Proc . Natl. Acad . Sci . USA Vol . 91 , pp. 10747-10751 , October 1994 Genetics DNA shuffling by random fragmentation and reassembly : In vitro recombination for molecular evolution WILLEM P. C. STEMMER Affymax Research Institute , 4001 Miranda Avenue , Palo Alto , CA 94304 Communicated by Peter Schultz , June 21 , 1994 -LRB- received for review December 20 , 1993 -RRB- ABSTRACT Computer simuations of the evolution of linear sequences have demonstted the importance of recombinatlon of blocks of sequence rather than point mutagenes alone . Repeated cycles of point mutagenesis , recombination , molecular evolution of and slosnhould allow in complex quences , suh as proteins . A method for the reassembly of genes from their random DNA gments , rting in in vitro recombini s reported . A 1-kb gene , after DNase I digesto and purification of 10 - to 50-bp random a nts , oal size wasr to dt and function . Similarly , a 2.7-kb plasmid could be fitly reambled . Complete recombination was obtand between two markers separated by 75 bp ; each marker was located on a separate gene . Oligonucleotides wIth 3 ' and 5 ' ends that are homologous to the gene cam be added to the frag mentiure N incorporated into the reassembled ne . Thus , mix of sthetic oligonuclotides and PCR a can be mixed into a gene at defined positions based on homolog . As an example , a library of cmera of the hunan and murine genes for interleukin 10 has been prepad . Shu g can also bi used for the in vitro genetic mapulaions , such a a equivalent of some s bakros with parental DNA . The advantages of recombination over exiting mut methods are likely to increase with the numbers of cycles of molecular evolution . The most widely used methods for protein mutagenesis are oligonucleotide-directed mutagenesis -LRB- 1-5 -RRB- and error-prone PCR -LRB- 6 , 7 -RRB- . Although recombination , with a low level of point mutation , was long ago demonstrated to be the preferred method for multiple cycles of mutagenesis -LRB- 8-10 -RRB- , so far no methods have been developed for general , homologous recombination of DNA in vitro . A technically simple approach to recombination is reported , and its application-to the design of linear sequences such as DNA , RNA , and proteins is explored -LRB- Fig. 1 -RRB- . The method involves digesting a large gene with DNase I to a pool of random DNA fragments -LRB- Fig. 2 -RRB- . These fragments can be reassembled into a full-length gene by repeated cycles of annealing in the presence of DNA polymerase . The fragments prime each other based on homology , and recombination occurs when fragments from one copy of a gene prime on another ` copy , causing a template switch . The first experiment was an attempt to reassemble a 1-kb gene from short random fragments and then to determine the rate of mutations that may have occurred -LRB- Fig. 2 -RRB- . Next I tested whether recombination could be obtained between two genes , each carrying a stop codon marker at positions that differ by 75 bp -LRB- Fig. 3 -RRB- . Oligonucleotides were added to the pool of random fragments to determine whether they became incorporated into the gene -LRB- Fig. 4 -RRB- . Finally , I attempted to create a library of chimeras from a pair of homologous interleukin 1P -LRB- IL-1 -RRB- genes from different species -LRB- Fig. 5 -RRB- . The publication costs of this article were defrayed in part by page charge payment . This article must therefore be hereby marked `` advertisement '' in accordance with 18 U.S.C. 1734 solely to indicate this fact . My interest is in applying repeated cycles of DNA shuffling and selection to the molecular evolution -LRB- 11-13 -RRB- of genes , operons , and biosynthetic pathways . I recently demonstrated the utility of this approach with a frlactamase system -LRB- 14 -RRB- . While many different library formats for molecular evolution have been reported for polynucleotides -LRB- 15-17 -RRB- and peptides and proteins -LSB- phage -LRB- 18-20 -RRB- , lacZ -LRB- 21 -RRB- , and polysomes -LRB- 22 -RRB- -RSB- , in