Download A general and rapid mutagenesis method using polymerase chain

Gene.91 (1990) 143-147
Elsevier
143
GENE 03618
A general and rapid mutagenesis method using polymerasechain reaction
(Recombinant DNA; rat muscle nicotinic acetylcholine receptor; flanking primer; fragment fusion; amplification; nucleotide
sequence)
Stefan Herlitze and Michael Kuenen
Abteilung Zellphysiologie,Max-Planck-Institut
fir MedizinischeForschung,Heidelberg (F. R. C.)
Received by J.A. EngPer:12 December 1989
Accepted: 26 December 1989
SUMMARY
The construction of deletions, insertions and point mutations in DNA sequences is a powerful approach to analysing the
function and structure of genes and their products. Here, we present a fast and efficient method using the polymerase chain
reaction to introduce mutations into cDNAs coding for the a-, ‘y-and s-subunit of the rat muscle acetylcholine receptor. Two
flanking primers and one mutant oligo, in conjunction with supercoiled plasmid DNA and a fragment of the target DNA
are sufficient to introduce the mutation by two PCR amplifications. Our method permits directing the location of mutations
anywhere in the target gene with a very low misincorporation rate, as no substitution could be detected within 9600 bp. The
utility of this approach is demonstrated by the rapid introduction and analysis of eleven mutations into three differentcDNAs.
Any kind of mutation can be introduced with an ef?iciency of at least 50%.
INTRODUCTION
Commonly, mutants have been constructed in vitro by
oligo-directed procedures, in which the appropriate oligos
are used to prime DNA synthesis on ss DNA templates (for
review, see Smith, 1985). Development of the polymerase
chain reaction (Saiki et al., 1985) offered a new powerful
tool for site-directed mutagenesis on any DNA (Higuchi
et al., 1988; Vallette et al., 1989; Kadowaki et al., 1989;
Kammann et al., 1989; Ho et al., 1989).However, a critical
Dr. M. Koenen, Abteilung Zellphysiologie,
Cmespondence
to:
Max-Planck-Institut flu Medixinische Forschung, Jahnstrasse 29,
D-6900 Heidelberg(F.R.G.)Tel. (06221)486-475;Fax (06221)486-351.
Abbreviations: AChR, acetylcholine receptor; a-AC& y-&WI and
s-AC%&cDNAs encoding the a-, ‘y-and s-subunits of the rat muscle
AChR; apl, ap2, flanking primers for the a-AChRcDNA; bp, base
pair(s);dNTP, deoxynucleotidetriphosphatc;ds, doublestrand(ed);fpl,
fp2, schematic flanking primers;mp, mutant primer;nt, nucleotide(s);
oligo, oligodeoxyribonucleotide;PCR, polymerase chain reaction; ss,
single strand(ed);wt, wild type.
0378-l119/90/503.50 0 1990Elsevier Science Publishers B.V. (Biomedical Division)
point when using PCR in mutagenesis is the low fidelity of
the T’uqpolymerase during replication (Tindall and Kunkel,
1988), possibly leading to replication errors during mutagenesis. Reiss et al. (1990)demonstrated the dependency of
misincorporation on fragment length and number of PCR
cycles. In addition, misincorporation caused by the minimal
homology requirement of PCR primers is possible (Sommer
and Tautz, 1989; Kwok et al., 1990).The aim of this study
was to develop a mutagenesis method allowingrapid introduction and identification of insertions, deletions and point
mutations at any position of a gene using one mutant oligo
and a minimal number of PCR cycles. Decreasing the number of replication cycles compared to Ho et al. (1989)
reduces the chance of misincorporation during PCR.
EXPERIMENTALAND DISCUSSION
(a) Strategy and mutagenesis
Complementary DNAs encoding the rat muscle AChR
subunits (V. Witzemann, E. Stein, B. Barg, T. Konno, M.
144
X
Y
W
p$PS4
8.
mp
"
b
M
l
X
W
|:l
,
I::
C
$'
d
Y
fpl
$,
=
M
3'
-I~
• . . . . . . . . . . 3'
-~ fp2
X
S'.,
3; I
z
I
l
M
M
i
y
3'
S'
Fig. 1. Schematicoutlineof the mutagenesis.(a) pSP64 plasmid(dashed lines)carryingthe cloned eDNA (blackened box), restrictionsites W, X, Y,
Z, and the sequencecomplementto the oligosusedin the first PCR. (b) Productof the first PCR (thinsolidlines)carryingthe introducedmutation(M).
