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.
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
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