none of these formats has recombination by random crossovers been used to deliberately create a combinatorial library . MATERIALS AND METHODS Substrate Preparation . The substrates for the shuffling reaction were 1.0-kb double-stranded -LRB- ds -RRB- DNA PCR products derived from pUC18 with the primer sequences AAAGCGTCGATTTTTGTGAT and ATGGGGTTCCGCGCACATTT -LRB- Fig. 2 -RRB- . The removal offree primers from the PCR product by Wizard PCR -LRB- Promega -RRB- was found to be very important . DNase I Digestion . About 2-4 pg of the DNA substrate -LRB- s -RRB- was digested with 0.0015 unit of DNase I -LRB- Sigma -RRB- per p1 in 100 p1of 50 mM Tris ` HCI , pH 7.4 / 1 mM MgC12 for 10-20 min at room temperature . Fragments of 10-50 bp were purified from 2 % low melting point ` agarose gels by electrophoresis onto DE81 ion-exchange paper -LRB- Whatman -RRB- , elution with 1 M NaCl , and ethanol precipitation . PCR Without Primers . The purified fragments were resuspended in PCR mixture -LRB- 0.2 mM each dNTP/2 .2 mM MgCl2/50 mM KCI/10 mM Tris-HCl , pH 9.0 / 0.1 % Triton X-100 -RRB- at a concentration of 10-30 ng/pl . No primers we 're added at this point . Taq DNA polymerase ' -LRB- Promega -RRB- was used at 2.5 units per 100 .1 A of reaction mixture . A PCR program of 940 for 60 s ; 940C for 30 s , 50-55TC for 30 s , and 72TC for 30 s -LRB- 30-45 times -RRB- ; and 72 C for 5 min was used in an MJ Research -LRB- Cambridge , MA -RRB- PTC-150 thermocycler . The PCR reassembly of small fragments into larger sequences was analyzed by taking samples after 25 , 30 , 35 , 40 , and 45 cycles of reassembly -LRB- Fig. 2 -RRB- . Whereas the reassembly of100to 200-bp fragments can yield a single PCR product ` of the correct ' size , 10 - to 50-base fragments typically yield some product of the correct size as well as products of heterogeneous molecular weights . Most of this size heterogeneity appears to be due to single-stranded sequences at the ends of the products , since after restriction enzyme digestion a single band of the correct size is obtained . PCR with P . After 1:40 dilution of this primerless PCR product into PCR mixture with 0.8 pM each prnier and `` 15 additional cycles of PCR -LRB- 94TC for 30 s , 500 -LRB- for 30 s , and 720C for 30 s -RRB- , a single product of the correct size is typically obtained -LRB- Fig. 2 -RRB- . Cloning and Analysis . After digestion of the PCR product with terminal restriction enzymes -LRB- BamHI and EcoO109 -RRB- and gel purification , the reassembled fragments were ligated into Abbreviations : IL , interleukin ; ds , double stranded X-Gal , 5-bromo4-chloro-3-indolyl P-D-galactoside . 10747 10748 Genetics : Stemmer A fragment with DNAsel m Proc . Natl. Acad . Sci . USA 91 -LRB- 1994 -RRB- B - reassemble fragments _ C select best D recombinants X m X X N mm X m X X M - X X X - XX , - M m NX X X * X NlXI repeat for multiple cycles __ XX 1 . % m N FIG. 1 . -LRB- A -RRB- A pool of homologous genes with different point mutations is fragmented with DNase I. -LRB- B -RRB- For simplicity , all mutations shown are considered beneficial and additive . -LRB- C -RRB- Reassembly of the random fragments into full-length genes results in frequent template switching and recombination . A recombinant gene containing the four crossovers -LRB- thick lines -RRB- can be selected from the library of recombinants based on its improved function . -LRB- D -RRB- Selected pool of improved recombinants provides the starting point for another round of mutation and recombination . The recombination process alone causes a low level of point mutations but , if desired , additional mutations could be introduced by error-prone PCR or UV mutagenesis of the pool of genes . pUC18 digested with BamHI and EcoO109 . After transformation and plating on plates with ampicillin , 5-bromo-4chloro-3-indolyl -LRB- 3-D-galactoside -LRB- X-Gal -RRB- , and isopropyl -LRB- 3Dthiogalactopyranoside , the resulting colonies were analyzed for the presence of the HindIII/Nhe I fragment , which is diagnostic