(e) Restrictionfragment(heavylines)of the targetgeneisolatedafter digestionwithrestrictionendonucleasesX and W. (d) Interstrandannealingallows
elongation(dashed arrows)of either the mutant-carryingDNA fragmentor the wt fragment.The elongationproduct is the templatefor synthesisio$the
filial PCR product with fpl and fp2. (e)The finalproduct of the second PCR (onlythe mutant-carryingfragmentis shown) van be digested with the
restriction endonucleasesZ and Y to isolate a DNA fragmentused for reinsertioninto the eDNA.
Criado, M.K., M. Hofmann and B. Sakmann, in preparation) were cloned into pSP64 (Melton et al., 1984) and a
derivative, pTV64N, to analyse the function of receptor
mutants after injection ofcRNA into Xenopus laevis oocytes
(Methfessel et al., 1986). Methods for mutagenesis using
PCR have been described employing oligo primers containing restriction sites (Kadowaki et al., 1989), or covering
regions containing restriction sites useful for replacing the
wt sequence (Vallette et al., 1989). In addition, the introduction of point mutations with high efficiency has been
described (Ho et al., 1989). These authors identified one
misincorporation in 3900 bp using two mutant oligos and
three PCR amplifications to introduce a mutation. The
method described here minimises misincorporation using
only one mutant oligo, two amplifications and a DNA
fragment replacing an additional PCR amplification. The
experimental design of our mutagenesis is presented schematically in Fig. 1. The flanking primers, fpl and fp2, are
21 nt long and used in all mutagenesis experiments with the
same eDNA. The fpl initiates DNA synthesis on the 5'
border of the sequence to be mutated and will, in conjunction with the rap, generate a mutated DNA fragment
(Fig. lb). We added a ten- to 20-fold excess ofthe mutated
DNA amplified in the first PCR to a restriction fragment
(Fig. lc) of the same eDNA. The use of this restriction
fragment replaced an otherwise necessary PCR amplification. The restriction fragment overlaps with the first PCR
product allowing interstrand annealing followed by enzymatic extension of the DNA at both 3' termini (Fig. ld)
(Yon and Fried, 1989; He et al., 1989; Horton et al., 1989).
The elongation results (Fig. ld) in a fragment carrying the
mutation and a wt fragment. The final product is amplified
(Fig. le) using fpl and fp2. DNA fragment fusion in combination with fpl and fp2 allows the introduction of the
mutation at any position of the eDNA. The internal restriction sites allow subcloning of the mutated DNA fragment.
For example, five different point mutations were incorporated separately into the eDNA encoding the ~-subunit
using ~pl and ~p2 and in each case a different mutant
primer. We used the GeneAmp kit (Perkin Elmer Cetus)
145
TABLE I
Parameters of mutagenesis
cDNA a
Mutant b
Mutation ©
Oligo d
(nt)
RFr ~
(bp)
Overlap(RFr/PCRt )r
(bp)
PCRI s
(bp)
Efficiency(M/C) h
% (A+T)'
~.-ACItR
e-G256Sx
L257Vx
V258Ax
L2591
e-G269( - )
Insertion
36
BstEll-EcoRl
(1130)
398
821
9/10
53
Deletion
30
367
790
5/10
37
262
529
2/4
53
288
40
555
371
515
3/5
33
24
365
377
377
377
322
322
720
732
732
732
3/4
6/8
2/4
2/4
2/4
2/4
52
48
?-ACkR
ot.AChR
3,-$257Gx
V258Lx
A259Vx
I260L
~-G270( - )
y-X268Q
Insertion
36
Deletion
Point
30
25
0t-S250G
0t-S246G
0t-S246A
0~-$246V
0t-T264A
0t-T264V
Point
Point
Point
Point
Point
Point
27
27
27
27
27
27
BstXI-EcoRl
(1140)
Nhel-$auI
(1700)
678
678
48
48
41
44
a cDNA used in mutagenesis (see section a).
b Name of the mutation.
