site-directed mutagenesis by overlap extension using...

Gene, 77 (1989) 51-59

Elsevier

51

GEN 02940

Site-directed mutagenesis by overlap extension using the polymerase chain reaction

(Genetic engineering; sequencing; recombinant DNA; Tag polymerase; oligodeoxyribonucleotide primers;

major histocompatibility complex mutants)

Steffan N. Hoa, Henry D. Hunt a, Robert M. Horton b, Jeffrey K. Pullen” and Larry R. Peasea

a Department of Immunology and b Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, MN 55905 (U.S.A.)

Received by M.R. Culbertson: 15 December 1988

Accepted: 19 December 1988

SUMMARY

Overlap extension represents a new approach to genetic engineering. Complementary oligodeoxyribo-

nucleotide (oligo) primers and the polymerase chain reaction are used to generate two DNA fragments having

overlapping ends. These fragments are combined in a subsequent ‘fusion’ reaction in which the overlapping

ends anneal, allowing the 3’ overlap of each strand to serve as a primer for the 3’ extension of the com-

plementary strand. The resulting fusion product is amplified further by PCR. Specific alterations in the

nucleotide (nt) sequence can be introduced by incorporating nucleotide changes into the overlapping oligo

primers. Using this technique of site-directed mutagenesis, three variants of a mouse major histocompatibility

complex class-I gene have been generated, cloned and analyzed. Screening of mutant clones revealed at least

a 98 y0 efficiency of mutagenesis. All clones sequenced contained the desired mutations, and a low frequency

of random substitution estimated to occur at approx. 1 in 4000 nt was detected. This method represents a

significant improvement over standard methods of site-directed mutagenesis because it is much faster, simpler

and approaches 100% efficiency in the generation of mutant product.

INTRODUCTION

The alteration of genes and/or the proteins they

encode through the substitution of specific nucleo-

tides within a gene sequence by site-directed muta-

genesis represents a fundamental tool of modern

recombinant DNA technology. It not only allows for

the analysis of the structural basis of gene and pro-

tein function, but also facilitates the generation of

novel gene products. Several techniques designed to

introduce specific mutations into cloned genes

through oligo-mediated mutagenesis have been de-

scribed (Smith, 1985; Botstein and Shortle, 1985;

Wu and Grossman, 1987). These techniques require

Correspondence to: Dr. L.R. Pease, Department of Immunology,

Mayo Clinic, Rochester, MN 55905 (U.S.A.) Tel. (507)284-8177;

Fax (507)284-1637.

Abbreviations: aa, amino acid(s); bp, base pair(s); dNTP, any

deoxynucleotide triphosphate; ds, double strand(ed); EtdBr,

ethidium bromide; kb, kilobase or 1000 bp; MHC, major

histocompatibility complex; nt, nucleotide(s); oligo, oligodeoxy-

ribonucleotide; PCR, polymerase chain reaction; SOE, splicing

by overlap extension; ss, single strand(ed); T,, melting tempera-

ture; wt, wild type.

0378-l 119/89/$03.50 0 1989 Elsevier Science Publishers B.V. (Biomedical Division)

52

multiple steps in the introduction of the target se-

quence into the appropriate bacteriophage or plas-

mid vector systems and the generation of hetero-

duplex substrates for mutagenesis. The mutant pro-

ducts often occur at low frequency and must be

isolated and reintroduced into the original vector for

expression.

An alternative method to create site-directed

mutations within the nucleotide sequence employs

the PCR (Saiki et al., 1985; Mullis and Faloona,

1987) which uses two synthetic oligos as primers to

amplify a nucleotide sequence of interest. These

primers anneal at either end of the targeted nucleo-

tide sequence and are oriented in opposite direc-

tions. Exponential amplification of the target se-

quence occurs over the course of multiple rounds of

denaturation, annealing and 3’ extension by DNA

polymerase. PCR can be used to introduce addi-

tional sequences, such as restriction sites, by incor-

porating these into the oligo primers (Mullis and

Faloona, 1987). However, this use of PCR as a

means of site-directed mutagenesis is limited

because all sequence alterations must be introduced

within the primer located at the ends of the targeted

sequence. Given that restriction sites must be located

at the ends of fragment to permit cloning, this

approach requires that the sites of mutagenesis be

located near these restriction sites. The introduction

of mutations at other sites within the amplified gene

sequence is therefore not possible.

