Use of exonuclease for rapid polymerase-chain-reaction-based in vitro mutagenesis

Gene, 97 (1991) l-6 Elsevier GENE 03823

Use of exonuclease (Site-directed

for rapid polymerase-chain-reaction-based

mutagenesis;

Venkatakrishna

kinased

primer;

phage 1 exonuclease;

Shyamala * and Giovanna Ferro-Luzzi

Division of Biochemistry

and Molecular

Biology,

in vitro mutagenesis

single mutated

primer;

histidine

transport

operon)

Ames

University of California, Berkeley,

CA 94720 (U.S.A.)

Received by M. Bagdasarian: 19 July 1990 Accepted: 17 August 1990

SUMMARY We describe a simple strategy for improving site-specific mutagenesis. We have combined the polymerase chain reaction (PCR) with a phage A exonuclease (ExoL) treatment to produce mutated fragments larger than 2.5 kb. The applicability of this approach has been proven with two overlapping mutated primers. The procedure has also been made more cost-effective by the use of a single mutated primer, which is referred to as SMP-PCR procedure. The entire procedure of kinasing the primer, amplification by PCR, Exo1 digestion and second step of PCR can be performed in less than 6 h. We have used this approach to generate a number of mutations in the Salmonella typhimurium hisP gene of the histidine transport operon.

INTRODUCTION Site-directed in vitro mutagenesis is a powerful tool in studies of gene function. The conventional in vitro mutagenesis technique requires the synthesis of a mutated oligomer which is annealed to the gene of interest presented as ss DNA, and used as primer for DNA synthesis to copy the entire gene plus the vector. With the advent of PCR this technology has been incorporated into a number of modifications of the conventional in vitro mutagenesis method resulting in protocols that are simpler and more time effective. Most of these modifications involve the synthesis of

Correspondence to: Dr. G.F.-L. Ames, Department of Biochemistry, UC Berkeley, CA 94720 (U.S.A.) Tel. (415)642-1979;Fax (415)643-5165. * Present address: Chiron Corporation, 4560 Horton Street, Emeryville, CA 94608 (U.S.A.) Tel. (415)655-8734;ext. 2279; Fax (415)655-9910.

Abbreviations: bp, base pair(s);

ds, double strand(ed); DTT, dithiothreitol; Exol, phage 1 exonuclease (5’-3’, ds specific); HisP, histidine permease; hisP, gene encoding HisP; IPCR, inverse PCR; kb, kilobase or 1000 bp; nt, nucleotide(s); oligo, oligodeoxyribonucleotide; PCR, polymerase chain reaction; PNK, polynucleotide kinase; RCPCR, recombinant circle PCR; SMP, single mutated 10 mM Tris

HCl pH 8.0/l mM EDTA;

0378-l 119/91/$03.50

0 1991 Elsevier

primer; ss, single strand(ed);

TE,

wt, wild type.

Science Publishers

B.V. (Biomedical

Division)

two overlapping mutated primers each of which carries the mutation of interest (Kadowaki et al., 1989; Higuchi et al., 1988; Horton et al., 1989; Kamman et al., 1989; Valette et al., 1989). The PCR-generated mutated fragment then needs to be restriction digested and cloned into expression vectors for functional studies. It is essential, therefore, to generate a fragment sufficiently large that is spans unique restriction sites present in the cloning vector. However, the small size of the final PCR product has been the major drawback of PCR-based in vitro mutagenesis. Until recently the longest amplified fragment obtained by the overlap technique has been less than 500 bp (Horton et al., 1989). Here we describe the use of ExoL to overcome this problem. We have developed two simple methods that allow the generation of mutated fragments larger than 2 kb. The first method uses two overlapping mutated oligos and an EXO~ digestion step to generate ss DNA, which then acts as template for amplification of a large fragment. This overlap extension method is very efficient for generating mutations, insertions or deletions. However, the expense of synthesizing two mutated oligos renders this approach expensive. The second method, SMP-PCR, uses a single mutated oligo along with the Exoil step and is therefore cost-effective.

