR, the transformatio and recombination of linear molecules is an inefficien
  • Mutant libraries are normall constructed by the mutagenesis of a small, defined area of a plasmid containing the gene or control region of interest. Methods for generating mutant libraries typically use synthetic oligonucleotides with random or biased mixtures bases in one or more positions along the oligonucleotide. variety of methods have been used to introduce these mutagen oligonucleotides into the expression vector.
  • t oligonucleotides are hybridized to a substantiall complementary strand of DNA and a polymerase is used to exten the length of the oligonucleotide into a polynucleotide whos length is dependant both on the length of the template and o the conditions of enzymatic extension.
  • This procedure permit the construction of large libraries of mutants havin mutations in one or more regions of the polynucleotide o protein sequence as compared with the template. From thes libraries, the transfectants or transformants can be screene for the desired characteristic.
  • rando mutagenesis employing PCR, and random mutagenesis in general are restricted in design by the choice of restrictio endonucleases traditionally employed for these procedures Often random mutagenesis has a relatively low efficiency suc that a significant number of individual mutations are los during primer extension and introduction of the polynucleotid into the host. Further, mistakes or unintended mutations ar often incorporated into the sequences resulting in a additional decrease in the efficiency. Selected mutations ma therefore be under or overrepresented in the library.
  • FIG. 1 is a schematic diagram outlining the steps of
  • FIG. 2 shows the design of EIPCR primers.
  • Line A shows a region of the PCR template (SEQ ID NO: 1) and two mutations to be made by EIPCR (indicated by small arrows) .
  • Line B shows how the primers (SEQ ID NO: 2; SEQ ID NO: 3) relate to th mutated product (line C) (SEQ ID NO: 4) .
  • This is not a actual reaction intermediate, but is a cartoon to draw whe designing the primers.
  • the primers are indicated in grey
  • the Bsa I recognition sequence SEQ ID NO: 5) is underlined Four or more bases are added 5' to the enzyme recognitio sequence of each primer to ensure efficient substrat recognition by the enzyme.
  • Line C shows the sequence of th mutated product.
  • the grey boxes show the parts of the prime that have been incorporated into the final product. Th overhangs of the two DNA ends are indicated, but th recognition sequences have been cut off and are not part o the final product.
  • Figure 3 is a list of class IIS restriction enzymes an the nucleotide sequence of their recognition sequences (SEQ I NOS: 5 through 20) .
  • Figure 4 is a schematic diagram showing the use of EIPC technology for generating single chain antibodies.
  • Line shows the template region (SEQ ID NO: 21) to be mutagenized t create a linker between heavy and light chain encodin sequences.
  • Line B shows the EIPCR primer design (SEQ ID NO 22; SEQ ID NO: 23) and line C shows the nucleotide (SEQ ID NO 24) and amino acid (SEQ ID NO: 25) sequence of an identified active single chain antibody sequence.
  • Figure 5 is a schematic of the 1.8 kb expression vecto pMCHAFvl for CHA255 Fv fragment expression.
  • the expressio cassette is located between Hind III and Eco Rl restrictio endonuclease sequences in pUC19.
  • Figure 6 is a schematic of EIPCR primer design.
  • Line shows the area of the wildtype leader sequence that wa replaced by a library of leader sequences.
  • Line B shows th design of the mutagenic primers relative to the template (SE ID NO: 26 and SEQ ID NO: 27) .
  • Line C shows the sequence o the identified, positive single chain Fv linker conferrin increased protein expression that was obtained from the. rando library (SEQ ID NO: 28) .
  • Figure 7 is a schematic illustrating EIPCR promote library mutagenesis.
  • Figure 7A is the template sequence.
  • T underlined regions in Figure 7B indicate the regions variability in the library.
  • the invention is directed to a method for generating recombinant mutagenesis library by introducing one or mor changes within a predetermined region of double strande nucleic acid, comprising providing a first primer populatio and a second primer population, each population having variable base composition at known positions along th primers, the primers incorporating a class IIS restrictio enzyme recognition sequence, being capable of directing chang in the nucleic acid sequence and being substantiall complementary to the double-stranded nucleic acid to allo hybridization thereto.
  • the method also comprises hybridizin the irst and second primer populations to opposite strands o the double-stranded nucleic acid to form a first pair o primer-templates oriented in opposite directions, performin enzymatic inverse polymerase chain reaction to generate a least one linear copy of the double stranded nucleic aci incorporating the change directed by the primer, cutting th double stranded nucleic acid copy with a class IIS restrictio enzyme to form a restricted linear nucleic acid molecul containing the change and introducing nucleic generate therefrom into compatible host cells.
  • the method additionall comprises the step of joining termini of the restricted linea nucleic acid molecule to produce double stranded circula nucleic acid.
  • the method preferably produces restricte linear nucleic acid molecules containing only the directed change in the nucleic acid sequence.
  • the double stranded nucleic acid is circular DNA.
  • the method can be performed on either eukaryotic or prokaryotic cells.
  • the double stranded nucleic acid encodes polypeptide.
  • the change in the nucleic acid can be introduced into the amino acid coding region of the polypeptide or into a regulatory region of the polypeptide. Thus changes may be introduced into promoter a enhancer regions of the double stranded nucleic acid.
  • T polypeptide encoded by the double stranded nucleic acid preferably expressed from the host cells.
  • t double stranded nucleic acid comprises a viral vector a compatible host cells comprise a helper virus packaging ce line that directs the packaging of viral particles containi the viral vector. The viral particles are preferab collected and the method additionally comprises the step infecting susceptible cells with the viral particles.
  • method for improving polypeptide expression fr a double-stranded nucleic acid sequence encoding polypepti comprising: measuring polypeptide expression from the doub stranded nucleic acid in a compatible host cell, providing first primer population and a second primer population, ea of the populations having a variable base composition at kno positions along the primers, the primers incorporating a cla IIS restriction enzyme recognition sequence, being capable directing change in the nucleic acid sequence and bei substantially complementary to t he double stranded nucle acid to allow hybridization thereto.
  • the method additional comprises hybridizing the first and second primer populati to opposite strands of the double stranded nucleic acid form a first pair of primer-templates orientated in opposi directions, performing enzymatic inverse polymerase cha reaction to generate at least one linear copy of the doub stranded nucleic acid incorporating the change directed by t primers, cutting the double stranded nucleic acid copy with class IIS restriction enzyme to form a restricted line nucleic acid molecule containing the change, introducing t nucleic acid from the cutting step or the PCR step into ho cells and measuring polypeptide expression from the modifi nucleic acid in the cells, and identifying cells wi expression levels greater than the expression levels measur in cells containing unmodified double stranded nucleic aci
  • the method preferably additionally comprises the step joining termini of the restricted linear nucleic acid molecu to produce modified double stranded circular nucleic acid a the method also preferably comprises the step of obtaini modified template from selected cells
  • t modified nucleic acid sequence is identified and transferr into another nucleic acid sequence.
  • the primers can dire changes in a regulatory sequence, including promoters, or t primers can direct changes in a polypeptide sequence. In preferred embodiment the primers direct changes in a riboso binding sequence.
  • method for generating a recombinant library usin wobble-base mutagenesis comprising: providing a first prime population and a second primer population, said primers bein substantially complementary to a region of double strande nucleic acid encoding polypeptide to allow hybridizatio thereto, the primers having a variable base composition in th third position of a least one nucleotide codon correspondin to the double stranded nucleic acid and a class II restriction enzyme recognition sequence.
  • the ' metho additionally comprises hybridizing the first and second prime populations to opposite strands of the double stranded nuclei acid to form a first pair of primer-templates orientated i opposite directions, performing enzymatic inverse polymeras chain reaction'to generate at least one linear copy of th double stranded nucleic acid incorporating the change directe by the primers, cutting the double stranded linear nuclei acid with a class IIS restriction enzyme to form restricte linear nucleic acid molecule containing the change an introducing nucleic acid generated therefrom into compatibl host cells.
  • the variable base codons preferably do not alte the corresponding animo acid sequence of the polypeptide.
  • the primers direct alterations in the leader sequence of the polypeptide.
  • the leade sequence is preferably the bacterial OmpA protein leader sequence of a fragment thereof and the leader sequence is preferably linked to polynucleotide encoding light and hea chain antibody fragments.
  • the invention provides a novel method for rapid a efficient site directed mutagenesis of double-stranded line or circular DNA.
  • the method termed Enzymatic Inver Polymerase Chain Reaction (EIPCR) , greatly improves t utility of previous PCR techniques enabling rapid screening selection of putative mutant to identify clones containi changes of interest.
  • EIPCR Enzymatic Inver Polymerase Chain Reaction
  • oligonucleotide primers containing t desired sequence changes are used to direct PCR synthesis a double-stranded circular DNA template ( Figure 1) .
  • T primers are designed so that they additionally contain a cla IIS restriction enzyme recognition sequence and a sequen complementary to the template for primer hybridization.