Mutagenesis by overlap extension, as described

here, employs the PCR as a means of creating altered

genes from cloned DNA. This method makes possi-

ble the introduction of specific mutations into the

nucleotide sequence directly from a cloned gene in its

original vector with essentially 100% efficiency in a

few simple steps. The requirement for restriction

endonucleases or DNA ligase is eliminated by

generating two PCR fragments having overlapping

ends that can be effectively fused by recombining

them in a subsequent PCR reaction. This method

can be used for the purpose of site-directed muta-

genesis or alternatively, as described in the accom-

panying paper by Horton et al. (1989), for generating

hybrid gene constructs between two independent

genes, a method we have designated SOE. In this

report we demonstrate the use of the overlap exten-

sion technique to create site-directed mutations in a

murine MHC class-I gene.

MATERIALS AND METHODS

(a) Reagents

Polymerase chain reactions were performed using

the GeneAmp kit (Perkin Elmer Cetus). Restriction

enzymes Hind111 and XhoI were obtained from

Boehringer-M~nheim. Sequencing of ds plasmid or

ss Ml3 subclones was performed by the dideoxy

chain-termination method using Sequenase (U.S.

Biochemicals Corp.). Sequencing of ds DNA was

performed as previously described (Kraft et al.,

1988).

(b) Generation of PCR products

PCRs were carried out using Taq polymerase as

specified by the manufacturer (Perkin Elmer Cetus).

Briefly, ~pl~cation of DNA fragments from the

plasmid template was achieved by adding 0.1 to

100 ng of template DNA, 50 mM KCljlO mM

Tris * HCI pH 8.3/1.5 mM MgCl,/O.Ol% (w/v)

gelatin/200 PM each of dNTP/l PM of each primer

and 2.5 units of Taq polymerase in a final volume of

100 ~1. These samples were overlaid with 100 ~1 of

light mineral oil (Sigma Co.) and subjected to 30

cycles of denaturation (1 min, 94”C), annealing

(2 min, 50 o C), and extension (3 min, 72 o C) using a

DNA Thermal Cycler (Perkin Elmer Cetus). The

products of the reaction were analyzed on an agar-

ose gel containing 3% Nusieve agarose (FMC),

1% Seakem agarose (FMC) and 0.5 pg EtdBr/ml

in Tris 3 acetate buffer/40 mM Tris - acetate/l mM

EDTA pH 8.0).

Although products were obtained by PCR using

template concentrations ranging from 1 pg to 1 pg,

subsequent sequence analysis from cloned PCR pro-

ducts of MHC class-11 mutant genes suggests that

there may be a higher observed error frequency

among clones generated from reactions using low

initial template concentrations as compared with

those from reactions using higher template concen-

trations (S.N. H., and D.J. McKean, unpublished

observations). The reasons for this are discussed

further below. Thus, to minimize undesired nucleo-

tide changes initial template concentrations of 1 pg

are recommended. Because high concentrations of

initial template are used, gel purification of the

reaction products is routinely performed to ensure

53

no carryover of the wt template and to avoid amplification by-products, as described below.

(c) Mouse class-f MHC gene constructs

The DNA template used in the mutagenesis ex- periments consisted of a 4.3-kb DNA fragment containing the H-Z Kb target sequence (Schulze et al., 1983; Weiss et al., 1983) cloned into the plasmid pUC18. The unique Hind111 and X&I restriction sites were introduced earlier by a conventional gapped heteroduplex mut~enesis procednre (3.K. P., data not shown).

(d) Synthetic oligos used as PCR primrs

Synthetic oligos were s~thesized by the phos- phoramidite method using a model 380A Applied Biosystems synthesizer. Oligos were p&Bed by column chromatography using Sephadex G50 (Pharmacia) equilibrate with distilled water. The sequences of the mutagenic oligos are shown in Fig. 3B. The oligos were designed so that the length of the portion of the oligo that was to overlap with the other fragment in the fusion reaction had a calculated denaturation temperature that matched the a~~~~~g tem.perature used in the PCR reaction f5O’C). The ‘melting’ temperat~e (Tm) was calculated as follows:

T, (“C) = 4(G + C) i- 2(A + T)

as described by Suggs et al. (198 1).