2

First step of amplification

J,

3’ 5’ &

-

Exo A 3’

3’* &

5’

Second step of amplification for cloning

+

D

3’ 5’

Asymmetric amplification for sequencing F

4 J.-e3 3’-

5’

T’ Fig. 1. Schematic primers. o-x+,

representation

The hatched kinased

mutated

A and B are kinased site of mutation

of in vitro mutagenesis

box represents primers;

chromosomal

Y and Z, indicate

(0) using PNK. Primers

and are used for confirming

by the modified

DNA. Symbols: restriction

following

RESULTS

AND DISCUSSION

(a) Modified overlap extension method with the Exol digestion step To generate large fragments we have modified the basic overlap extension method (Higuchi et al., 1988) by introducing an Exol digestion step. Our intent was to combine the functions of polymerase and EXO~ to generate sufficient amounts of large, mutated ss DNA. The modified method is schematically depicted in Fig. 1. Two separate simultaneous amplifications are carried out on chromosomal DNA with each amplification reaction using a 5’-kinased mutated primer (A and B, respectively) and an end primer (C and D, respectively). Since the two mutated primers are complementary to each other, the products from the two amplifications overlap each other over this region. The strands obtained by the elongation of the kinased primers are preferentially digested by Exo1 (Higuchi and Ochman, 1989) thus resulting in amplified ss products after the first step of amplification and ExoA digestion. These ss DNA

method.

Mutation

with the arrow indicating

enrichment

is introduced

by the use of two mutated

the direction

of ss DNA by Exol digestion

of the unique restriction

asymmetric

Both these methods have been used to generate a number of site-specific mutations in the HisP protein of the S. typhimurium, using chromosomal DNA or whole cells as the source of template DNA (Joshi et al., 1990).

extension

primers

sites. To facilitate

C and D are oligos outside the mutation

overlap

arrows,

site used for cloning.

of extension;

x, mutation;

of ds DNA, mutated Primers

primers

E and F border

the

amplification.

products overlap and thus can hybridize with each other at their 3’OH ends. This modification of the earlier procedure (Higuchi et al., 1988) is critical to allow annealing of this relatively short overlapping region during the second step of amplification. A second amplification is carried out on this ExoA-digested material using the two end primers (C and D). The polymerase initially extends each ss DNA to fulllength products which then can hybridize with the end primers and facilitate continued cycles of amplification. An aliquot of the final product is used to confirm the mutation by asymmetric amplification and the rest is used for cloning at restriction sites Z and Y into the desired vector (Shyamala and Ames, 1989a). We have used this method to introduce mutations into the h&P gene (Fig. 2, top). Table I summarises the specific oligos and their relevant positions in Figs. 1 and 3. Either chromosomal DNA or intact bacterial cells were used to provide template; kinased oligos P22 and P23 and end oligos M5 and P21 were employed to amplify DNA. Oligos P22 and P23 were kinased at the 5’-end using PNK. We have simplified the next step by using the kinasing mixture without any further purification as a source of the oligo for the PCR reaction. Products of 1.0 kb (M5/P23) and 1.3 kb (P22/P21) were obtained, respectively (Fig. 2, left panel, lanes A and C). The PCR reaction mix could be used directly for digestion with Exol thus enriching for the ss

3 product

of second

exonucleased Primers

step of amplification

products

P23 and P22 were kinased

of 1.3 ~1 of 10 x MgCl,/S PNK

kinase

mM DTT)/l

(Pharmacia).