  • T primers are hybridized to opposite strands of the circul template and direct the amplification of each strand to fo linear molecules containing the desired mutations.
  • the en of the linear molecules are filled in with Klenow polymera or T4 DNA polymerase and restricted with the appropriate cla IIS restriction enzyme to produce compatible overhangs f circularization and ligation.
  • EIPCR uses class IIS restriction enzyme recogniti sequences in the mutated or non-mutated PCR primers.
  • Thi type of recognition sequence is used because the cleavage sit is separated from the recognition sequence and therefore doe not introduce extraneous sequences into the final product Restriction of the PCR products with a class IIS enzy removes the recognition sequence and produces homogeneou termini for subsequent ligation.
  • Class IIS recognitio sequences therefore circumvent problems associated wit ligating heterogeneous PCR termini since such termini will b cleaved off using a class IIS recognition enzyme. If th primers are designed with complementary cleavage sites, th resulting termini will have complementary overhangs which ca be used for circularization of the linear molecules.
  • EIP allows efficient mutagenesis and production of homogeneo termini of any DNA template without incorporating extraneo sequences.
  • EIPCR also allows mutagenesis at any location within a circular template independent of convenie restriction sequences.
  • the term "predetermined change” refers t a specific desired change within a known nucleic aci sequence. Such desired changes are commonly referred to i the art as site directed mutagenesis and include, for example additions, substitutions and deletions of base pairs. Specific example of a base pair change is the conversion o the first A/T bp in the sequence AGCA to a G/C bp to yield th sequence GGCA. It is understood that when referring to a bas pair, only one strand of a double-stranded sequence.or on nucleotide of a base pair need be used to designate th referenced base pair change since one skilled in the art wil know the corresponding complementary sequence or nucleotide
  • class IIS restriction enzym recognition sequence refers to the recognition sequence o class IIS restriction enzymes.
  • Class IIS enzymes cleav double-stranded DNA at precise distances from thei recognition sequence.
  • the recognition sequence is generall about four to six nucleotides in length and directs cleavag of the DNA downstream from the recognition sequence. Th distance between the recognition sequence and the cleavag site as well as the resulting termini generated in th restricted product vary depending on the particular enzym used.
  • the cleavage site can be anywhere from one to many nucleotides downstream from the 3* most nucleotide of the recognition sequence and can result in either blunt cuts or 5* and 3 1 staggered cuts of variable length.
  • complementary cleavage sites refers to complementary nucleic acid sequences at such single-stranded overhangs.
  • Class IIS restriction enzy recognition sequences suitable for use in the invention c be, for example, Alw I, Bsa I, Bbs I, Bbu I, Bsm Al, Bsr Bsm I, BspM I, Ear I, Esp 31, Fok I, Hga I, Hph I, Mbo II, P I, SfaN I, and Mnl I. It is understood that the recogniti sequence of any enzyme that utilizes this separation betwe the recognition sequence and the cleavage site is includ within this definition.
  • the term "substantially complementar” refers to a nucleotide sequence capable of specificall hybridizing to a complementary sequence under conditions kno to one skilled in the art. For example, specifi hybridization of short complementary sequences will occu rapidly under stringent conditions if there are no mismatche between the two sequences. If mismatches exist, specifi hybridization can still occur if a lower stringency is used Specificity of hybridization is also dependent on sequenc length. For example, a longer sequence can have a greate number of mismatches with its complement than a shorte sequence without losing hybridization specificity. Suc parameters are well known and one skilled in the art wil know, or can determine, what sequences are substantiall complementary to allow specific hybridization.
  • a primer capable of directing when used in reference to nucleic acid sequence changes refer to a primer having a mismatched base pair or base pairs withi its sequence compared to the template sequence. Suc mismatches correspond to the mutant sequences to b incorporated into the template and can include, for example additional base pairs, deleted base pairs or substitute bas pairs. It is understood that either one or both primers use for the PCR synthesis can have such mismatches so long a together they incorporate the desired mutations into the wild type sequence.
  • the invention provides methods of introducing a least one predetermined change in a nucleic acid sequence o a double-stranded DNA.
  • Such methods include: (a) providin a irst primer and a second primer capable of directing sa predetermined change in said nucleic acid sequence, said ir and second primers comprising a nucleic acid sequen substantially complementary to said double-stranded DNA so to allow hybridization, a class IIS restriction enzy recognition sequence and cleavage sites; (b) hybridizing sa first and second primers to opposite strands of said doubl stranded DNA to form a first pair of primer-templates orient in opposite directions; (c) extending said first pair primer-templates to create double-stranded molecules; ( hybridizing said first and second primers at least once said double-stranded molecules to form a second pair primer-templates; (e) extending said second pair of prime templates to produce double-stranded linear molecul terminating with class IIS restriction enzyme recogniti sequences; and (f) restricting said double-stranded line molecules with a class IIS restriction enzyme to fo restricted linear
  • EIPCR Enzymatic Inverse Polymerase Chain Reaction
  • the invention is described with particular reference t introducing a predetermined change into a circular templat and recircularizing of the product to generate mutant copie of the starting template.
  • the primers designe for use on linear templates are similar to those used fo circular templates.
  • Appropriate modifications of primers f use on linear templates are known to one skilled in the a and will be determined by the intended use of the final muta product. For example, when generating circular product either from a linear or circular starting template, it beneficial to use primers containing complementary cleava sites downstream from the class IIS recognition sequenc Such complementary sites greatly increase the efficiency intramolecular ligation.
  • the primers contain class IIS recognition sequences which produce singl stranded overhangs at their cleavage sites, such cleava sites need not be complementary.
  • the produ is a linear molecule for subcloning into a vector
  • cleava sites which are not complementary can be used for direction cloning of the product.
  • a blunt cleavage si can be used to eliminate sequence r> irements for subclonin
  • the cleavage sit within the primers can be complementary or non-complementar EIPCR primers are synthesized having three basic sequen components.
  • the first sequence componen of the primers is the region which directs the predetermine changes. This region contains the desired mutations which ar to be introduced into the template. The length and sequenc of this region will depend on the number and locations o incorporated mutations. For example, if multiple and adjacen mutations are desired, then the primer will not contain an nucleotides within this region identical to the wild-typ sequence. However, if the mutations are not located a adjacent positions, then the nucleotides in between suc mutations will be identical to the wild-type sequence an capable of hybridizing to the appropriate complementar strand. Thus, the region can be from one to many nucleotide in length so long as it contains the desired mismatches wit the wild-type sequence.
  • a strategy for designing EIP primers is outlined in Figure 2. This strategy shows example of a pair of primers which can be used for mutagenes at two nonadjacent locations.
  • One skilled in the art can u this strategy and the teachings described herein to design a use primers that incorporate essentially any desired mutati into a double-stranded DNA.
  • the template containing the wil type sequence is shown in Figure 2A (SEQ ID NO: 1) . Al shown are the desired nucleotide substitutions (arrows) .
  • T actual primers are depicted in Figure 2B as the shad sequence (SEQ ID NO: 2; SEQ ID NO: 3).
  • the region of ea primer containing the desired substitutions is complementa and corresponds to the opposite strand at the same location within the template ( Figure 2C) (SEQ ID NO: 4) .
  • the mutan region would consist of the sequence GTTCC and its complement respectively.
  • the second sequence component of EIPCR primers is th region containing the class IIS restriction enzyme recognitio sequence.
  • the location of the recognition sequence is 5' t the mutant region and thus is incorporated at the termini o any extension products. Since recognition sequences ar located at the ends of linear extension products, they ca also contain additional 5 1 sequences to facilitate recognitio and cleavage by a class IIS enzyme.
  • sequences included within the recognition sequenc component of EIPCR primers are the nucleotides between th recognition sequence and the cleavage site.
  • the number o nucleotides will correspond to the distance between these tw sites and therefore will vary for different enzymes.
  • the primers of Figure 2 contain a Bsa I recognitio sequence which is cleaved by Bsa I on opposite (SEQ ID NO: 5) strands one and five nucleotides, respectively, 3' to th recognition sequence, leaving a four nucleotide single-stran overhang.
  • overhang sequences within th primers are completely complementary to each other but ca include limited mutations.
  • Primers are synthesized wit filler nucleotides placed 5' to the first cleavage site. Th number of filler nucleotides corresponds to the distanc between the particular class IIS recognition sequence used an its cleavage site.
  • the sequence of such spacer nucleotide can, for example, correspond to wild-type or non-wild-typ sequences or to predetermined mutations. For generating jus a few point mutations, it is beneficial to match thes nucleotides to the wild-type sequence to increase th hybridization stability of the adjacent mutant primer region.
  • Types of restriction enzyme recognition sequences to b used in the invention are those recognized by class IIS enzymes. These enzymes recognize the DNA through a sequence specific interaction and cleave it at a discrete distance downstream from the recognition sequence. The ability t cleave such sequences downstream provides a useful means t remove heterogeneous ends and to produce complementary termini for circularization while at the same time removing the recognition sequence from the final product.