(e) Joining DNA fragments by overlap extension

PCR-generated DNA fragments from an initial set of reactions were either used directly in a subsequent overlap extension reaction or fust purified. In the former case, 10 ,LLI (or dilutions thereof) from the two PCR reactions containing the overlapping fragments were mixed and subjected to PCR amplification using the external oligo primers (analogous to ‘a’ and ‘d’ in Fig. 1). iteratively, the DNA fra~ents were fist purified to eliminate contalnil~ating PCR-amplified products generated in the first PCR reactions. Purification of PCR products was performed by size- fractionating the DNA by electrophoresis through an agarose gel as described for the analysis of PCR reaction products. The band of the ap~r~p~ate size

was cut from the gel and purified using the Gene-

Clean kit (BiolOl) according to the m~ufacturers specifications. The GeneClean kit is based on a previously described method (Vogelstein and Giiespie, 1979). The reaction conditions used for the fusion of the two PCR-generate ~agmen~s were identical to those used to generate the fragments.

(f) Hybridization analysis

DNA fragments were transferred onto nylon membranes (MSI) and hybridized at 70°C with the mutagenic oligo probe 5’ end-labeled with 32P by polynucleotide kinase (NEN) as described (Duran and Pease, 1988).

RESULTS AND DISCUSSION

(a) description of the overlap extension technique

mutagenesis by overlap extension involves the generation of DNA fragments that, by virtue of having incorporated complementary oligo primers in independent PCR reactions, can be effectively ‘fused’ anywhere along the gene sequence by combining them in a second primer extension reaction. The method is illustrated in Fig. 1. In separate PCRs two fragments of the target gene sequence are amplified. Each reaction uses one flanking primer that hybridizes at one end of the target sequence (primer ‘a’ or ‘d’ in Fig. 1) and one internal primer that hybridizes at the site of the mutation and contains the mis- matched bases (primer ‘b’ or ‘c’ in Fig. 1). Since the product generated in a PCR incorporates lhe primers, the wt sequence will not be amplified. By using two internal primers that overlap, the two fragments AB and CD, generated in the first PCR, can be fused by denaturing and annealing them in a subsequent primer extension reaction. ‘The overlap allows one strand from each fragment to act as a primer on the other, and extension of this overlap results in the mutant product ~mutant fusion product’ in Fig. 1). Even though the annealing of the short overlap between the two fragments may occur at low frequency, the inclusion of additional flanking primers (‘a’ and “d’ in Fig. 1) allows the ‘fusion’ product that is formed to be spliced by PCR.

54

L A=- . . T -ii-

(2) c+d

AB . f

CD . .

A&CD

a, _____________________ -----------__________

a+d Y

f .

. MUTANT FUSION PRODUCT

Fig. 1. Schematic diagram of site-directed mutagenesis by over-

lap extension. The ds DNA and synthetic oligos are represented

by lines with arrows indicating the 5’-to-3’ orientation. The site

of mutagenesis is indicated by the small black rectangle. Oligos

are denoted by lower-case letters and PCR products are denoted

by pairs of upper-case letters corresponding to the oligo primers

used to generate that product. The boxed portion of the figure

represents the proposed intermediate steps taking place during

the course of reaction (3), where the denatured fragments anneal

at the overlap and are extended 3’ by DNA polymerase (dotted

line) to form the mutant fusion product. By adding additional

primers ‘a’ and ‘d’ the mutant fusion product is further amplified

by PCR.

As an additional advantage, site-directed muta-

genesis by overlap extension is extremely flexible in

the variety of sequence alterations that can be

achieved. In addition to point mutations, insertions

and deletions can be incorporated into the over-

lapping oligo pair (Fig. 2). Insertions are possible

because the ‘b’ and ‘c’ overlapping oligos need only

be complementary to the template in the 3’ portion

of the oligo. Additional sequence present in the 5’

portion of these oligos will be incorporated into the

AB and CD fragments and, upon generation of the

AD fusion product, will represent an insertional

mutation (Fig. 2A). Deletions are similarly obtained

by using.paired ‘b’ and ‘c’ oligos that correspond to

the sequence after the deletion (Fig. 2B).

