k. 1992 Oxford University Press
624 Nucleic Acids Research, Vol. 20, No. 3
A POR method for introducing mutations into cloned DNA by joining an internal primer to a tagged flanking primer Jorg Stappert, Johann Wirsching and Rolf Kemler Max-Planck-Institut fOr Immunbiologie, StObeweg 51, D-7800 Freiburg, FRG Submitted September 4, 1991 We have developed a simple, rapid method for introducing mutations into cloned and sequenced DNA. Our method allows introducing several mutations in virtually any position on either ds or ss DNA in one step. To do this we have combined extension of mutated internal primers by T4 DNA polymerase with selective amplification of the mutated DNA strand by PCR from addedon external primers. This scheme (Fig. 1) overcomes the problem of having to have suitable restriction sites near the mutated nucleotides. We introduced two new restriction sites into the full-length cDNA of the uvomorulin-gene as silent mutations, using Primer I and/or II (1). The primers are respectively 18 or 21 nt long. Each primer contains two mismatches relative to the cDNA template. A total of 6 nt exact homology to the template is included on each end to ensure proper hybridization to the template. The third primer used in the first step has a length of 42 nt and contains 24 nt at the 5'-end which are not homologous to the template. Within this homologous region a suitable restriction site for BssHll is available. The distance between Primer I and Primer A is 200 nt; Primer HI and Primer A are 560 bases apart. Step 1. The primers were annealed to ss or alkaline denatured ds template DNA in a volume of 10 M1 containing 0.1 pmol template DNA, 1O xexcess of Primers I, H and A and Annealing Buffer (10 xAnnealing Buffer is 200 mM Tris-HCI pH 7.4, 20 mM MgCl2, 500 mM NACl). In both cases, the mixture was then heated to 65°C for 3 min and allowed to cool slowly to room temperature for 30 min. Synthesis of the complementary DNA strand was done in a volume of 20 Md containing the same annealing mixture plus 2.5 U T4 DNA polymerase, 1U T4 DNA ligase and Synthesis Buffer (lOxSynthesis Buffer is 5 mM each dNTP, 10 mM ATP, 1 00 mM Tris-HCI pH 7.4, 50 mM MgCl2, 20 mM DTT). After incubating at 37°C for 90 min, we stopped the reaction by adding Stop Buffer (10 mM Tris-HCl pH 8.0, 10 mM EDTA) to it. Step 2: For amplification of the mutated DNA strand we used the external Primers Autg and B* (B* with a restriction site for EcoRI) at 10 AM with 4 Ml from the 'extension' reaction, 100 MM of each dNTP 2.5 U of Taqpolymerase in Taq-Buffer (10 xTaq-buffer is 100 mM Tris-HCI pH 8.4, 500 mM KCl, 20 mM MgCl2, 1 mg/ml gelatin). The PCR mixture was subjected to 30 cycles of amplification: 1 min at 950C, 1 min at 550C, 2.5 min at 720C. After chloroform treatment 5 Ml of the PCR mixture was analyzed on a 1% TBE Agarose-gel and EtBr stained; a strong single band of the expected 1.2 kb size was visible. The ethanol precipitate was suspended in 30 tul TE. Then 5 M1 of each purified fragment was digested with the appropriate restriction enzymes MroI and Spel to determine the efficiency of incorporation of the mutated primers. Cloning of the amplified DNA fragments, restriction digests, ,
ligation and transformation were done using accepted protocols (2). To identify positive clones bearing the newly introduced restriction sites, DNA of each of 100 clones was digested with the corresponding enzymes and electrophoresed to look for cleavage. The results are summerized in Table I. The higher efficiency of incorporation for primer II ( 560 nt from the external primer Atag versus 200 nt for primer I) suggests little problem of increasing the distance from the tag primer. DNA sequence analysis of several positive clones gave no indication for any nucleotide misincorporation by Taqpolymerase. Bacteriophage T4 DNA polymerase (3, 4) or Sequenase (5) should be used in the polymerization/extension reaction. Unlike the Klenow fragment of E. coli DNA polymerase I, neither of these enzymes is able to displace the mutagenic oligonucleotide from its template (6).
REFERENCES 1. Kemler,R., Ozawa,M. and Ringwald,M. (1989) Current Opinion in Cell Biology 1, 892-897. 2. Sambrook,J., Fritsch,E.F. and Maniatis,T. (1989) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor. 3. Nossal,N.G. (1974) J. Biol. Chem. 245, 5668-5672. 4. Kunkel,T.A. (1985) Proc. Natl. Acad. Sci. USA 82, 488-502. 5. Schena,M. (1989) United States Biocheniical Corp. 15, 23. 6. Perlack,F.J. (1990) Nucleic Acid Res. 18, 7457-7458. x
5Step 1. Extension from
_.|
Y
internal primersI
P 11 Prim
Prom A
Prim
Step 2. Selective ampli f ication of the mutated DNA strana by PCR
P
6' _ 33_ 31F -
_
_
_
_
--GENEvEMED.-
Y
r;B
_-
__== =L -dMlMM:MlMo.-,.;w _
_
_
Prim
5
Atag
Figure 1. In the first step, two or even more mutagenic primers (0, 0) and Primer A are annealed to the template. Primer A has a suitable restriction site (y) and a 5'add-on sequence which is not complementary to the template. After primer extension and ligation reaction the mutated DNA strand is selectively amplified by PCR in the 2nd step by using the two outer Primers Atag and B*. Primer Atag is identical to the 5' add-on sequence of Primer A, whereas primer B* is complementary to the other end of the wild-type DNA sequence also containing a suitable restriction site (x) to reclone the mutated fragment. Primer Incorporated
Template ss.DNA
i
SO dsDNA50
II 92 66
and II | 75 r40
Table I. The percentage of clones which were positive for Primer I and/or Primer II as assayed by looking at size and number of fragments in restriction digests. A total of 100 clones each from ds or ss templates were tested for incorporation of each Primer.