  • class IIS recognition sequences have been listed previously and are also listed in Figure 3 along with their nucleotide sequences and cleavage sites (SEQ ID NOS: 5 through 20) . Although recognition sequences having complementary cleavage sites associated with them are preferred, those which have blunt ended cleavage sites can also be used in the invention.
  • the third sequence component of EIPCR primers is the region to be hybridized to the template DNA. This region must be sufficient • in length and sequence to allow specific hybridization to the template.
  • the hybridized portion of the primers must also form a stable primer-template which can used as a substrate for polymerase extension. It is typical found 3 ' to the mutant primer region and its sequence determined with respect to the location of the desir mutations. For example, for the primers shown in Figure (SEQ ID NO: 2; SEQ ID NO: 3), the hybridization region twenty nucleotides in length and found 3* to the muta region. However, the hybridization region can also be 5' the mutant region.
  • the mutant regi must form a stable primer-template which can be used as substrate for polymerase extension.
  • Longer or short hybridization sequences can be used in this region so long they are appropriately located with respect to the muta region and also specifically hybridize to the templa molecule.
  • One skilled in the art knows or can readil determine the specificity of such hybridization regions fo use in EIPCR primers.
  • the invention also provides a synthetic primer fo introducing at least one predetermined change in a nuclei acid sequence of a double-stranded circular DNA.
  • the prime includes: (a) a class IIS restriction enzyme recognitio sequence; (b) said predetermined change in said nucleic aci sequence; and (c) a nucleic acid sequence substantiall complementary to said double-stranded DNA.
  • the preferre orientation of the above regions (a) through (c) is in a 5 1 t 3 - direction.
  • primers can be, for example hybridized to a double-stranded circular or linear DN molecule which has first been denatured. Denaturation can b performed, for example, using heat or an alkaline solution Other methods known to one skilled in the art* can also b used.
  • Hybridization of the primers occurs on opposite strand of the circular template and in a location where the single stranded overhangs of each primer's complementary cleavag site can be joined together by restriction and ligation.
  • such joining should occur so that the wild-typ sequence is reformed except for the incorporation of t desired mutations.
  • One way to ensure proper sequen reconstruction is to design the primers such that the complementary cleavage sites overlap and are either identic to the template sequence or contain some or all of the desir mutations.
  • Such primers once hybridized to a double-strand circular DNA, form primer-templates and can be extended wi a polymerase.
  • the first extension reactions of circul templates result in the synthesis of double-stranded circul products which can be concatenated.
  • t " e concatemers can be either partially completely double-stranded.
  • the plasmid pVX is a specifi example of a 902 bp vector, Seed, B. , Nuc. Acids Res. 11:2477 2444 (198.”*), which is incorporated herein by reference.
  • Suc vectors can be further modified by the addition of, fo example, promoters, terminators and the like to achieve th desired end.
  • Complete extension of a circular DNA of abou 5.0 kb can be achieved using the conditions described herein however, alternative conditions used by those skilled in th art to achieve complete extension of larger circular DNAs ca also be used to practice the invention.
  • the* first extension reaction produces double-stranded linear molecule known in the art as the lon product.
  • the double-strande products After one extension reaction, the double-strande products, whether they exist as circular or linear molecules have incorporated at one of their ends the EIPCR primer wit its associated class IIS restriction enzyme recognitio sequence and the desired mutations.
  • These double-strande molecules can be used for a second cycle of hybridization an extension to produce double-stranded linear molecules whic terminate at both ends with EIPCR primers. Further cycle will result in the exponential amplification of templat sequence located between each primer on the circular DNA Thus, the location of the hybridized primers defines th termini of template sequences to be amplified.
  • Polymerases which can be used for the extension reactio include all of the known DNA polymerases. However, i multiple cycles of hybridization and extension are to b performed, such as required for PCR amplification, the preferably a thermostable polymerase is used.
  • Thermostabl polymerases include, for example, Taq polymerase, Ven polymerase and PFU polymerase. Vent and PFU polymeras advantageously exhibit a higher fidelity than Taq due to their 3* to 5' proofreading capability.
  • the products are restricted with the appropriate class IIS restriction enzyme to remove the class IIS recognition sequence and heterogeneous termini and to create cohesive termini used for circularization.
  • the resulting termini correspond to the single-strand overhangs produced after restriction of each primer's complementary cleavage site.
  • the linear products can be pre ⁇ treated with a polymerase, such as Klenow, under conditions which create blunt ends. This procedure will fill in any uncompleted product ends produced during amplification and allows efficient restriction of essentially all of the products.
  • the cohesive termini can be joined to recircularize the linear molecule. Covalently closed circles can subsequently be formed in vitro with a ligase.
  • in vivo ligation can be accomplished by introducing the circularized products into a compatible host by transformation or electroporation, for example.
  • Transformation or electroporation of the circularized products can additionally be used for the propagation and manipulation of mutant products.
  • Such techniques and the uses are known to one skilled in the art and are describe for example, in Sambrook et al., Molecular Cloning: Laboratory Manual, Cold Spring Harbor, Cold Spring Harbor, (1989), or in Ausubel et al., Current Protocols in Molecul Biology, John Wiley and Sons, New York, NY (1989), both which are incorporated herein by reference.
  • Propagation a manipulation procedures do not have to be performed at the e of all EIPCR reactions. The need will determine whether su procedures are necessary. For example, transformation and D preparation can be eliminated if two consecutive EIP reactions are to be performed where the product of the fir reaction is used as the template for the second reaction.
  • a that is necessary is that the first reaction products a circularized and ligated prior to hybridization with t second reaction primers. Additionally, primers for EIPCR c be used without purification. EIPCR is not as sensitive other methods to the presence of primers of incomplete leng because the non-uniform DNA ends are removed by restriction the class IIS recognition sequence.
  • the invention further provides methods of producing least two changes located at one or more positions within nucleic acid sequence of a double-stranded circular DNA.
  • T methods include: (a) providing a first population of prime and a second population of primers capable of directing sa changes in said nucleic acid sequence, said first and seco populations of primers comprising a nucleic acid sequen substantially complementary to said double-stranded DNA so to allow hybridization, a class IIS restriction enzy recognition sequence, and cleavage sites; (b) hybridizing sa first and second populations of primers to opposite strands said double-stranded DNA to form a first pair of prime template populations orientated in opposite directions; ( extending said first pair of primer-template populations create a population of double-stranded molecules; ( hybridizing said first and second populations of primers least once to said population of double-stranded molecules form a second pair of primer-template populations; (e extending said second pair of primer-template populations t produce a population of double-strand
  • Also provide is a population of synthetic primers for producing at leas two changes located at one or more positions within a nuclei acid sequence of a double-stranded circular DNA comprising (a) a class IIS restriction enzyme recognition sequence; (b) said changes within said nucleic acid sequence; and (c) nucleic acid sequence substantially complementary to sai double-stranded circular DNA.
  • the method for producing at least two changes located a one or more positions is similar to that described above fo site-directed mutagenesis except that the primers can hav more than one nucleotide at a desired position. For example, if it is desirable to produce mutations incorporating from two to four different mutant nucleotides at a particular position, then a population of primers should be synthesized such that all mutant nucleotides are represented within the entire population. Each individual primer within the population will contain only a single mutant nucleotide. The proportion of primers containing identical mutant nucleotides will determine the expected frequency of that mutation being correctly incorporated into the final product.
  • primer populations should be synthesized which contain individual primers having each of the desired mutations. Primer populations can also be synthesized which direct single mutations at one position and multiple mutations at another position by incorporating one or more mutant nucleotides a the appropriate position.
  • primers are identical to tha previously described for introducing at least on predetermined change into a double-stranded circular DNA. Th only difference is that instead of hybridizing a first prime and a second primer to form a pair of primer-templates hybridization is with a first population of primers and second population of primers to form a pair of primer-templat populations.
  • Each primer-template within the population ca include, for example, one of the desired mutant sequences t be incorporated into the resultant products.
  • Amplification o the primer-template population will produce a population o linear products containing all desired mutations. Th products can be restricted, circularized and screened fo individual mutant clones.
  • Screening can be performed, fo example, by sequencing or by expression of polypeptide Selection can be performed by linking polypeptide expressio with the expression of a suitable marker such as an antibioti resistance gene, luciferase, or the like. Only colonie containing the gene are selected. Following selection positive colonies can then be screened for a particula characteristic.
  • Expression screening or selection offers th advantage of screening or selecting a large number of clone in a relatively short period of time. These assays permit th identification of clones of interest. Examples of screenin and selection assays are well known to those with skill in th art. Each assay is designed and modified for that particula application. Examples of these assays are found in th examples below.
  • the methods and primers described herein can be used t create essentially any desired change in a nucleic aci sequence.
  • Templates can be linear or circular and result i products containing only the desired changes since class II recognition sequences allow the removal of extraneous an unwanted sequences.