ABCDEM

kb

ATGC

(0). A final volume of 13 ~1 consisted

buffer

The reaction

(70 mM mixture

chain reaction

25”C/5.0

ATGC

mM MgCl,/16 (Perkin

Elmer-Cetus).

glycine . NaOH

2:

30 min aliquots

0.5

at 37°C.

primers, of

and relevant

S. fyphimurium.

restriction (Bottom)

h.bP DNA.

mutagenized and P22/P21,

respectively;

overlap

extension

sites in the histidine Left

panel:

method transport

amplification

Lanes: A, C, amplification

of

products

B, D, Exol digests of amplified

operon in

vitro

The

Nucleotide

Exol-digested

spin column

to remove products

was

volume

was 2min.

strain

passed

were used as primers

was carried out as described amplification

to 5 ~1 of 67 mM

The presence (Shyamala

LT2 was used in these

for

through

the buffer constituents.

primers. The amplification by asymmetric

at

at 72°C for

with 5 units of Exol

DNA

in a 50+1 final reaction

time which

unit of

were denatured

DNA was used directly for Exol

43 ~1 was added

P4/P8 which flank the site of mutation.

of M5/P23 products;

The samples

a

Five-n1 for second

with the external above, except for of mutations and Ames,

studies.

(Bottom)

was

1989b). Right

panels: sequence of the asymmetrically amplified in vitro mutagenized DNA. The product ofM5/P21 was asymmetrically amplified with primers for sequencing.

E,

mutant

The sequence

The limiting primer P4 was used

of the wt is shown in panel A and of the

in panel B. The mutated

region is marked.

factors: the effticiency of kinasing and the efficiency of Exol digestion. The Exo,I-digested DNA was passed through Sephacryl-S300 spin column for removing the buffer salts and used directly for the second step of PCR without further purification. The second step of amplification

DNA initiated by end primers. Following Exol treatment, a differentially moving band typical of ss DNA could be seen (lane D). Since the mobility of ss DNA is dependent on the nt composition of the DNA, it may not always be identifiable (lane B). The yield of ss DNA depends on two TABLE

DNA

PM of each primer

the extension S. typhimurium

to the

of the hisA4 and hisP genes,

DNA

of the Exo,l-digested

ascertained

representation

units of

for 30 min at

out using either 0.1 ,ng

sulfate/l

buffer pH 9.4, and incubated

PCR amplification

of the modified

was incubated

at 55°C for 1 min, and extended

Of the amplified

Sephacryl-S300

Fig. 2. Application

7.6/10 mM

mM of each deoxynucleotide/0.5

1 min for 20 cycles. The PCR-amplified digestion.

hisP genes. (Top) Schematic

pH

cells as source of template

mM ammonium

(one of which was kinased)/0.5 94°C for 1 min, annealed

-

Tris*HCl

was carried

DNA or intact bacterial

Taq polymerase

-

with the

(Shyamala and Ames, 1989b; Joshi et al, 1990). The final volume was 50 ~1, in a reaction mixture containing 67 mM Tris . HCl pH 8.8, at

B

A

M5/P21,

M, Hind111 digest of 3, DNA.

nl of 0.2 M ATPjlO ~1 of 30 PM primer/5

37°C. The polymerase of chromosomal

using primers

serving as template;

I sequence

and alignment

of the oligomers Figure’

Oligo (symbol)

Sequence a

M5

TATAGATC_TGAAGAGGGCGGATTTGTGAT(T)1633

M7

TGACGCTGAATACCTGCGCGTACA(T)1974

P4

AGCTCAAAGTGGCGGATAAA(T)2602

P8

TATC-TCGAGCGCCGATGTGGGTTCAT(B)2920

P9 P21

AGGCCTGATAGGGCGTC(B)4042

P22

TCAACCTCTEAGCCACATGA(T)267

P23 P24

TCATGTGGCTgAGAGGTTGA(B)269

P25

TATCTCGGG-TTATTTCAGCGAGCCTTTCA(B)3

P34

TCAG&&TTCATGACCG(B)2433

Primer c

TATCTCG-AGCACCGTCATGTGGCTCCAG(B)2698

1 1

TATAGATCTGGTTGCAGCACGTGTCCT(T)2330 143

a Nucleotides to the left of the dash indicate bases that are absent in the chromosomal DNA but have been added to create restriction sites. Letters T or B in parentheses indicate whether the sequence corresponds to the top or bottom strand. The underlined nt represent the change introduced, multiple substitutions are indicated with a slash, and the nt on top of them indicate ’ Numbers refer to the figures in this paper. ’ Letters

refer to Figs. 1 and 3, and to sections

a and b.

the wt. The numbers

indicate

the corresponding

nt (Higgins

et al., 1982).