for the + / + recombinant . Whole Plasmid Reassembly . A 1:1 mixture of two 2.7-kb whole plasmids , pUC18 + / - and pUC18 - / + , containing stop codons at different locations of lacZa -LRB- Fig. 3 -RRB- was digested with DNase I and 100 - to 200bp fragments were purified as described above . The reassembly program was 60 cycles of 940C for 30 s , 550C for 30 s , and 720C for 30 s plus 3 s per cycle . Gel analysis showed that most of the PCR product was >> 20 kb . After digestion with a unique enzyme -LRB- EcoO109 -RRB- , most of the product consisted of a single band of the expected size , which was gel purified , ligated , transformed , and plated on LB/ampicillin/X-Gal plates as described above . IL-1 P Experiments . A murine IL-1p3 gene -LRB- BBG49 -RRB- and a human IL-1 -LRB- 3 gene with Escherichia coli codon usage -LRB- BBG2 ; R & D Systems -RRB- were PCR amplified from plasmid templates using the primers TTAGGCACCCCAGGCTTT and ATGTGCTGCAAGGCGATT , resulting in two 0.8-kb PCR fragments . The areas of sequence identity between the human and the murine IL-1 -LRB- 3 sequences are on average only 4.1 bases long -LRB- Fig. 5 -RRB- . To force recombination based on such short sequence identity , a very low effective annealing temperature was used . After the denaturation step , the tube was rapidly cooled in dry ice/ethanol and reheated to the annealing temperature . Then 1 unit of the Klenow fragment of DNA Ff5 , l -RRB- -RRB- t ~ P -LRB- ER polymerase I was added . Because the enzyme is heat-labile , it needs to be added at every cycle . Preparation of dsDNA PCR products , DNase I digestion , and purification of fragments was as described for lacZ . Only the first 15 PCR cycles of the reassembly were performed with the heat-labile polymerase -LSB- manual PCR program : 940C for 1 min , 10 s on dry ice/ethanol -LRB- until frozen -RRB- , incubation for -20 s at 250C , addition of 1 unit of Klenow fragment , reaction for 2 min at 250C -LRB- 15 times -RRB- -RSB- . After these 15 manual cycles , Taq polymerase was added and an additional 22 cycles of PCR without primers were performed -LRB- 940C for 30 s , 350C for 30 s , no 720C -RRB- . The reaction mixture was then diluted 1:20 , primers were added at 0.8 ILM -LRB- AACGCCGCATGCAAGCTTGGATCCTTATT and AAAGCCCTCTAGATGATTACGAATTCATAT -RRB- , and plus primer PCR was performed as described for lacZ . The second primer pair differed from the first pair only because a change in restriction sites was preferred . After digestion of the PCR product with Xba I and Sph I , the fragments were ligated into Xba I/Sph I-digested pUC18 . The sequences of the inserts from several colonies were determined by dideoxynucleotide DNA sequencing -LRB- United States Biochemical -RRB- . RESULTS AND DISCUSSION Reassembly of the kcZa Gene . Fig. 2 shows a DNase I digest of a 1.0-kb sequence carrying the gene for the lacZae fragment . Gel-purified 10 - to 50-bp fragments were reassembled -LRB- initially without primers -RRB- to a single PCR product of the correct size . Of the resulting colonies , 84 % -LRB- n = 377 -RRB- are TI R ' R EN ; ni -LRB- Jei PIrlt \ 1JIf -RRB- - f -RRB- rd ! ' I -RRB- \ ~ ~ c ; ltvlT ~ ; ' 1W , ' , , Ill.ll 1 '' ' ' I o O iLaclphau - up ! : ^ t * : FI I bp k r t : ' 8s i kh h I1 -LRB- 1 -RRB- 1 -RRB- h Jp FIG. 2 . Reassembly of a 1.0-kb gene from 10 - to 50-bp random fragments . -LRB- a -RRB- A 1-kb DNA fragment encoding lacZa was amplified by PCR . After digestion of the gene with 0.15 unit of DNase I for 15 min at 200C -LRB- b -RRB- , fragments of 10-50 bases were purified from an agarose gel -LRB- c -RRB- . -LRB- d -RRB- Purified fragments were reassembled into a full-length gene at a high fragment concentration -LRB- 30 ng/pJ -RRB- in the absence of primers . The average size of the PCR products increases gradually via the priming of one product on another . The heterogeneous appearance of the product is largely due to the partially single-stranded nature of the product . -LRB- e -RRB- After addition of primers and additional