  • Product termini which are homogeneous i nature are also produced using the class IIS recognitio sequences.
  • Use of circular templates allows the incorporati of mutations at any desired location along the template wi subsequent recircularization of the mutant products. Thu additions, deletions and substitutions of single base pair multiple base pairs, gene segments and whole genes can rapid and efficiently be produced using EIPCR. A specific use EIPCR would be in the mutagenesis of antibodies or antibo domains.
  • CDR antibody complementary determini regions
  • EIPCR can also be used for produci chimeric and/or humanized antibodies having desir immunogenic properties.
  • the efficiency of incorporating correct mutations int the product using EIPCR can be, for example, greater tha about 90%, preferably about 95 to 99%, more preferably abou 100%. This efficiency is routinely obtained when using abou 0.5 to 2.0 ng of template in a 25 cycle PCR reaction
  • the efficiency directl correlates with the number of amplification cycles an inversely with the amount of template used. For example, th more amplification cycles which are performed, the greater th amount of mutant product present and therefore a large fraction of mutant sequences will be present within the tota sequence population.
  • EXAMPLE I This example shows the use of EIPCR for site-direct mutagenesis of two bases located on a 2.6 kb pUC-based plasm (designated pl86) .
  • the 3 s end of the primer is an exact match of 20 base
  • the 5' ends of the primers comprise the enzyme recogniti site and the enzyme cut site, which was designed to fo complementary overhangs.
  • Four additional bases were added to the enzyme recognition sequence to facilitate recogniti and digestion of the PCR product by the enzyme.
  • T complementary mutations were designed into each of t primers.
  • Bsa 1 was the enzyme used to make the overhan
  • T reaction was then extracted with an equal volume o phenol/chloroform (1:1), ethanol-precipitated, and the pelle was washed and dried.
  • the blunt end product was the restriction digested with Bsa I (New England Biolabs, Beverly MA) as recommended by the manufacturer.
  • DNA ligase (Gibco-BRL) for one hour at room temperature. Gel purification of the digested DNA before ligation was n necessary. After ligation, the DNA was transformed in competent DH10B cells recommended by the manufacturer (Gibc BRL) . Approximately 400 colonies were obtained from transformation using 10 ng of DNA into 30 ul of froz competent cells. The transformation efficiency was 4xl cfu/ug of DNA. Seven colonies were randomly picked a plasmid DNA was prepared for restriction digests. differences in restriction pattern were seen. The mutat areas of the plasmids of these seven colonies were sequence Double-stranded dideoxy sequencing was performed on a Dupo Genesis 2000 automated sequencer using the Dupont Genesis 20 sequencing kit. The sequences of all seven plasmids contain the desired mutation.
  • This example shows the use of EIPCR for constructi large libraries of protein mutants.
  • the binding site of an antibody called the Fv fragmen normally consists of a heavy chain and a light chain, eac about 110 amino acids long.
  • Fv fragmen normally consists of a heavy chain and a light chain, eac about 110 amino acids long.
  • scFv single chain Fv fragment
  • the single chain construct wa shown to be much more stable than the two chain Fv.
  • EIPCR wa used to make a large library of different linkers and scree for a scFv clone that is not only active but also expressed a a high level.
  • This plasmid was used as the template for EIPC in which the DNA between the c-terminus of the first chain an the n-terminus of the mature second chain was replaced by random mixture of bases, encoding «-, library of random linkers
  • the design of the primers is shown in Figure 4B in the shade region where N represents an equal proportion of all fou nucleotides at the position within the primer population.
  • Synthesis of the two primer populations used to construc the library was performed on a Milligen/Biosearch 8700 DN synthesizer.
  • the mixed base positions were synthesized usin a 1:1:1:1 mixture of each of the four bases in the reservoir.
  • the oligonucleotides were made trityl-on and were purified with Nensorb Prep nucleic acid purification column (NEN-Dupont, Boston, MA) as described by the manufacturer.
  • PCR reactions were performed in 100 ⁇ l volumes containin
  • T digested DNA was then extracted with phenol/chlorofor ethanol precipitated, and resuspended in 20 ul lx NEB ligati buffer, containing 1 mM ATP and 10 units T4 DNA ligase and t reaction was incubated for 2 hours at room temperature.
  • the labelled chelate was prepared incubating 10 ul of 0.075 mM Eotube chelate with 50 uCi buffered 111 Indium Chloride in a metal free tube. Colony lif of the petri plates containing the protein library we prepared using BA83 nitrocellulose filters (Schleicher an Schuell, Keene, NH) .
  • the filters were blocked by incubatio in Blotto (7% non-fat milk in PBS) for 10 minutes, washed wit PBS, followed by incubation in Blotto containing 10 uCi o m Indium Chloride per filter for 1 hour at room temperature The filters were then washed repeatedly with PBS for a tota of 15 minutes, dried and exposed to Kodak X-omat A autoradiography film for several hours.
  • the quality of the protein library was determined by DN sequencing of the linker of several unscreened clones Sequencing was performed as described in Example I. Th composition of the mixed site residues was 19% G, 31% A, 25
  • the size of the library was determined by plating. In typical electroporation, 30,000 cfu's were obtained fro electroporation of l ul of ligation mixture into 20 ul o cells. The ligation contained 0.1 ug of DNA in 20 ul. Th library size was about 3 l0 5 recombinants and th electroporation efficiency was 6xl0 6 cfu/ug. Approximate 30,000 clones were screened, and about 60 colonies gave range of signals on the primary screen (0.2%) . Those with t strongest signal were colony purified and the DNA sequence the linker was determined. The sequences of one inker fr an identified scFv clone is shown in Figure 4C. LIBRARY MUTAGENESIS
  • Library mutagenesis using a heterogenous prim population permits incorporation of a large number mutations into a population of host cells to generate recombinant library.
  • the resulting mutations are typical introduced into a polynucleotide suitable for cell deliver " a polynucleotide can additionally be adapted for expressio
  • These polynucleotides may contain anges in either t regulatory region of the polynu ieot ' .-:.*-. or in a translatab region.
  • the directed mutations in the lynucleotide sequen may alter levels of protein expressiw.i, alter a function characteristic of a protein, or confer a particular ce phenotype.
  • library mutagenesis The incorporation of a large number of mutatio into a host population is termed library mutagenesis.
  • libraries can be prepared and screened for c an ⁇ es any measurable cell property.
  • the tranr or? ad transfected ' cells containing the altered nuc- court ac sequences can be screened or selected for & desir polynucleotide sequence independent of polypeptide expressio
  • Enzymatic Inverse PCR amplifies the enti plasmid, a portion of the plasmid or linear sequence of polynucleotide.
  • EIPCR Enzymatic Inverse PCR
  • EIPCR library mutagenesis i that any plasmid or DNA fragment can be used to create library of mutations.
  • the only limitation is the efficienc of the PCR process.
  • the generation of a complementary stran is limited by the length of the template and by the elongatio rate of the polymerase. It is likely that advances in the PC technology, in particular, enzyme efficiency, will permit lon DNA fragments to be used in this invention.
  • the librar mutagenesis methods disclosed herein are rapid and efficien and permit one of skill in the art to generate severa libraries in a day. For example, once primers are prepared libraries such as those prepared in Example III can b generated in 6 to 10 hours.
  • EIPCR library mutagenesis the entire plasmid i amplified using mutagenic primers.
  • the simple design of EIPC results in a high efficiency of ligation of mutant plasmids, thus generating a high level of diversity in the library. Th higher the level of genetic diversity in a recombinan library, the more likely the library will contain a mutant o interest readily identifiable by methods known to one of skil in the art .
  • Another important benefit of EIPCR over othe methods for library mutagenesis is that, as in EIPCR site directed mutagenesis, mutations can be made in any area of th sequence independent of available restriction sequences. Restriction endonuclease recognition sites are not incorporated into the final construct.
  • EIPCR for library mutagenesis
  • Example III The usefulness of EIPCR for library mutagenesis, is described in Example III and illustrated in Figure 5.
  • a method for performing library mutagenesis to generate a recombinant library by introducing changes within a predetermined region of linear or, preferably, circular double stranded DNA is contemplated herein.
  • the method comprises ( providing a first primer population and a second prim population, each having at least one variable base at kno complementary positions along the primers capable of directi a change in the nucleic acid sequence, the first and seco primer populations being substantially complementary to t double-stranded nucleic acid to allow hybridization there and having a class IIS restriction enzyme recognition sequen and cleavage sites, (b) hybridizing the first and seco primer populations to opposite strands of the double strand nucleic acid to form a first pair of primer-templates orient in opposite directions, (c) performing enzymatic PCR as here before described, (d) cutting the double stranded line molecules with a class IIS restriction enzyme to fo restricted linear polynucleotide sequences containing t change in said nucleic acid sequence, thereby removi restriction endonuclease recognition sites, (e) optional joining termini of the restricted linear molecules of step ( to produce a double-stranded circular polynucleotide sequenc and
  • primer population is used to describe the po of primers that have identical base compositions except certain predetermined locations along the sequence th contain a variable composition.