© Type of the mutation.
d Size of the oligo.
e RFr (restriction fragment) of the cDNAs used in the second amplification.
r Overlap of the RFr and the first amplification product (PCRt).
s Size of the first amplification product (PCRI).
h Efficiency of mutagenesis: identified mutants (M) among analysed clones (C).
i Yo (A + T) of the oligo.
and the conditions recommended by the supplier. The first
PCR was performed in 100/d using 10 ng of supercoiled
plasmid carrying the ~-AChR, 1 pM ~p l, 1/~M mp, 200 p M
of each dNTP, reaction buffer and 2.5 units of AmpliTaq
DNA polymerase. Each cycle consisted of a l-min step at
94°C, 2 min of annealing at 37°C arid a synthesis step for
3 min at 72 ° C. After 25 cycles the DNA product (Fig. lb)
was purified on a 1 ~o agarose gel and isolated using the
liquid nitrogen elution technique (Koenen, 1989). The
second PCR was started with 125 rig purified first PCR
product, 10 ng ofa 1700-bp fragment produced by digestion
of the o~-AChRclone using Nhel + Saul, 1/~M of both apl
and 0cp2, 200 #M of each dNTP, reaction buffer and 2.5
units AmpliTaq polymerase in 100/d. The mutated DNA
fragment amplified by 0~pl and ~p2 was digested with
Ball + BstXI, purified on a 1 ~o agarose gel and ligated into
the dephosphorylated ~-AChR clone. Replacement of the
wt fragment by a restriction fragment carrying the mutation
reduced the expenditure in DNA sequencing work, as only
the subcloned DNA fragment had to be sequenced. Furthermore isolation era restriction fragment from the second
PCR product overcomes possible artefacts (small de-
letions) generated at the ends of the synthesised fragments
as described by Hemsley et al. (1989). The DNA fragment
produced during the mutagenesis has to be sequenced to
recognise additional alterations, introduced by PCR. In our
experiments no misiacorporation could be detected within
9600 bp. Differences in the efficiency in mutagenesis (50~0
to 100~o) using the 0~-,e- and ?-AChRhave to be noticed
(Table I). We are unable to explain the different efficiencies
and none of the p~ameters listed in Table I shows any
correlation to efficiency. In five out of eleven experiments
the efficiency ranged from 60 to 100% (Table I). Singletrack sequencing analysis was initially made of ten subclones, but reduced when four subclones were found to be
sufficient to identify all mutants.
(b) Identification of the mutant clones
Plasmid DNA isolated from small cultures (IshHorowicz and Burke, 1981) were redissolved in 50 #! water,
and 25 #1 were used for purification on a 1% agarose gel.
Supercoiled DNA was eluted according to Koenen (1989)
and fmally redissolved in 3.5/d of water. Sequencing followed the conditions recommended by the supplier, using
146
G, G2 G3 G4
A,, % C.
mmm
.
.
.
.
~
the flanking primers amplify a mutant fragment of
1000-1500 bp containing restriction sites for reinsertion
into the cDNA. If no useful restriction sites exist, it is
possible to place the flanking primer in the polylinker present on both sides of the cloned D N A fragment.
(d) Restriction fragments used in the second PCR
In the second PCR, we mixed the product of the fn'st
amplification carrying the mutation with a restriction fragment ofthe same cDNA, purified on a 1% agarose gel. This
DNA fragment was selected: (1) to overlap in its proximal
5' region with the first PCR product allowing efficient
interstrand annealing necessary for elongation of the
mutated fragment (Fig. ld); (2) to replace a PCR amplification for minimising the regeneration of additional replication errors; (3)to contain restriction sites useful for reinsertion into the cDNA.
.~
m
ACKNOWLEDGEMENTS
m
4J~
malt
Fig. 2. Singie-track analysis of mutant clones. Only the G-specific nt
sequence reaction of four isolated plasmid DNAs (numbered 1-4) is
present in lanes Gn-G4. Lanes G,, Aw, I', and C, represent the nt
sequence of the wt DNA control. Arrows indicatethe nt positions of the
C and T replaced by G in the mutant ~-$246G. A 6-h exposure (RT) of
a 6% denaturing sequencinggel is shown. In this sequence analysis all
clones carried the mutation. In the mutagenesis of ~-$246G six out of
eight analysed plasmids carried the mutation.
the Sequenase kit version 2.0 (United States Biochemical
Corporation). We used single-track sequencing (Fig. 2) to
identify the mutant clones using a sequencing primer initiating DNA synthesis 80-100 bp upstream from the mutation.
The complete nt sequence of the mutated fragment was
obtained to recognise any misincorporation. Interestingly,
during the mutagenesis experiments we identified three
plasmids carrying alterations in the sequence homologous
to the mutant oligos. In the first plasmid one nt was deleted
5' and one inserted 3' of the 7-GIy deletion (3'-G270(-)).
The other alterations exchanged beside the point mutations
(~-$246A, ~-T264A) one nt in the 5' end of the oligo. These
replication errors can be explained by mismatches in the
oligo template complex.