cycles of PCR , a single PCR product of the correct size is typically obtained . Cloning of this product into a plasmid yielded -84 % light - to dark-blue colonies , reflecting mutations that occurred during the reassembly process . Genetics : Stemmer Template pUC18 - + pUC18 + - Proc . Natl. Acad . Sci . USA 91 -LRB- 1994 -RRB- Phenotype BamHI Polylinker 10749 LacZ alpha gene XhoI NcoI NheI Ecrl EcoOl09 EcoOO09 White BamHI Polylinker HindM Scal 0 White Lo -- 75bp b -RRB- Hpal EcrV Lo Hindm-NheI diagnostic fragment J Fspl _ _ Sall XhoI NcoI PstI ATCCGTCGT.ACTG BstEII ACCCTG NheI EcoRI - BssHII Pvul . TCCGACC + ms ___ TTACCC ^ ^ CTTAkTCG : CCTTGCTGCGCATCCACCT T TCGCGktfa - - GCG I I - IGTCG&CCIXKGGCATGCARWTTAOC kCTTOCTGTA + ACTGCAGCGTCGTGACTGGGAAA ; LCCCTGG = TTACCCk & CTTAATCGCCTTGCTr.CGCATCCACCTTTCGCTA #A4ZAATTAA -LRB- * AAbATAT -LRB- * CGCG SailI PstI SphI HinDII Scal PstI BstEtI EcrV BssHII PvuI FspI HpaI FIG. 3 . Efficiency of recombination between two inactive lacZa genes , each carrying a single marker at a position that differs by 75 bases . Each marker is a 20 - to 30-bp nonhomologous sequence with four stop codons , of which two are in the lacZ frame -LRB- small boxes -RRB- , with one in each of the other two frames -LRB- underlined -RRB- . The two 1-kb templates were mixed at a 1:1 ratio and shuffled . After cloning , 24 % -LRB- n = 386 -RRB- of the colonies had an active lacZ gene , resulting in blue colonies on X-Gal plates , which is near the theoretically expected value for complete recombination -LRB- 25 % -RRB- . All 10 of the blue colonies contained the expected HmdIII/Nhe I restriction fragment -LRB- data not shown -RRB- . LacZ + , versus 94 % without shuffling . When a high concentration of fragments -LRB- 10-30 ng/0l -RRB- was used , the reassembly reaction was surprisingly reliable . By sequencing of random clones , it has been shown that the reassembly process introduces point mutations at a rate of 0.7 % -LRB- n = 4437 bases ; Table 1 -RRB- , which is similar to error-prone PCR -LRB- 6 , 7 -RRB- ; 11/12 types of substitutions were found with no frameshifts -LRB- Table 1 -RRB- . Since the only difference compared to normal PCR was the use of small fragments , the rate of point mutagenesis may depend on the size of the fragments that are used in the reassembly . In contrast to PCR , DNA reassembly is an inverse chain reaction . In PCR the number of polymerase start sites and the number of molecules grows exponentially , whereas in DNA reassembly the number of start sites and the number -LRB- but not the size -RRB- of the molecules decreases over time . This reaction may perhaps be used for reassembly of genes from the highly fragmented DNA of fossils -LRB- 23 -RRB- . LacZ alpha gene BanAH NheI Ecl EcoOO09 L - Z Spike : 18 F 4 - l 18 I HitindU HindM-NheI diagnostic fragment Control -LRB- no spiked oligo -RRB- Top strand spike Bottom strand spike Top and bottom strand spike % blue colonies 0.0 % -LRB- N = > 1000 -RRB- 8.0 % -LRB- N = 855 -RRB- 9.3 % -LRB- N = 620 -RRB- 2.1 % -LRB- N = 537 -RRB- FIG. 4 . Mixtures of synthetic oligonucleotides can be added to the random fragments derived from a gene . In the reassembly reaction , these oligonucleotides can be incorporated instead of the original sequence . A 1-kb PCR fragment containing lacZ - from the pUC18 - / + plasmid -LRB- see Fig. 3 -RRB- was digested with DNase I and small fragments were purified . A 66-mer oligonucleotide , containing 18 bases of homology to lacZ at both the 3 ' and 5 ' ends , was mixed in at a 4-fold molar excess . This oligonucleotide was designed to replace the 25-base heterologous lacZ-inactivating marker containing four stop codon mutations with wild-type lacZ sequence . The degree of incorporation of such oligonucleotides can be varied over a wide range by adjusting the molar excess , the annealing temperature , or the melting temperature of the homologous flanking sequence . Incorporation was less when both the top and bottom strand oligonucleotides were added , presumably because of