  • the primers for EIPCR libra mutagenesis are otherwise designed similar to those prime used for EIPCR site-directed mutagenesis.
  • Primer pairs f EIPCR mutagenesis are designed to hybridize to the top a bottom strands of a double stranded template and to extend opposite directions.
  • the primers are chosen to substantially complementary to that region of the nucleic ac template to be mutagenized. These primers may be overlappi on the template, contiguous, or non-overlapping.
  • T h primer pairs are substantially complementary to the templa to facilitate hybridization during the PCR proces
  • the primer contains at least a 15 base region the 3 ' end of the primer that is complementary to t template. Other regions of complementarity may b interspersed throughout the length of the primer.
  • the prime additionally contains a class IIS restriction endonucleas recognition sequence and a region containing noncomplementar bases that confers the desired variable mutation.
  • Th variable region can be of any length, the only restriction o length being the ability of the primers to hybridize to th template and direct synthesis of a substantially complementar strand of DNA. Further, the variable region or regions may b interspersed between complementary regions along the prime strand.
  • Filler base regions can additionally be added to th primer at the 5' end of the primer, before the class II recognition sequence, and between the class IIS recognitio sequence and the class IIS cleavage site. Any final prime length is contemplated within the scope of the invention. Primer length is limited only by the efficiency of th oligonucleotide synthesizer. Primers may be prepared b methods known to those of skill in the art. Those with skil in the art will be readily able to determine if a given prime adequately hybridizes to a given template and is thus suitabl for amplification using EIPCR.
  • primer variability desirable for library mutagenesis is determined during primer synthesis.
  • the addition of trinucleotide fragments during synthesis provides direct control over amino acid mixtures.
  • the nucleotide mixture is formulated to contain a predetermined percentage of each of the four bases. These percentages may vary from 0% to less than 100% for any one base and from 0 to 100% for each of the 64 amino acid encoding trimers.
  • the frequency of a given sequence is determined by the desired probability that a particular base or trimer will be present at a particular position along the primer.
  • the library is to contain variable mutations at position 6 of the primer oligonucleotide corresponding to a 75% average likelihood that position 6 is guanosine and a 25% average likelihood that position 6 will b adenosine
  • the elongating primer will be exposed to mixture of 3/4 guanosine and 1/4 adenosine at position 6
  • These mixtures can also be prepared in proportions such tha for a region of 10 bases it is likely that on average only on of the 10 bases in any primer is different from the templat sequence. This provides a primer pool that theoreticall represents every possible permutation in each nucleotid position over a 10 base pair sequence.
  • the primer pairs contain complementary region at the class IIS restriction endonucleas cleavage site.
  • this overlappin region preferably does not contain a mutation. This ensure that recircularization of the template can occur following PC amplification.
  • class II restriction endonuclease Bsal is used to generate a four bas overhang at each end of the nucleotide sequence.
  • Figure provides an exemplary list of other class IIS restrictio endonucleases, contemplated within the scope of thi invention.
  • Library mutagenesis can be used to alter any regio within a nucleic acid sequence. These mutagenesis procedure are particularly useful for generating a library of mutation within the mature region of a protein sequence, within leader sequence, or within sequences that do not encod protein. Sequences that do not encode protein may influenc or regulate protein expression. These include, but are no limited to non-coding regions on the DNA, for example, enhancer sequences, promoter regions, sites for DNA bindin proteins such as repressors, Z-DNA formation, matri associated regions, telomeres, origins of replication an recombination signals.
  • non-coding regions RNA additionally contemplated include, but are not limited snRNP's, spliceosomes, ribosome binding sites, regions secondary structure, terminators, stability sites and c sites. It is additionally contemplated within the scope this invention that EIPCR library mutagenesis can be used generate recombinant libraries containing altered sequenc corresponding to tRNA or rRNA. Mutations in regulatory regio of a nucleic acid sequence can effect the level of prote expression, while in-frame substitution mutations within t nucleic acid sequence encoding protein can effect prote function. It is therefore contemplated that the proceedur described herein will be useful for generating recombinan libraries having mutations in any of these aforementione regions of the nucleic acid.
  • EIPCR library mutagenesis can be used to alter th functional characteristics of a particular protein.
  • a protei sequence engineered into an expression construct can be use as a nucleic acid template for EIPCR library mutagenesis
  • this procedure can b used, for example, to mutagenize a binding region on polypeptide, thereby generating an expression library that ca be screened or selected for altered binding characteristics
  • EIPCR mutagenesis can also be employed to mutate a region o a polypeptide sequence that influences intra-molecula binding.
  • a polypeptide region that links tw protein domains involved in ligand binding can be mutated using the methods disclosed herein, to optimize th interactions between the protein domains.
  • Wobble base library mutagenesis incorporates mutations within th primer population in positions that correspond to the thir position of a nucleotide codon. Most mutations in the thir position of a codon do not alter the amino acid sequence o the resulting polypeptide.
  • Alterations in the nucleotide sequence that do not affe the protein sequence may alter the level of protein synthes or expression within a given host.
  • alteratio in the nucleic acid sequence of the leader portion of polypeptide can influence levels of protein synthesis from o protein to another or from one host to another.
  • An example two primers designed to confer alterations in the OmpA leade sequence that result in increased levels of antibody F fragment expression from E. coli is found in Figure 6.
  • Onc a leader sequence is optimized for the expression of on particular polypeptide, using EIPCR library mutagenesis within a given host, it is further contemplated that thi leader sequence can then be linked to other gene sequence encoding polypeptide to optimize expression of othe polypeptide.
  • regulatory regions can b optimized using EIPCR library mutagenesis and that thes optimized regions can be engineered into other expressio constructs for maximal expression of other polypeptides i vitro or in vivo.
  • the invention is preferably designed to incorporate on or more random changes within predetermined regions of circular template, such as a vector.
  • Vector choice i determined first by the choice of host cell used to create th desired library. It is well known to those of skill in th art that vectors are commercially available for protei expression in prokaryotic and eukaryotic systems. Expressio vectors are available for bacteria, yeast and mammalia systems.
  • viral vectors for both eukaryotic an prokaryotic cells are also contemplated within the scope o this invention.
  • Expression vectors are required when th translation products from the mutated nucleic acid sequence are to be assayed. An analysis of random mutations in nuclei acid may not require the use of an expression vector whe mutations can be screened using polynucleotide probes or t like. Those with skill in the art will be able to choose appropriate commercially available vector, create their o vector, or recreate the exemplary vector described in Examp V below.
  • EIPCR library mutagenesis could be performed one region of nucleic acid within a construct, and a seco (and/or subsequent) mutagenesis procedure be performed another region of a construct or on a separate nucleic ac construct. Following amplification, these sequences can th be combined to produce a construct with two or more regions o random mutagenesis.
  • a general description of the hybridization of aliquots o the first and second primer pools to the nucleic acid templat as well as a general description of EIPCR are disclosed in th detailed description of site-directedmutagenesis beginning o page 16.
  • inverse in enzymatic inverse polymeras chain reaction is used to describe the primer pair orientatio during the PCR process such that at the initiation o elongation the 3• end of the primers are directed away fro one another.
  • the mechanics of hybridization and nucleic aci sequence amplification in library mutagenesis are similar to if not identical to, those employed in EIPCR site-directe mutagenesis and will not be repeated here.
  • the ter “performing EIPCR” as a step in the production of a library o mutations following the hybridizing step of the primers to th template comprises 1) extending the first pair of primer templates to create double stranded molecules; 2) denaturin the primer templates; 3) hybridizing the first and .secon primers at least once to the double stranded molecules to for a second pair of primer-templates; 4) extending the secon pair of primer-templates following hybridization to produc double-stranded linear molecules terminating with class II restriction enzyme recognition sequences; and 5) repeatin steps 1-3 as needed.
  • the appropriate class IIS restricti enzyme is used to cleave the nucleic acid to create termi compatible for ligation. Ligation of the linear molecules i performed under conditions that favor recircularization of t plasmid. These conditions are well known to those with skil in the art and exemplary conditions are described in Exampl III.
  • the nucleic acid is next introduced into the desired hos cells.
  • the nucleic acid can be introduced into the host cell by any means known to those of skill in the art.
  • Thes methods include, but are not limited to methods to prepar competent bacterial cells including CaCl 2 treatment, an methods to tran ⁇ fect eukaryotic cells including CaP precipitation, liposome mediated transfection, vira infection, or electroporation.
  • the method for introducin nucleic acid into the host cell will, in part, be determine by the host cell type. Descriptions of each th transformation and transfection procedures are found i recombinant methodology handbooks including those of Sambroo et al. or Ausubel et al. (supra.
  • the cells are expanded an screened for the desired cell function.