4

A 5’

First step of amplification

n3’ J, Second step of amplification for cloning C

5’ 3’/

3’ 5’

‘c3’

5’

:

: 2,

I 2

&

Asymmetric amplification for seq ue nci ng &3’

2, Fig. 3. Schematic

representation

of in vitro mutagenesis

using an SMP. Mutation

for the first as well as second polymerase

chain reactions.

The hatched

the direction

o-x+,

kinased

of extension;

x, mutation;

mutated

5’

T?

is introduced

box represents

primer;

carried out with end primers M5/P21 yielded a unique product of the expected size of 2.4 kb (Fig. 2, lane E). The presence of the mutation in the final product was ascertained by performing an asymmetric amplification reaction with primers P8/limiting P4 (E and F respectively in Fig. l), flanking the site of mutation and sequencing the product with the limiting primer P4 (Fig. 2, right, panels A and B). The final mutated DNA fragment spanned the sites for restriction enzymes KpnI and AvaI, which were utilized for cloning into a compatibly digested expression vector for structure-function studies. All final recombinant colonies carried the desired mutation. The conventional overlap extension method has been the technique of choice for generating PCR-based in vitro mutations (Higuchi et al., 1988; Valette et al., 1989), site-specific insertions (Kamman et al., 1989; Higuchi et al., 1988), deletions (Valette et al., 1989) and hybrid genes (Horton et al., 1989; Valette et al., 1989). One of the advantages of the overlap extension method is that only mutated fragments are used in the second step of PCR, therefore eliminating the possibility of contamination with wt sequences. This approach is the equivalent of destroying preferentially the wt DNA in the conventional mutagenesis through the incorporation of phosphorothioated nt during elongation (Sayers et al., 1988). The main drawback of the system as described has been the limited size of the final product (Horton et al., 1989). In our hands the largest fragment obtainable was

by the use of an SMP with chromosomal

chromosomal

Y and Z, restriction

DNA. Symbols:

DNA as template

--f, primers with the arrow indicating

sites.

937 bp which was not large enough to span the unique restriction sites in our expression vector (data not shown). A recent report describes the synthesis of a 997-bp fragment (Horton et al., 1990). The inability to get large fragments by this method lies in the fact that the complementary strands of each of the two products from the first step of PCR anneal to each other faster and more efficiently than the short overlap between the two products, thus making it difficult to generate a fragment that spans the length of both products. This imposes a serious limitation on the use of the final mutated fragment for cloning at unique restriction sites in specific expression vectors. A possible solution to overcome this problem involves amplifying several small fragments and directionally ligating them to obtain a suitably sized fragment for cloning (Horton et al., 1989; Valette et al., 1989). Such an approach is cumbersome and costly. Other investigators have overcome the problem by performing IPCR (Hemsley et al., 1989) or RCPCR (Jones and Howard, 1990). However, these methods require cloning the gene of interest into a circular plasmid template of reasonably small size and entail amplification of a large section of unwanted vector template. Higuchi and Ochman (1989) have described a method to sequence amplified DNA following Exo,l digestion to create ss DNA. We have extended the use of this method to overcome the problems described above in site-directed mutagenesis by enriching for ssDNA after the first step of