(b) Site-directed mutagenesis of a mouse class-I MHC gene by overlap extension

To demonstrate this method we have introduced

mutations into the mouse major histocompatibility

A. INSERTIONAL MUTATION

AB . . ..h.

. . . . . . CD . . . . . . . K.“”

t

INSERTION PRODUCT

B DELETION MUTATION

2L 4. I_.___._______________~

. _______________________ v--_,

b -=i-

AB i ___

. ____ CD --~~~~~~~--~~_-____-\ -_____________________

t _~_~~~~~~~~~~~~_,~~

. ___________----______ DELETION PRODUCT

Fig. 2. Schematic diagram showing how insertion and deletion

mutations may be generated by the overlap extension method.

(A) Insertional mutation. The inserted sequence is depicted by

the dotted line. Oligo primers ‘h’ and ‘c’ are complementary to

each other in their 5’ ends, which corresponds to the insertion

sequence. The 3’ ends of these primers are complementary to

template target sequences on either side of the point at which the

insertion is to be made. (B) Deletion mutation. The sequence to

be deleted is depicted by the heavy lines. The solid and broken

lines indicate the template sequence on either side of the point

ofdeletion. Primers ‘b’ and ‘c’ are designed such that their 3’ ends

hybridize to template sequence on one side of the deletion, and

the 5’ ends are complementary to template sequence on the other

side of the deletion. The AB and CD products generated from the

PCR reaction using these primers are therefore overlapping at

the deletion point.

complex class-I Kb gene that correspond to the 5 aa

changes found in the naturally occurring Kb mutant

bml0 (Nathenson et al., 1986; recent sequence

analysis of the bml0 mutation has demonstrated the

presence of an additional aa change at position 167,

in which the wt tryptophan is replaced by a serine;

J. Schneck and D. Margulies, personal communi-

cation). A schematic representation of the class-I results in the substitution of Glu for Lys. These gene illustrating the regions targeted for PCR amplili- 16-mer oligos are completely overlapping, in contrast cation and mutagenesis is shown in Fig. 3A, with the to the other oligo pairs which consist of a 35mer oligo primers labeled as in Fig. 1. The external (‘b2’) and a 26-mer (‘~2’ and ‘~3’) that overlap by 16 primers ‘a’ and ‘d’ were located approx. 100 bp nt. The oligo pair ‘b2’ and ‘19’ introduced 4 aa sub- outside of the unique Hind111 and Hz01 restriction stitutions with 5 nt changes to give rise to the mutant sites, thus making it possible to easily ligate the designated bmlO-2, while the oligo pair “b2’ and ‘~3 fusion product from the overlap extension reaction introduced 5 aa substitutions with 6 nt changes to back into the expression vector containing the give rise to the bml0 mutant (Fig. 3B). These con- remainder of the wt class-1 gene construct. The over- structs are designed to investigate the functional lapping oligo pair used to introduce the mutation for importance of specific amino acid residues hypothe- the mutant designated bmlO-1 (oligos ‘bl’ and ‘cl’) sized to be involved in antigen and/or T cell receptor

A. MC” 2

-_-

It -_-

d a b/c t d

Hindlll XhOl AD

55

I I IOlbp ’ 561bp ' lc@bp

i

A6

454bp CD

332bp

B. Kb 160 165 170 175 I&U Glu Gly Thr CyS Val Glu Trp I&U Arg Arg Tyr X&U Lys Asn Gly Asn

Kbm10 ___ ___ ___ Ala ___ MSt ___ SSr ___ ___ ___ --_ -__ Glu LS" ___ ___

binlO- ___ ___ ___ ___ ___ ___ ___ _-- ___ ___ ___ ___ ___ G.JU ___ --- ___

t Bl 3' T ATG GAC CTC TTG CCC 5'

Glu Cl 5' A TAC CTG GAG AAC GGG 3'

*

bmlO-2 ___ ___ ___ A&, ___ )fSt ___ ,+r --- --- --- --- ___ ___ L.S" ___ ___

* * * 82 3'C CTC CCG CGC ACE; TAC CTC ATC GAG GCG TC.T ATG G 5'