  • screening assays that are available to the investigator Assay design should reflect the desired goal of mutagenesis
  • the assay disclosed in Example III below i designed to detect increased levels of expression of particular antibody fragment in E. coli.
  • Assays can also b designed to detect increases in the binding constants (K a ) o an antibody or receptor to its antigen or ligand.
  • Othe assays can be designed" to detect changes in the level o protein expression or changes in the functional activity of protein.
  • the increase ability of a protein to promote growth or stimulate particular cellular function can be measured by removing cel supernatants from mutated cells or their progeny, adding thi supernatant to susceptible cells, and assaying for growt promoting activity.
  • Those with skill in the art will be abl to select an appropriate screening or selection assay for particular library to identify a particular clone of interest
  • EIPCR library mutagenesis can b used to alter the expression of one polypeptide in relation t a second polypeptide.
  • rando mutagenesis is used to increase the level of Fv heavy chai expression, thereby equalizing levels of heavy and light chai Fv fragment expression.
  • a contemplated method within th scope of this invention is one that identifies an optimize nucleic acid sequence derived from EIPCR library mutagenesi to promote an increase in the level of protein expression a compared with wildtype sequence.
  • EXAMPLE III This example illustrates a preferred embodiment of EIPCR library mutagenesis, wobble base mutagenesis.
  • wobble base mutagenesis mutations are introduced into the nucleic acid sequence without altering the amino acid sequence of the target protein.
  • the leader or signal sequence of a protein is variably mutated in the third base position of at least one codon to generate - a recombinant library that can be screened for colonies with increased levels of eukaryotic protein expression as compared with non- mutated controls.
  • the expression level of foreign proteins in E. coli is determined by a large number of factors, and expression level optimization is normally a slow and tediou process.
  • Fv fragmen expression of an anti-metal-chelate antibody wa optimized in E. coli.
  • the Fv fragment was expressed in activ form in the periplasm of E. coli.
  • Both the heavy and ligh chains of the Fv fragment, each with its own leader peptide were placed under the control of a Lac promoter on a 1.8 k plasmid.
  • the CHA255 antibody binds a chelated radioactiv metal ( 111 Indium or 90 Y chelate complex) to provide a simpl screening assay to permit detection of functional antibod fragments.
  • other screening systems may b useful.
  • any expression vector that can be amplified together wit its insert is contemplated within the scope of this invention
  • pMCHAFvl the 1.8 kb expression vector used for EIPCR mutagenesis and F expression, is shown in Figure 5.
  • the nucleic acid sequenc encoding light chain of the Fv fragment is 5' to the nuclei acid sequence encoding the heavy chain of the Fv fragment.
  • Each chain has its own OmpA signal peptide, and both chain are driven by a single Lac promoter.
  • the OmpA signal sequenc and Lac promoter sequence are ; t vided in references fro Mowa et al. and Reznikoff et •; respectively, which ar hereby incorporated by reference (Mowa et al., J. Biol. Che 255:27-29, 1980, J. Mol. Biol. 143:317-328 (1980) an Reznikoff et al. (1980) "The Lac Promoter". The Operon. Mille et al. Eds. Cold Spring Harbor Press, NY.)
  • the antibody gene for CHA255 are the same as those used in Example I above. T codons of the light and heavy chain are those obtained fr the original mouse antibody sequence.
  • the Om leader sequence is the native sequence obtained from the Om protein nucleic acid sequence as described in Example pMCHAFvl was constructed from pMINI3 ( Figure 5) .
  • pMINI3 is 1.0 kb expression vector which contains a ' synthetic L promoter, supF (derived from tRNA-tyr, Huang et al. , supra as the selectable marker, and a rop " ColEl origin, obtain from pUC (Pharmacia, Piscataway, N.J.).
  • the supF vectors a designed to be used with commercially available chemically electro-competent E.coli MC1061/P3 cells (Invitrogen Inc., S Diego, CA) .
  • These cells contain amber mutations in both t ampicillin and tetracycline drug resistance genes, located a P3 incompatibility group plasmid.
  • the P3 plasmid c co-exist with ColEl incompatibility group plasmids such pUC.
  • the P3 plasmid is too large to interfere with p plasmid purification.
  • Transformants are selected on plat with 25 ug/ml ampicillin and 7.5 ug/ml tetracycline.
  • the two oligonucleotides used to construct the librar are shown schematically in Figure 6B.
  • the oligonucleotide are designed to hybridize to opposite DNA strands of th pMCHAFvl template adjacent to the OmpA leader sequence.
  • Th resulting DNA and mRNA derived from this pool of mutate oligonucleotides is a library of sequences, all encoding th same OmpA protein sequence.
  • the X in Figure 6B corresponds t the variable positions within the primer population.
  • Th sequences are provided as SEQ ID NO: 26 and SEQ ID NO: 27
  • the. N corresponds " to the X in Figure 6B.
  • Prime oligonucleotides also contain R and Y base designations.
  • Th R indicates the incorporation of a purine and the Y indicate the incorporation of a pyrimidine.
  • the limitation of purine or pyrimidines in the third position of the codon ensures tha the amino acid sequence is not modified by the incorporatio of random nucleotides.
  • Constant regions within the primer ar coded by the appropriate base designation.
  • the prime (moving 5' to 3)' contain, as indicated, filler sequence, Bsal class IIS restriction endonuclease recognition site filler sequence, a Bsal cleavage site that forms the cohesi termini for circularization, a region comprising random bas positions in the third position of the nucleotide codon, an a complementary region to anchor the primer to the templat during hybridization.
  • Oligonucleotide synthesis was performe on a Milligen/Biosearch 8700 DNA synthesizer (Milligen Burlington, MA) .
  • the mixed base positions were synthesize using a fresh 1:1:1:1 molar mixture of each of the four base in the U reservoir.
  • the oligonucleotides were made trityl-o and were purified with Nensorb Prep nucleic acid purificatio columns (NEN-Dupont, Boston, MA) as described by th manufacturer.
  • PCR was performed in a 100 ⁇ l volume. Each reactio contained 0.5 ⁇ M of each purified primer, 0.5 ng pMCHAFv template plasmid DNA, lx Taq buffer, 200 ⁇ M of each dNTP an 1 ⁇ l Taq polymerase (Perkin-Elmer-Cetus)
  • the thermo-cyclin parameters were: 94 c C/3 min for 1 cycle; 94 C C/1 min, 50°C/ in, 72°C/2 min for 3 cycles; 94°C/1 min, 55°C/1 min, 72°C/ min, with autoextension at 5 sec/cycle for 10 cycles; 94°C/ min, 55°C/1 min, 72°C/3 min, 1 with autoextension at sec/cycle for 12 cycles; followed by one 10 min cycle at 72°C
  • the primers direct the amplification of linear DNA sequence of equal length to the template plasmi with an additional 11-14 bp extensions at each end of the DN that includes the class IIS restriction sequence.
  • the DNA obtained from 2-4 100 ⁇ l PCR reactions wa flushed by addition of dNTPs to 200 ⁇ M, 50 units DN Polymerase Klenow fragment and 30 units T4 DNA Kinase an incubated at 37 ⁇ C for 30 minutes. After phenol/chlorofo extraction and precipitation, the DNA was digested with Bs
  • the digested DNA was g purified, ethanol precipitated, and ligated at l concentration and without polyethylene glycol to favo intramolecular interactions, thus favoring circularization o the nucleic acid as opposed to concatamer formation.
  • Th ligation was ethanol precipitated using ammonium acetate washed twice with 80% ethanol, vacuum dried and resuspended i 20 ul 0.1 x TE (Sambrook et al. , supra.) for electroporation After digesting the 12-14 bp overhang with Bsal, the resultin cohesive termini were ligated intramolecularly, and th ligation was electroporated into E. coli for expressio analysis.
  • MC1061/P3 cells Invitrogen, Sa Diego, CA
  • Invitrogen electroporator Cells were plated on 23 x 23 cm plates as described above.
  • the cells were plate on 23x23 cm plates with CS agar (48 g/1 yeast extract, 24 g/1 tryptone, 3 g/1 NaH2P04, 3 g/1 Na2HP04, 15 g/1 agar) with 0.5 ug/ml isopropylthiogalactoside (IPTG) (Boehringer Mannheim, Indianapolis, IN) for induction of protein expression.
  • CS agar 48 g/1 yeast extract, 24 g/1 tryptone, 3 g/1 NaH2P04, 3 g/1 Na2HP04, 15 g/1 agar
  • IPTG isopropylthiogalactoside
  • the Fv expressing constructs were grown at 30°C.
  • CS broth permits the use of higher levels of IPTG before over-expression of t foreign protein causes bacterial death.
  • CS bro most of the Fv protein can be found in the media rather th in the bacterial periplasm.
  • the actual size of the library w determined by plating. In a typical electroporation, 5 x colony forming units (cfu) were obtained from electroporati of 1 ⁇ l of ligation mixture into 20 ⁇ l of cells. The ligati contained 0.5 ⁇ g of DNA in 20 ⁇ l. The library size is th about 1 x 10 7 and the efficiency was 2 x 10 7 cfu/ug. For th particular example, the screening assay was found to be mo limiting than library size.