competitive hybridization with the complementary oligonucleotide . Crossover Between Two Markers in the lacZ Gene . Crossover between the two inactive lacZa genes from plasmids pUC18 - / + and pUC18 + / - , each of which contains a single stop codon marker at positions that differ by 75 bases , was measured after shuffling -LRB- Fig. 3 -RRB- . The ratio of active , recombinant colonies was 24 % -LRB- n = 386 -RRB- , close to the theoretically expected value of 25 % for complete recombination . All 10 of the blue colonies assayed contained the diagnostic HindIII / Nhe I restriction fragment . The whole 2.7-kb plasmids containing these same LacZmarkers were also efficiently reassembled from random 100to 200-bp fragments . For reassembly of fragments derived from whole plasmids the theoretical end point is a single , large concatemeric molecule . As expected , concatemers of > 20 kb were obtained , which , after digestion with a unique restriction enzyme , yielded the expected 2.7 kb ; 11 % -LRB- n = 328 -RRB- of the resulting plasmids had recombined in the 75-bp area between the markers , resulting in a LacZ + phenotype . Recombination of a Pool of Point Mutants . When 14 different point-mutated LacZ - colonies , obtained from the experiment described in Fig. 2 , were recombined as a pool , 34 % -LRB- n = 291 -RRB- of the resulting colonies were LacZ + . These colonies presumably arose by recombination of the DNA from different colonies . Assuming that the point mutations are spread out throughout the gene and that each of the 14 mutant plasmids contains only one knockout mutation , then for each marker there is a 13/14 chance of getting the wild-type version and the probability of getting all 14 wild-type versions in a single clone is -LRB- 13/14 -RRB- 14 = 35 % , close to the observed value of 34 % . The close match between the predicted and the observed values of both LacZ shuffling experiments suggests that the fragments reassort freely . The 34 % value contrasts with the expected rate of reversal of a single point mutation by error-prone PCR , which should be close to the published mutagenesis rate of 0.7 % -LRB- 7 -RRB- . However , a control experiment using error-prone PCR instead of shuffling was not feasible , because the 1-kb fragment proved too large to be generated by error-prone PCR . Incorporation of Synthetic Oligonucleotides . Synthetic oligonucleotides can be added to the reassembly mixture and incorporated into the gene based solely on 15-20 bases of homology at the 5 ' and 3 ' flanking sequences -LRB- Fig. 4 -RRB- . Single-stranded DNA was incorporated more efficiently than dsDNA -LRB- 8-9.3 % versus 2.1 % -RRB- , presumably because of competitive hybridization . Mixtures of synthetic oligonucleotides , PCR fragments , or even whole genes can be mixed into another sequence library , and the insertion of one sequence cassette is independent from the insertion of a cassette in another part of the template -LRB- unpublished data -RRB- . Thus , the 10750 Genetics : Stemmer Proc . Natl. Acad . Sci . USA 91 -LRB- 1994 -RRB- G M AT G H ATGGC M H AGG AGG GCCA A TOC TCTGCAAGT ~ frCG A GMpT ; GT GSIMMG CA CGTAC AGCTCTGCA TJGCCA 2CA T A. a AAC AAC ATGAGC CGTh AAGGTG TC TC G ATGAG TCGT AAGGT CGAC ACAIaTCC TCC TmCft TG TC CCT B TGAAA AAAGAC2 CTGffDTARG C T TGAAA TG CC TAGAAAGAC M GACrq H GACU a GTACC Ct C ~ AGACCCspAA ~ 2 G G CAA fG GG A CCG TGGA CAA GG TCGAGTCT GGTTC TCGAGTCTJ aGTTC M CCMA GG CAT GTCrC.GTCCTAA CGTMTAA C GCTAA CGGTTSAEGGTCAGGATATCAPACTTCA ToFEGT GCTAACCTGTVYTCCO H CAGGGTAT - I '' ~ I - - i FIG. 5 . Shuffling of a murine gene -LRB- M -RRB- with a human gene -LRB- H ; with E. coli codon usage -RRB- for IL-1 , B represents an extreme example of shuffling , since the average length of sequence identity is only 4 bases . Crossovers could be obtained only by ensuring a low annealing temperature , which required the use of a manual PCR with a heat-labile , low-temperature DNA polymerase , such as the Klenow fragment of DNA polymerase I. Shown are the 17 crossovers that