(c) Flanking primers
For each cDNA we positioned the flanking primer to
permit the constru.~tion of all mutations. For the cDNAs
encoding the ~-, 3'- and e-subunit of the rat muscle AChR
We thank Dr. B. Sakmann for suggestions and helpful
discussion, Drs. F. Edwards and P. Seeburg for critical
reading the manuscript. The work was supported by the
Leibnitz-F6rderprogramm of the Deutsche Forschungsgemeinschaft to B. Sakmann.
REFERENCES
Hemsley, A., Arnheim,N,, Toney, M.D., Cortopassi, G. and Galas, DJ.:
A simple method for site-directedmutagenesisusingthe polymerase
chain reaction. Nucleic Acids Res. 17 (1989)6545-6551.
Higuchi, R., Krummel, B. and Saiki, R.K,: A general method of in vitro
preparation and specific mutagenesis of DNA fragments: study of
protein and DNA interactions. Nucleic Acids Res. 16 (1988)
7351-7367.
Ho, S.N., Hunt, H.D., Horton, R.M., PuUen,J.K. and Pease, L.R.: Sitedirected mutagenesis by overlap extension using the polymerase
chain reaction. Gene 77 (1989) 51-59.
Horton, R.M., Hunt, H.D., Ho, S.N., Pullen, J.K. and Pease, L.R.:
Engineeringhybridgeneswithoutthe use ofrestriction enzymes:gene
splicing by overlap extension. Gene 77 (1989)61-68.
Ish-Horowicz, D. and Burke, J.F.: Rapid and efficient cosmid cloning.
Nucleic Acids Res. 9 (1981) 2989-2998.
Kadowaki, H., Kadowaki,T., Wondisford, F.E. and Taylor, S.I.: Use of
the polymerasechain reactioncatalyzed by Taq DNA polymerasefor
site-specific mutagenesis.Gene 76 (1989) 161-166.
Kammann,M., Laufs,J., Schell,J. and Gronenborn, B.: Rapid insertional
mutagenesis of DNA by polymerase chain reaction (PCR). Nucleic
Acids Res. 17 (1989) 5404.
Koenen, M.: Recoveryof DNA from agarose gels using liquid nitrogen.
Trends Genet. 5 (1989) 137.
Kwok, S., Kellogg,D.E., McKinney,N., Goda, L., Spasic, D., Levenson,
C. and Sninsky,J.J." Effects of primer-~.emplatemismatches on the
polymerase chain reaction: human immune-deficiencyvirus type 1
model studies. Nucleic Acids Res. 18 (1990) 999-1005.
Melton, D.A., Krieg, P.A., Rebagliati, M.R., Maniatis, T., Zinn, K. and
Green, M.R.: Efficient in vitro synthesis of biologically active RNA
147
and RNA hybridisation probes from plasmids containing a bacteriophage SP6 promoter. Nucleic Acids Res. 12 (1984) 7035-7056.
Methfessel, C., Witzemann, V., Takahashi, T., Mishina, S., Numa, S. and
Sakmann, B.: Patch clamp measurements on Xenopus iaevis oocytes:
current through endogenous channels and implanted acetylcholine
receptor and sodium channels. Ptl0gers Arch. 407 (1986) 577-588.
Reiss, J., Krawczak, M., Schloesser, M., Wagner, M. and Cooper, D.N.:
The effect of replication errors on the mismatch analysis of PCRamplified DNA. Nucleic Acids Res. 18 (1990) 973-978.
Saiki, R.K., Scharf, SJ., Faloona, F., Mullis, K.B., Horn, G.T., Ehrlich,
H.A. and Arnheim, N.: Enzymatic amplification of/~-globin genomic
sequence and restriction site analysis for diagnosis of sickle cell
anemia. Science 230 (1985) 1350-1354.
Smith, M.: In vitro mutagenesis. Annu. Rev. Genet. 19 (1985) 423--46Z
Sommer, R. and Tautz, D.: Minimal homology requirements for
primers. Nucleic Acids Res. 17 (1989) 6749.
Tindall, K.R. and Kunkel, T.A.: Fidelity of DNA synthesis by Thennus
aquaticus DNA polymerase. Biochemistry 27 (1988) 6008-6013.
Vallette, F., Mege, E., Reis, A. and Adesnik, M.: Construction of mutant
and chimeric genes using the polymerase chain reaction. Nucleic
Acids Res. 17 (1989) 723-733.
Yon, J. and Fried, M.: Precise gene fusion by PCR. Nucleic Acids Res.
17 (1989) 4895.