Ala Met ser Leu c2 5' TAG CTC CGC AGA TAC CTG AAG CTC GG 3'

* **

bml0 ___ ___ ___ Ala -__ MSt ___ Ser ___ ___ ___ ___ ___ Glu -u ___ ___

* * * 82 3'C CTC CCG CGC ACG TAC CTC ATC GAG GCG TCT ATG G 5'

AlEI Met ser Glu I&u c3 5' TAG CTC CGC AGA TAC CTG GAG CTC GG 3'

* * **

Fig. 3. Mutagenesis strategy for the class-I MHC gene. (Panel A) The oligos used as primers for the PCR reactions are shown in relation

to the map ofthe II-2 Kb class-1 gene. The locations of the unique Hind111 andXho1 sites are shown. The flanking primers corresponding

to ‘a’ and ‘d’ in Fig. 1 anneal to sequences within the second exon and third intron, respectively, outside the unique Wind111 and XhoI

restriction sites. The sequences of these oligos are as follows: ‘a’, 5’-GGCTACTACAACCAGAG-3’; ‘d’ 5’-TGGGAGAGGCC-

ATGGCT-3’. The products of the first two PCR reactions (AB and CD) and the fusion reaction (AD) are shown in relation to their

location within the class-1 gene. Digestion of the AD fragment with Hind111 + XhoI results in the formation of three fragments of 101,

561 and 108 bp in length. (Panel B) The complementary oligo pairs used to introduce the mutations were either entirely or partially

overlapping, although in each case the overlap consisted of 16 bp.

56

738 bp-

4Q2 bp-

369 bp-

246 bp-

123 bf-

- 564 bp

-369 bp

-- 246 bp

- 123 bp

Fig. 4. Generation of a mutant fusion product from overlapping PCR-generated fragments by overlap extension. (Panel A) Electro-

phoretic analysis of PCR amplification products forming mutant 6&O-1. 123-bp ladder markers (BRL), wt AD product generated by

PCR using primers ‘a’ and ‘d’ and the K” template (AD wt), fragment AB generated by PCR using primers ‘a’ and ‘bl’ (refer to Fig. 2B),

fragment CD generated by PCR using primers ‘cl’ and ‘d’, AD fusion product generated by combining fragments AB and CD directly

with additional primers ‘a’ and ‘d’(AB + CD titration; these samples were generated by adding ten-fold dilutions ofthe samples containing

AB and CD, starting with 10 ~1 of each undiluted sample). (Panel B) Eiectrophoretic analysis of PCR amplification products forming

mutant 6m10-2. AD wt fragment as above, fragment AB generated by PCR using primers ‘a’ and ‘b’, fragment CD generated by PCR

using primers ‘~2’ and ‘d’, AD fusion product generated by combining fragments AB and CD that had been gel puritied, fusion fragment

AD digested withH~dII1, fusion fragment digested with HindI + XhoI, wt HindIII-XhaI insert, 123-bp ladder marker, HindIII-digested

phage I DNA marker (New England Biolabs). (Panel C) Southern-blot analysis ofthe PCR-amplified products. The gel shown in Fig. 4B

was probed with the oligo ‘~2’.

binding to the class-1 molecule. The results of

functional studies using cell lines transfected with

these mutant class-I constructs will be reported else-

where.

The products from the PCR using primer ‘a’ or ‘d’

in combination with the overlapping oligo primers

‘bl’ and ‘cl’ (used to introduce the mutations desig-

nated b&O-l) are shown in Fig, 4A. The sizes of the

AB product and the CD product are consistent with

the map shown in Fig. 3A. The products from the

first set of PCR reactions appeared relatively homo-

geneous based on EtdBr staining, although addi-

tional PCR-generated products of differing sizes are

visible at much lower concentrations. The fusion

reaction in which dilutions of the first PCR contain-

ing the AB and CD fragments were mixed directly,

with no further purification, resulted in the gener-

ation of multiple products including the AD fusion

product of the predicted 770-bp length (Fig. 4A;

AB + CD titration). For comparison, the AD wt

fragment generated by PCR using the original tem-

plate and primers ‘a’ and ‘d’ gave rise to a 770-bp

fragment (Fig. 4A, AD wt). The presence of a pre-

dominant 350-bp product generated in this fusion

reaction suggests that under these conditions the

fusion or amplification of minor PCR products may

be favored in the second reaction. To increase the

efficiency and yield of the fusion reaction, the input

fragments AB and CD were gel-purified. The product

of the PCR fusion reaction using gel-puked AB and

CD fragments contained predominantly the AD

fusion product and no visible 350-bp band (Fig. 4B).