amplification. This permits the two mutated ss strands to anneal efficiently at the short overlap irrespective of their size. Using this approach we have generated final fragments of up to 2.4 kb, and there is no obvious reason why the size of the final product obtained by this method could not be considerably larger. In any case, a product of 2.5 kb should suffice for spanning unique restriction sites in most structural genes, which should enable easy subcloning. (b) In vitro mutagenesis using a single mutated primer The modified overlap extension method is very efficient in yielding mutated fragments. However, due to the cost factor involved in synthesizing two mutated oligos for every mutation, as required by all overlap extension methods, we pursued the use of a SMP oligo for in vitro mutagenesis. Fig. 3 is a schematic representation of this method (SMP-PCR). In the first step of amplification a kinased mutated primer (A) and an end primer (B) are used to get a mutated amplified fragment. The kinased strand is then digested with EXO~ to generate a mutated ss region spanning from the end primer to the mutation site. In the second step of amplification this mutated ss fragment and a second end primer (C) are used to amplify the entire region spanning the restriction sites (Y, Z) for cloning. An aliquot of the final product is used for confirming the mutation by asymmetric amplification and sequencing, while the rest is used for cloning. The efficiency of in vitro mutagenesis by SMP-PCR also depends on efficient removal of the first end primer (B) prior to second step of amplification. Since chromosomal DNA is used as template, presence of the first end primer (B) in combination with the second end primer (C) will result in significant contamination with wt DNA. While Sephacryl S-300 effectively retains a primer of 24 nt, for larger primers Biogel P60 has been found to be necessary. Fig. 4 (left panel) shows the results obtained with the hisP gene: in the first amplification a 440-bp fragment was generated (lane A). The amplified DNA was digested with Exol (lane B) and used as a primer along with an end primer (P25) on chromosomal DNA as a template in a second amplification reaction, yielding a final 1.16-kb product (lane C). The presence of the mutation in the final product was ascertained by carrying out asymmetric amplification with P9Jimiting P24 (D and E, respectively, in Fig. 1) concentration and sequencing the product with P24 (Fig. 4, right panels). The final fragment was digested with KpnI, which is present inside of the M7 primer-binding site, and with AvaI, which is tailored into the primer P25, and used for inserting the fragment into the expression vector. Nelson and Long (1989) were able to use an SMP for mutagenesis; however, to improve the specificity of the final product a total of four oligos were employed. While this

MABC

kb

B

A ATGC

ATGC

I-

0.5 Fig. 4. Application panel:

of the SMP-PCR

amplification

of in vitro

Lanes: A, amplification product;

C, product

and the product

product

sequence

was asymmetrically

Left SMP.

amplified

using primers P25

DNA as the template; obtained

amplified

P24. Panels

DNA, respectively.

M, in

after the second

amplified using primers

The asymmetrically

with the limiting primer

quence of the wt and mutated

using

B, Exol-digested

of asymmetrically

DNA. The 1.16-kb product

which flank the site of mutation. sequenced

of M7/P34;

of M7/P34 with chromosomal

step of amplification

hi.rP DNA

ofthe second step of amplification

~/Hind111 digest. Right panel: vitro mutagenized

to the hisP operon.

method

mutagenized

P24/P9,

amplified DNA was A and B show the seThe region of mutation

is marked.

work was in progress, a paper using three oligos, one of which is an SMP, was published (Sarkar and Sommer, 1990). However, in these studies the largest first-step amplification product that was used as a primer for the second step was only 340 bp. Our modification of providing ss products enables the use of at least a 931-bp amplification product as a primer for the second step (data not shown) and at this time we do not know the size limit of the fragment that can be used. This enables the introduction of mutations at regions located far away from the restriction sites. Also, in previous studies cDNA and plasmid DNA enriched for the gene(s) of interest were used as templates for the second step of amplification. This poses a prerequisite that the gene(s) of interest be available in a specialized form. By using chromosomal DNA as the template for the second step of amplification we have demonstrated that the SMP method can function on a complex mixture of DNA and is applicable to any general situation. Using these two rapid methods we have been able to create a large number of mutations in the hisP gene of the histidine permease operon to study the ATP-binding properties of the mutated HisP protein. (c) Conclusions (1) Through a combination of PCR and EXO~ we have generated mutated ds DNA fragments of up to 2.5 kb.

6 (2) The applicability of this approach has been demonstrated for overlap extension as well as SMP-PCR methods. (3) For site-directed mutagenesis, the SMP-PCR method is inexpensive and effective. For more complex manipulations, such as gene splicing by overlap extension, site-specific deletions, and insertions, extension is preferable.

the modified overlap

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R.M., Hunt,

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