were obtained from DNA sequence analysis of nine colonies . Boxes show mismatched bases ; thick bars represent crossovers . Surprisingly little sequence identity was sufficient to yield crossovers , presumably because of the tolerance for mismatched bases at the low annealing temperature . degree of recombination , the homology required , and the composition of the mixture can be independently and simultaneously varied along the length of the reassembled gene . Creation of a Library of Chimeras of Two Related Genes . We attempted to create a library of chimeras between a human -LRB- with E. coli codon usage -RRB- and a murine IL-1p gene . Since the areas of sequence identity are on average only 4.1 bases long -LRB- Fig. 5 -RRB- , this represents an extreme case , exploring the minimum requirements for crossover . Crossovers could not be obtained by several approaches using Taq polymerase , even when a 10-fold excess of one of the IL-1 , B genes was used -LRB- data not shown -RRB- . The explanation is that with any heat-stable polymerase , the cooling time of the PCR machine -LRB- 94 C to 25 C at 1-2 C/s -RRB- causes the effective annealing temperature to be higher than the set annealing temperature . To force efficient crossovers based on such a low degree of homology , a very low effective annealing temperature is needed . By using the Klenow fiagment of DNA polymerase I , a heat-labile polymerase , a low annealing temperature was obtained . Under these conditions , a total of 17 crossovers by DNA sequencing of nine colonies was obtained -LRB- Fig. 5 -RRB- . One of the crossovers was based on only 1 or 2 bases of uninterrupted identity , suggesting that some mismatches are tolerated if sufficient flanking homology is present . Technial Issues . The removal of free primers from the PCR product before fragmentation proved to be very important . Without adequate primer removal , contamination with fulllength template can lead to a low frequency ofrecombination . However , even restriction fiagments or whole plasmids can be efficiently reassembled from their fragments , avoiding the potential contamination of PCR products with primers . The preferred fragment size depends on the intended number of crossovers , which differs for various applications . For the in vitro evolution of a gene , for example , larger Table 1 . Mutations introduced by mutagenic shuffling Transition Transversion Frequency Frequency A-T 1 2 A - C C A 1 0 C G 3 G C G T 2 T A 1 T G 2 A total of 4437 bases of shuffled lacZ DNA were sequenced . The mutation rate was 0.7 % ; 11/12 types of base substitutions were found , but there were no frameshifts . G A C T A G T C 6 4 7 3 fragments , which result in one or two crossovers per gene , may be preferred -LRB- 14 -RRB- . The stringency of annealing during the reassembly PCR depends on the minimum degree of homology that should still yield crossovers . If the degree of homology is high , Taq polymerase can be used with an annealing temperature of between 45 C and 65 C. Crossovers occur when a partially reassembled PCR product primes on the homologous position of a related but not identical template -LRB- template switch -RRB- . Crossovers based on identity of < 15 bases appear more difficult to obtain . Control over the stringency of recombination is expected to be much greater with in vitro than with in vivo recombination . Comparison to Other Mutagenesis Techniques . The two most widely used methods for protein mutagenesis are errorprone PCR and oligonucleotide-directed mutagenesis . Error-prone PCR uses low-fidelity polymerization conditions to introduce a low level of point mutations randomly over a long sequence -LRB- 6 , 7 , 24 , 25 -RRB- . Error-prone PCR can be used to mutagenize a mixture of fragments of unknown sequence . However , computer simulations -LRB- 8-10 -RRB- have suggested that point mutagenesis alone may often be too gradual to allow the block changes that are required for continued sequence evolution . The published error-prone PCR protocols -LRB- 