  • Colony lifts of 23cmx23cm plates with 0.3-1 x 1 colonies were prepared using BA83 nitrocellulose filte (Schleicher and Schuell, Keene, NH) .
  • the filters were block by incubation in 3% non-fat milk in 25 mM Tris-HCI pH7.5 f 10 minutes, washed with 25 mM Tris, followed by incubation 25 mM Tris containing 50 uCi of chelated 11 Indium or 90 Yttri per filter for 1 hour at room temperature.
  • the filters we then washed with 25 mM Tris for a total of 15 minutes, dri and exposed to Kodak X-omat AR autoradiography film f several hours.
  • the sequence of the 130 bp fragment, containing t mutation that conferred increased protein expression wa determined by double stranded dideoxy sequencing on a Dupon Genesis 2000 automated sequencer using the Dupont Genesis 200 sequencing kit.
  • the DNA sequence of the 130 bp fragmen differed from the wildtype sequence only at the targete wobble bases, confirming that the amino acid sequence was no
  • Fv expression level guantitation The expression level of Fv fragments was determined assaying cell free supernatants. Wildtype and purified muta colonies were grown under expression conditions in CS broth described above. Dilutions of antibody containing sampl were incubated with radiolabelled metal-chelate. Aft incubation for one hour, the free, unbound metal chelate w separated from the antibody-bound metal chelate centrifugation through a Millipore ultrafree filter (molecul wight cut-off of 10,000 MW, Millipore, Bedfo; , MA) Sampl of the filtrated and the pre-filtration mixture were count for radioactivity, yielding a "fraction bound". A standa curve of "fraction bound' ersus known amounts of antibody w constructed. The usine,c of Fv in an unknown sample w determined from the standard curve. The results of the ass indicated that the mutants reproducibly expressed 10 tim more active Fv fragment than the original construct.
  • the protein sequence of the antibody fragments in th example is not altered by wobble base mutagenesis. Therefo any difference in signal strength in the screening assay due to differences in expression levels. However, t expression level may be affected by the mutation in sever ways. The mRNA stability could be improved by the mutatio Similarly, initiation and translation from the ribosome may improved.
  • wobble base mutagenesis can potentially influen polypeptide expression in a number of ways depending on whe the mutagenic primers bind to nucleic acid and which rand mutations are conferred upon the sequence.
  • EIP is used to create a promoter library for gene expression in coli.
  • Fv fragment expression of the anti-met chelate antibody (CHA255) is optimized using a population primers with variable sequences in the promoter region (Figu).
  • the plasmid used is pCCHAVll, a 2.4 plasmid containing the Lac promoter followed by an OmpA lead sequence linked to the antibody light chain fragment sequenc and followed by an optimized OmpA sequence linked to th antibody heavy chain fragment. Both antibody chain sequence are driven by a single Lac promoter.
  • This optimized Omp sequence (SEQ ID NO: 26) is derived from Example III. Plasmi pCCHAVll is LacI negative, chlora phenicaol resistance gen positive with a Rop " CoIE 1 origin. In this example second copy of the Lac promoter region is placed in front o the antibody heavy chain fragment sequence.
  • the nucleic aci sequence is provided in Figure 7A (ID SEQ NO: 30) and th inserted promoter sequence is provided in Figure 7B and as I SEQ NO: 33.
  • the inserted region includes the Lac promote library region followed by the wildtype Lac operator followe by the ribosome binding site.
  • the sequence including th ribosome binding site is provided in ID SEQ NO: 34.
  • the primers used to create the recombinant promote library are provided as ID SEQ NO: 31 and ID SEQ NO: 32.
  • I SEQ NO: 31 directed mutations to the ribosome binding sit while ID SEQ NO: 32 directed changes to the Lac promote region-
  • ID SEQ NO: 32 directed changes to the Lac promote region-
  • the ribosome binding site; the -10 an the -35 regions of the Lac promoter are underlined and th sequence is provided as ID SEQ NO: 34 and ID SEQ NO: 3 respectively.
  • the bold underlining in Figure 7B correspond to the primer regions in ID SEQ NO: 31 and ID SEQ NO: 32 tha are underlined.
  • the underlined portions are those position along the primer that contain variability.
  • the first underlined position is a cytosine.
  • T expected bias of the primer population at this position i 75%:C, 8.3%:G, 8.3%:T, 8.3%:A.
  • Libraries were created usi primer populations based on ID SEQ NO: 31 and ID SEQ NO: 3 Other libraries were created using one biased prim population while the other member of the primer pair contain no variability.
  • a recombinant library w created using ID SEQ NO: 31 to prepare a variable first prim pool, while the second primer corresponded exactly with ID S NO: 32 and therefore contained no variability.
  • the libra generated from these primers contains mutated sequences at t ribosome binding site and a constant Lac promoter sequenc
  • the oligonucleotides comprise a Bsal restriction endonuclea recognition site, a region of variability reflected in t underlined portion of ID SEQ NO: 31 and ID SEQ NO: 32, and region complementary to the template.
  • Example III Colony Screening Assay and Identification of Positive Clon
  • the screening assay is described in Example III Colonies with increased levels of hapten binding a identified and colony purified. These colonies are expand and analyzed for the. presence of unintended mutations Optimized promoter sequences are identified by sequencing t expression plasmids from positive colonies.
  • EIPCR is employed to create a eukaryotic mutagenesis library Similar to EIPCR in E. coli.
  • any region of a eukaryotic vect can be modified.
  • Eukaryotic expression vectors may modified in regulatory regions or within translated regions a particular gene.
  • a retroviral expressi vector pLN is used to generate a library of mutations with the ribosome binding site of the Neomycin resistance gene.
  • T ribosome binding site also known as a Kozak sequence (Koza M. , Nuc. Acids. Res. 12(2) :857-72, 1984 which is here incorporated by reference) is a highly conserved region eukaryotic cells comprising the consensus sequen CCACCATG(G) .
  • the retroviral expression plasmid pLN was obtained fro A.D. Miller and is described in a publication by Miller et al
  • the vector contains two Moloney Murin
  • LTR Leukemia Virus
  • MeMuLV Leukemia Virus
  • LTR long terminal repeats
  • MeMuLV Leukemia Virus
  • Betwee the LTR regions is the Neomycin resistance gene (Neo r ) .
  • Th Neo r ribosome binding site is targeted for library mutagenesi to confer increased resistance to G418 in the eukaryotic cel line NIH 3T3 (ATCC) .
  • the plasmid has a final size of 6 kb.
  • Oligonucleotide synthesis Oligonucleotides are prepared that are similar in desig to those described for Example I above.
  • the primers ar designed to flank the Neo r ribosome binding site and ar substantially complementary to both strands of DNA.
  • a shor (4-10 bp.) variable region is designed to overlap the ribosom binding site.
  • the oligonucleotides contain a class II recognition site, the -variable region, and a twenty bas complementary region that anchors the oligonucleotides to th pLN plasmid.
  • Reaction tubes are prepared for PCR in a final 100 ul. reaction volume. Reaction conditions are optimized fro initial reaction conditions as outlined in Example II Following PCR, the DNA is purified, cleaved with the desir class IIS restriction endonuclease, recircularized a ligated.
  • Ligated product from the PCR reaction is electroporat into the helper virus packaging cell line PE501 obtained fr A.D. Miller and described by Miller et al. , supra. Mutated p is transiently packaged into retroviral particles using PE50 Cell supernatant containing viral particles is harvested fr the packaging cell line and titered on virus susceptible NI 3T3 cells (ATCC) .
  • PE501 obtained fr A.D. Miller and described by Miller et al. , supra.
  • Mutated p is transiently packaged into retroviral particles using PE50
  • Cell supernatant containing viral particles is harvested fr the packaging cell line and titered on virus susceptible NI 3T3 cells (ATCC) .
  • Colonies expressing mutations are selected with elevat levels of G418, preferably between 0.75 -2.5 mg/ml
  • Thes colonies are expanded, lysed, and if desired, the DNA i purified.
  • the optimized promoter region is retrieved from th selected cells by PCR.
  • This new Kozak sequence can then b reintroduced into pLN to verify that the new sequence confer elevated G418 resistance.
  • the region is sequenced to identif the selected nucleic acid sequence. The results from this wor permits the identification of sequences conferring increase G418 resistance and facilitates the identification of Koza sequence requirements and the isolation of improved sequence that can be transferred to other constructs to improve th expression of other protein sequences.
  • Neomycin resistance i just one of a variety of selection systems useful for EIPC library mutagenesis applications. For example, as a selectio procedure, transfected cells can be screened by a Fluorescen Activated Cell Sorter (FACS) and positive colonies expand from these cells for further analysis.