Because this modification in the protocol eliminated

the 350-bp fragment, the band was not characterized

further.

To determine whether the product from the fusion

reaction contained the intended nucleotide substitu-

tions, the reaction products shown in Fig. 4B were

transferred to a nylon membrane and hybridized

with end-labeled oligo ‘~2’. The CD fragment and the

AD fusion product hyb~dized strongly to the mutagenic oligo (Fig. K, AD, AD ~~~dII1; AD NkzdIII- Xfioi), while the PCR product using primers ‘a’ and ‘d’ on wt template and the wt k&zdIXI-X&I insert showed faint hybrid~ation. Hyb~idiz~tioi~ to CD

was faint due to the lower concentrative of DNA loaded. F~a~rn~nt AI3 showed no hybridization because it only matched 16 nt of the 26-m oligo ‘~2’. These results indicate that the fusion product contains the mutagenic sequence. By gel-p~~fying the AB and CD fra~ents, the wt template is not carried over into the PCR fusion reaction, thereby ~l~~~na~ing the possibility of also ~plifyin~ wt sequences.

To q~~~ltitativ~ly assess the effkiency of this mut~e~esis procedure, colony lifts from bacteria tr~sf~~~d with the ligation of the vector with the PCR fusion produet were hyb~di~ed with the mutagenic oligo. Among 168 colonies probed there were only three colonies that did not hybridize with the mutagenic oligo (data not shown). This represents at least a 98% efficiency of ~~uta~enesis. The three

175

bmlO-1 bm 1 O-2

negative colonies were not analyzed to determine whether thev represented failures in ligation or muta- . genesis. Furthermore, complete sequence analysis of PCR-generated DNA fragments revealed no clones containing the wt sequence. Thus, the efficiency of this n~utage~esis pso~edure is essentially lOOo/, in terms of generating altered product.

(c) Sequence analysis of cloned mutant genes gener-

ated by overlap exte&oa

The mutage~c AD fragment was then cut sequen- tially with ITtndIII and X/WI (Fig. 4B) and ligated back into the original plasmid vector. The resulting mutant clc~~s wese either sequenced directly by ds sequences of the mutant plasmid or by subcloning into M 13. All clones sequenced cont~ned the desired mutations introduced by the paired mutagenic oligas. Sequence data demonstrating the mutations forming bml0, brraf&1 and 6&O-2 in comparison with the wt sequence is shown in Fig. 5. In sequencing the bmlC> PCR-venerated mutant clones as well as three addi-

bmlU wt

175

Fig. 5. Sequence an&& of~~~~rnut~ts generated by the overfap extension technique. The three &zlO site-directed mutants generated

by the overlap extension method and the wt were sequenced from ss Ml3 templates. The region shown encompasses nt encoditlg aa

160 through 175, where the mutations were directed. The altered nt are indicated by the bor~~ontal dashes along the right ma&n of

each mutant sequence.

58

tional mutants, there was one undesired nucieotide change, detected out of more than 3900 nt sequenced. This represents sequence data from a total of nine independently derived clones generated from five separate overlap extension reactions needed to produce the three bml0 mutant genes and three additional mutant genes.

(d) Analysis of error frequency

The method of generating site-directed mut~ts described here has recently been suggested independently by Higuchi et at. (1988) who used direct sequencing analysis of the PCR-generated DNA fragments to demonstrate the presence of molecules containing an introduced nucleotide change. How- ever, these mutated products were not cloned and no conclusions could be made concerning the frequency of undesired nucleotide changes resulting from the misincorporation rate per nucleotide per cycle of the Taq polymerase. Here we have analyzed cloned, PCR-spliced DNA fragments generated using the overlap extension technique and have observed that the 0.026% error frequency (frequency of undesired nucleotide changes observed in the cloned PCR product) derived from the results presented here (one error in over 3900 nt sequenced) is much lower than the error frequency of 0.25% previously reported (Saiki et al., 1988). Assuming this error frequency of 0.25% applies here, the probability of obtaining one or fewer undesired mutations in 3900 nt is 0.0006, calculated by the binomial ~stribution (3900 x 0.9975 exp 3899 x 0.0025). Therefore, the error frequency observed here is significantly less than previously reported.