6 , 7 -RRB- do not allow amplification of DNA fragments > 0.5-1.0 kb , limiting their practical application . In contrast , DNA shuffling can be applied to sequences > 1 kb , has a mutagenesis rate similar to error-prone PCR , and also works with pools of unknown sequence . Repeated cycles of any mutagenesis strategy lead to an accumulation of neutral mutations , which , for example , may make a protein immunogenic . Only with DNA shuffling is it possible to remove such neutral mutations by backcrossing with excess parental or wild-type DNA -LRB- 14 -RRB- . In oligonucleotide-directed mutagenesis -LRB- 1-5 -RRB- , a short sequence is replaced with a synthetically mutagenized oligonucleotide . This approach does not generate combinations of distant mutations and is combinatorial only within the limits of the oligonucleotide . The limited library size relative to the vast sequence space -LRB- 13 -RRB- means that many rounds of selection are unavoidable for protein optimization . Mutagenesis with synthetic oligonucleotides requires sequencing of individual clones after each selection round followed by grouping into families , arbitrarily choosing a single family , and reducing it to a consensus motif , which is resynthesized and reinserted into a single gene followed by additional selection -LRB- 3 -RRB- . This process constitutes a statistical bottleneck ; it is labor intensive and not practical for many rounds of mutagenesis . Genetics : Stemmer Error-prone PCR and oligonucleotide-directed mutagenesis are thus useful for single cycles of fine tuning but rapidly become limiting when applied for multiple cycles . One apparent exception is selection of an RNA ligase ribozyme from a random RNA library using many rounds of amplification by error-prone PCR and selection -LRB- 15 -RRB- . Based on the work of Holland -LRB- 9 -RRB- , it is surprising that such a complex structure could be evolved without recombination . However , the occurrence of recombination in these experiments has not been ruled out . Using the LacZ assay -LRB- Fig. 3 -RRB- , recombination has been found to occur even during normal PCR at a frequency of -0.03 % per 25 cycles . Others have reported a rate of 5.4 % recombinants under standard PCR conditions -LRB- 26 -RRB- . Considering that > 1000 cycles of PCR were required for selection of the RNA ligase , even such a low rate of recombination may have contributed significantly to evolution of the ribozyme . A method that was developed for assembly of synthetic oligonucleotides into a full-length gene -LRB- 27 -RRB- could be used for recombination but differs fundamentally from DNA shuffling in that it uses known sequences and fixed junction sites . The chain shuffling of the heavy - and light-chain genes of antibodies is also conceptually different since it uses a single , fixed crossover site -LRB- 28 -RRB- . Implications for Molecular Evolution . The goal of applied molecular evolution -LRB- 11-13 -RRB- is to mimic the natural design process and speed it up by directed selection in vitro toward a simple , specific goal . Computer simulations of evolution , called genetic algorithms -LRB- 8-10 -RRB- , have shown that recombination -LRB- with a low level of point mutagenesis -RRB- between individuals is sufficient for the evolution of complex linear sequences . Similarly , by mimicking the mechanisms by which bacteria have evolved a variety ofresistance genes , the combination of episome transfer , recombination , point mutation , and selection should allow one to efficiently redesign genes , operons , pathways , and perhaps whole bacterial genomes for specific applications -LRB- 14 -RRB- . Limitations in genetic algorithms become more pronounced when more cycles are performed . The advantages of recombination , as well as the differences between different recombination formats , may become more pronounced with increasing numbers of cycles . The ability to perform the molecular equivalent of some standard genetic matings by recombination in vitro , such as a molecular