  • FACS Fluorescen Activated Cell Sorter
  • EIPCR library mutagenesis is a reliable a efficient method for obtaining optimized nucleic ac sequences.
  • EIPCR reactions have an efficiency of 95% better in reactions designed to measure the efficiency mutagenesis.
  • EIPCR library mutagenesis is general applicable for de novo design or redesign of protein nucleic acid sequences.
  • NAME ISRAELSEN, NED A.
  • MOLECULE TYPE cDNA

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Abstract

L'invention se rapporte à un procédé selon lequel on génère une banque recombinée en introduisant une ou plusieurs modifications dans une région prédéterminée d'un acide nucléique double-brin, et qui consiste à utiliser une première et une seconde populations d'amorces, chacune des populations présentant une composition de base variable au niveau de positions connues sur les amorces, ces amroces incorporant une séquence de reconnaissance d'enzyme de restriction de classe IIS, pouvant opérer la modification dans la séquence d'acide nucléique et étant sensiblement complémentaires à l'acide nucléique double-brin afin de permettre une hybridation avec celui-ci. Le procédé consiste en outre à hybrider les première et seconde populations d'amorces avec des brins opposés de l'acide nucléique double-brin pour former une première paire de modèles d'amorces orientées dans des directions opposées, à effectuer une amplification enzymatique inverse pour produire au moins une copie linéaire de l'acide nucléique double-brin comprenant la modification dirigée par les amorces, à couper cette copie par l'intermédiaire d'une enzyme de restriction de classe IIS pour former une molécule d'acide nucléique linéaire à restriction contenant la modification, à réunir les terminaisons de cette molécule pour produire un acide nucléique circulaire double-brin, et à introduire ce dernier dans les cellules hôtes compatibles. Un procédé permettant de produire une banque recombinée par l'intermédiaire de la mutagénèse à balancement est également décrit.
PCT/US1992/010647 1991-12-12 1992-12-10 Mutagenese produisant une banque par amplification enzymatique inverse WO1993012257A1 (fr)

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Cited By (13)

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WO1996038591A1 (fr) * 1995-06-02 1996-12-05 Incyte Pharmaceuticals, Inc. Procede ameliore pour obtenir des sequences d'adn complementaire longues
WO1997037014A1 (fr) * 1996-04-02 1997-10-09 Provost, Fellows And Scholars Of The College Of The Holy And Undivided Trinity Of Queen Elizabeth Near Dublin Suppression et remplacement genetiques
EP0871778A1 (fr) * 1995-12-08 1998-10-21 Stratagene Mutagenese amelioree, dirigee sur un site d'un adn circulaire
WO1999035260A1 (fr) * 1997-12-31 1999-07-15 Adprotech Limited Genes non identiques et leur application dans des adjuvants moleculaires ameliores
EP1044281A1 (fr) * 1998-01-09 2000-10-18 University Of Utah Research Foundation Procede d'amplification in vitro d'adn circulaire
WO2001032932A2 (fr) * 1999-11-02 2001-05-10 Curagen Corporation Procede d"identification d"une sequence d"acide nucleique
WO2001077324A1 (fr) * 2000-04-08 2001-10-18 Adprotech Limited Vecteurs d'immunisation par adn
WO2002046450A2 (fr) * 2000-12-04 2002-06-13 Genencor International, Inc. Procede servant a generer une banque d'oligonucleotides mutants au moyen d'une reaction d'amplification cyclique lineaire
US6713457B2 (en) 1995-09-21 2004-03-30 Gwenyth Jane Farrar Strategy for suppressing the expression of an endogeneous gene by using compounds that are able to bind to the non-coding regions of the gene to be suppressed
US6958213B2 (en) 2000-12-12 2005-10-25 Alligator Bioscience Ab Method for in vitro molecular evolution of protein function
US7662551B2 (en) 2000-12-22 2010-02-16 Alligator Bioscience Ab Synthesis of hybrid polynucleotide molecules using single-stranded polynucleotide molecules
US20120252701A1 (en) * 1994-02-17 2012-10-04 Codexis, Inc. Methods for generating polynucleotides having desired characteristics by iterative selection and recombination
US8551970B2 (en) 1996-04-02 2013-10-08 Optigen Patents Limited Genetic suppression and replacement

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EP0414134A1 (fr) * 1989-08-17 1991-02-27 Japan Tobacco Inc. Méthode de production d'une protéine antivirale par transformation de E.coli, gène codant pour cette protéine et vecteur de E.coli utilisé dans cette méthode

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US4959312A (en) * 1985-05-31 1990-09-25 The University Of Tennessee Research Corporation Full spectrum mutagenesis
EP0414134A1 (fr) * 1989-08-17 1991-02-27 Japan Tobacco Inc. Méthode de production d'une protéine antivirale par transformation de E.coli, gène codant pour cette protéine et vecteur de E.coli utilisé dans cette méthode

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GENE. vol. 84, 1989, AMSTERDAM NL pages 143 - 151 J.D. HERMES ET AL. *
NATURE. vol. 344, 19 April 1990, LONDON GB pages 793 - 794 JONES D.H. ET AL. *
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Cited By (25)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20120252701A1 (en) * 1994-02-17 2012-10-04 Codexis, Inc. Methods for generating polynucleotides having desired characteristics by iterative selection and recombination
WO1996038591A1 (fr) * 1995-06-02 1996-12-05 Incyte Pharmaceuticals, Inc. Procede ameliore pour obtenir des sequences d'adn complementaire longues
US6713457B2 (en) 1995-09-21 2004-03-30 Gwenyth Jane Farrar Strategy for suppressing the expression of an endogeneous gene by using compounds that are able to bind to the non-coding regions of the gene to be suppressed
US7132265B2 (en) 1995-12-08 2006-11-07 Stratagene California Circular site-directed mutagenesis
EP1760161A1 (fr) * 1995-12-08 2007-03-07 Children's Medical Center Corporation Méthode améliorée de mutagénèse dirigée sur ADN circulaire.
US7176004B2 (en) 1995-12-08 2007-02-13 Stratagene California Circular site-directed mutagenesis
US6713285B2 (en) 1995-12-08 2004-03-30 Stratagene Circular site-directed mutagenesis
EP0871778A4 (fr) * 1995-12-08 2002-10-02 Stratagene Inc Mutagenese amelioree, dirigee sur un site d'un adn circulaire
EP0871778A1 (fr) * 1995-12-08 1998-10-21 Stratagene Mutagenese amelioree, dirigee sur un site d'un adn circulaire
US8551970B2 (en) 1996-04-02 2013-10-08 Optigen Patents Limited Genetic suppression and replacement
US7138378B1 (en) 1996-04-02 2006-11-21 Optigen Patents Limited Genetic suppression and replacement
WO1997037014A1 (fr) * 1996-04-02 1997-10-09 Provost, Fellows And Scholars Of The College Of The Holy And Undivided Trinity Of Queen Elizabeth Near Dublin Suppression et remplacement genetiques
WO1999035260A1 (fr) * 1997-12-31 1999-07-15 Adprotech Limited Genes non identiques et leur application dans des adjuvants moleculaires ameliores
US6770631B1 (en) 1997-12-31 2004-08-03 Adprotech Limited Non-identical genes and their application in improved molecular adjuvants
US6620597B1 (en) 1998-01-09 2003-09-16 University Of Utah Research Foundation Method for in vitro amplification of circular DNA
EP1044281A4 (fr) * 1998-01-09 2002-07-10 Univ Utah Res Found Procede d'amplification in vitro d'adn circulaire
EP1044281A1 (fr) * 1998-01-09 2000-10-18 University Of Utah Research Foundation Procede d'amplification in vitro d'adn circulaire
WO2001032932A3 (fr) * 1999-11-02 2002-06-13 Curagen Corp Procede d"identification d"une sequence d"acide nucleique
WO2001032932A2 (fr) * 1999-11-02 2001-05-10 Curagen Corporation Procede d"identification d"une sequence d"acide nucleique
WO2001077324A1 (fr) * 2000-04-08 2001-10-18 Adprotech Limited Vecteurs d'immunisation par adn
WO2002046450A3 (fr) * 2000-12-04 2002-09-06 Genencor Int Procede servant a generer une banque d'oligonucleotides mutants au moyen d'une reaction d'amplification cyclique lineaire
WO2002046450A2 (fr) * 2000-12-04 2002-06-13 Genencor International, Inc. Procede servant a generer une banque d'oligonucleotides mutants au moyen d'une reaction d'amplification cyclique lineaire
US6958213B2 (en) 2000-12-12 2005-10-25 Alligator Bioscience Ab Method for in vitro molecular evolution of protein function
US7282334B2 (en) 2000-12-12 2007-10-16 Alligator Bioscience, Ab Method for in vitro molecular evolution of protein function
US7662551B2 (en) 2000-12-22 2010-02-16 Alligator Bioscience Ab Synthesis of hybrid polynucleotide molecules using single-stranded polynucleotide molecules

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