This low error frequency may be due to the use of cloned template rather than genomic DNA template. In the latter case the template sample actually contains only a few copies of the target sequence. A misincorporation in an early amplification cycle would be propagated through subsequent cycles and represent a significant portion of the final product. Starting with more of the specific target template allows for the generation of the final product after fewer cycles of PCR amplification, thereby decreas- ing the chance of an incorrect base being introduced by the polymerase. Assuming the misincorporation rate per nucleotide per cycle of the Taq polymerase is constant, the only other variable a_tTecting the error

frequency is the number of cycles of ~p~~cation. Thus, our observed error frequency of 0.026% may be much lower than the previously reported error frequency-of 0.25% because, by using cloned template, the number of cycles of PCR amplification required is much less. Whatever the explanation for the low error frequency of 0.026% observed here may be, it is clear from these results that the error frequency of 0.25% published previously (Saiki et al., 1988), which wodd have been prohibitively high for purposes of cloning, does not apply in this case. Therefore, the overlap extension technique is a practical alternative to conventional methods of mutagenesis or gene splicing, as described below.

(e) Advantages of the overlap extension technique

The use of overlap extension for site-directed mutagenesis represents a significant technical improvement over current methods. The standard methods (Smith et al., 1985) involve annealing the mutagenic oligo to an ss wt sequence. This requires a cloning step to transfer the wt sequence into an ss vector system such as M13. The wt sequence then serves as a template and the mutagenic oligo serves as the primer for the synthesis of a new mutagenic strand by DNA polymerase. Despite the use of selec- tion systems, the efficiency of this process (i.e., the % of output molecules that are not of the wt sequence) is significantly less than 100% because of the presence of the wt template throughout the mutagenesis procedure. This makes it necessary to screen the products using the mutagenic oligo as a hybridization probe. The major advantages of site-directed mutagenesis by overlap extension are its simplicity and virtual 100% efficiency. Site-directed mutagenesis by overlap extension eliminates the need for ss template and viral vector intermediates, thus elimi- nating a cloning step. The virtually 100% efficiency forgoes the need to screen the products of the mutagenesis for the presence of the nt changes. The elimi- nation of these steps reduces the time and effort required to generate site-directed mutations.

Although the method described here represents a novel approach to site-directed mutagenesis, an essential feature of this method is the ability to fuse or recombine two independent fragments of DNA without the use of a DNA ligase. The method of overlap extension can be used to generate recombi-

59

nant DNA praducts from two independent genes without the use of restriction endonucleases. Gene splicing by overlap extension eliminates the need for the introduction of restriction sites and is independent of the targeted sequences. We have used this technique to construct chimeric genes comprised of segments of different class-1 genes (Horton et al., 1989) and are currently involved in constructing additional fusion proteins. These constfucts have involved the fusion of as many as four DNA fragments having a combined size of more than one thousand bases. Given the substantial savings in time, the increased versatility in design strategies for ~n~oducing site-directed mutations and recombining gene sequences, and the low rate of undesired nucleotide changes, gene splicing by overlap extension represents a significant advance in recombinant DNA technology.

ACKNOWLEDGEMENTS

We would like to thank Dr. S.S. Sommer and his Lab for the use of their DNA Thermal Cycler and Drs. R.T. Abraham, B.N. Beck, D.J. McKean and E.D. Wieben for reviewing the manuscript. S.N.H. was supported by a medical scientist scholarship from the tife and Health Insurance Medical Research Fund. H.D.H. and J.K.P. were supported by NIR training grant CA-09127. Additiond fund- ing for this work was obtained through NIH grants AI-22420, AI-00706 and CA-42199.

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site-directed mutagenesis by overlap extension using...

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