backcross , is likely to be useful . A molecular backcross is performed by repeated `` mating '' with the desired background while selecting for the mutations of interest -LRB- 14 -RRB- . As in traditional breeding , this approach can be used to combine phenotypes from different sources into the background of choice . Shuffling requires the presence of homologous regions separating regions of diversity . Scaffold-like protein structures may be particularly suitable for shuffling . The conserved scaffold determines the overall folding by selfassociation while displaying relatively unrestricted loops that mediate specific binding . Examples of such scaffolds are the immunoglobulin -LRB- - barrel , and the four-helix bundle -LRB- 29 -RRB- . It is of interest to note that the crossover , the essence of sexual recombination and a presumably complex behavior , Proc . Natl. Acad . Sci . USA 91 -LRB- 1994 -RRB- 10751 actually occurs in naked DNA . Since sexuality appears to be a behavior that is inherent in the building blocks -LRB- 13 -RRB- , recombination and sexuality are probably as old as DNA itself . I thank Dr. William Dower , Dr. Peter Schatz , and Dr. Larry Mattheakis for valuable discussions and for comments on the manuscript and Andreas Crameri for assistance with DNA sequencing . The communicating member is chairman of the Affymax Research Institute Scientific Advisory Board . 1 . Reidhaar-Olson , J. F. & Sauer , R. T. -LRB- 1988 -RRB- Science 241 , 53-57 . 2 . Stemmer , W. P. C. , Morris , S. K. & Wilson , B. S. -LRB- 1992 -RRB- BioTechniques 14 , 256-265 . 3 . Arkin , A. & Youvan , D. 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C. -LRB- 1994 -RRB- Nature -LRB- London -RRB- 370 , 389-391 . 15 . Bartel , D. P. & Szostak , J. W. -LRB- 1993 -RRB- Science 261 , 1411-1418 . 16 . Bock , L. C. , Griffin , L. C. , Latham , J. A. , Vermaas , E. H. & Toole , J. J. -LRB- 1992 -RRB- Nature -LRB- London -RRB- 355 , 564-566 . 17 . Tuerk , C. & Gold , L. -LRB- 1990 -RRB- Science 249 , 505-510 . 18 . Cwirla , S. E. , Peters , E. A. , Barrett , R. W. & Dower , W. J. -LRB- 1990 -RRB- Proc . Natd . Acad . Sci . USA 87 , 6378-6382 . 19 . Scott , J. K. & Smith , G. P. -LRB- 1990 -RRB- Science 249 , 386-390 . 20 . McCafferty , J. , Griffiths , A. D. , Winter , G. & Chiswell , D. J. -LRB- 1990 -RRB- Nature -LRB- London -RRB- 348 , 552-554 . 21 . Cull , M. G. , Miller , J. F. & Schatz , P. J. -LRB- 1992 -RRB- Proc . Natd . Acad . Sci . USA 89 , 1865-1869 . 22 . Mattheakis , L. , Bhatt , R. & Dower , W. D. -LRB- 1994 -RRB- Proc . Natd . Acad . Sci . USA 91 , 9022-9026 . 23 . Cano , R. J. , Poinar , H. N. , Pieniazek , N. J. , Acra , A. & Poinar , G. 0 . -LRB- 1993 -RRB- Nature -LRB- London -RRB- 363 , 536-538 . 24 . Gram , H. , Marconi , L. , Barbas , C. F. , Collett , T. A. , Lerner , R. & Kang , A. S. -LRB- 1992 -RRB- Proc . Natl. Acad . Sci . USA 89 , 3576-3580 . 25 . Hawkins , R. E. , Russell , S. J. & Winter , G. -LRB- 1992 -RRB- J. Mol . Biol . 226 , 889-896 . 26 . Meyerhans , A. , Vartanian , J. P. & Wain-Hobson , S. -LRB- 1990 -RRB- Nucleic Acids Res . 18 , 1687-1691 . 27 . DiDonato , A. , De Nigris , M. , Russo , N. , DiBiase , S. & D'Alessio , G. -LRB- 1993 -RRB- Anal . Biochem . 212 , 291-293 . 28 . Waterhouse , P. , Griffiths , A. D. , Johnson , K. S. & Winter , G. -LRB- 1993 -RRB- Nucleic Acids Res . 21 , 2265-2266 . 29 . Osterhout , J. J. , Handel , T. , Na , G. , Toumadje , A. , Long , R. , Connolly , P. J. , Hoch , J. C. , Johnson , W. C. , Live , D. & DeGrado , W. F. -LRB- 1992 -RRB- J. Am . Chem . Soc . 114 , 331-337 .

O O O O O O O O O O O O O O O O O O B O O O B O O O O O O O B B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O B B O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O B B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O B O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B B O O O O O B B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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assembly fragmentation evolution dna molecular