624 Nucleic Acids Research, Vol. 20, No. 3
A POR method for introducing mutations into cloned DNA by joining an internal primer to a tagged flanking primer Jorg Stappert, Johann Wirsching and Rolf Kemler Max-Planck-Institut fOr Immunbiologie, StObeweg 51, D-7800 Freiburg, FRG Submitted September 4, 1991 We have developed a simple, rapid method for introducing mutations into cloned and sequenced DNA. Our method allows introducing several mutations in virtually any position on either ds or ss DNA in one step. To do this we have combined extension of mutated internal primers by T4 DNA polymerase with selective amplification of the mutated DNA strand by PCR from addedon external primers. This scheme (Fig. 1) overcomes the problem of having to have suitable restriction sites near the mutated nucleotides. We introduced two new restriction sites into the full-length cDNA of the uvomorulin-gene as silent mutations, using Primer I and/or II (1). The primers are respectively 18 or 21 nt long. Each primer contains two mismatches relative to the cDNA template. A total of 6 nt exact homology to the template is included on each end to ensure proper hybridization to the template. The third primer used in the first step has a length of 42 nt and contains 24 nt at the 5'-end which are not homologous to the template. Within this homologous region a suitable restriction site for BssHll is available. The distance between Primer I and Primer A is 200 nt; Primer HI and Primer A are 560 bases apart. Step 1. The primers were annealed to ss or alkaline denatured ds template DNA in a volume of 10 M1 containing 0.1 pmol template DNA, 1O xexcess of Primers I, H and A and Annealing Buffer (10 xAnnealing Buffer is 200 mM Tris-HCI pH 7.4, 20 mM MgCl2, 500 mM NACl). In both cases, the mixture was then heated to 65°C for 3 min and allowed to cool slowly to room temperature for 30 min. Synthesis of the complementary DNA strand was done in a volume of 20 Md containing the same annealing mixture plus 2.5 U T4 DNA polymerase, 1U T4 DNA ligase and Synthesis Buffer (lOxSynthesis Buffer is 5 mM each dNTP, 10 mM ATP, 1 00 mM Tris-HCI pH 7.4, 50 mM MgCl2, 20 mM DTT). After incubating at 37°C for 90 min, we stopped the reaction by adding Stop Buffer (10 mM Tris-HCl pH 8.0, 10 mM EDTA) to it. Step 2: For amplification of the mutated DNA strand we used the external Primers Autg and B* (B* with a restriction site for EcoRI) at 10 AM with 4 Ml from the 'extension' reaction, 100 MM of each dNTP 2.5 U of Taqpolymerase in Taq-Buffer (10 xTaq-buffer is 100 mM Tris-HCI pH 8.4, 500 mM KCl, 20 mM MgCl2, 1 mg/ml gelatin). The PCR mixture was subjected to 30 cycles of amplification: 1 min at 950C, 1 min at 550C, 2.5 min at 720C. After chloroform treatment 5 Ml of the PCR mixture was analyzed on a 1% TBE Agarose-gel and EtBr stained; a strong single band of the expected 1.2 kb size was visible. The ethanol precipitate was suspended in 30 tul TE. Then 5 M1 of each purified fragment was digested with the appropriate restriction enzymes MroI and Spel to determine the efficiency of incorporation of the mutated primers. Cloning of the amplified DNA fragments, restriction digests, ,
ligation and transformation were done using accepted protocols (2). To identify positive clones bearing the newly introduced restriction sites, DNA of each of 100 clones was digested with the corresponding enzymes and electrophoresed to look for cleavage. The results are summerized in Table I. The higher efficiency of incorporation for primer II ( 560 nt from the external primer Atag versus 200 nt for primer I) suggests little problem of increasing the distance from the tag primer. DNA sequence analysis of several positive clones gave no indication for any nucleotide misincorporation by Taqpolymerase. Bacteriophage T4 DNA polymerase (3, 4) or Sequenase (5) should be used in the polymerization/extension reaction. Unlike the Klenow fragment of E. coli DNA polymerase I, neither of these enzymes is able to displace the mutagenic oligonucleotide from its template (6).
REFERENCES 1. Kemler,R., Ozawa,M. and Ringwald,M. (1989) Current Opinion in Cell Biology 1, 892-897. 2. Sambrook,J., Fritsch,E.F. and Maniatis,T. (1989) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor. 3. Nossal,N.G. (1974) J. Biol. Chem. 245, 5668-5672. 4. Kunkel,T.A. (1985) Proc. Natl. Acad. Sci. USA 82, 488-502. 5. Schena,M. (1989) United States Biocheniical Corp. 15, 23. 6. Perlack,F.J. (1990) Nucleic Acid Res. 18, 7457-7458. x
5Step 1. Extension from
_.|
Y
internal primersI
P 11 Prim
Prom A
Prim
Step 2. Selective ampli f ication of the mutated DNA strana by PCR
P
6' _ 33_ 31F -
_
_
_
_
--GENEvEMED.-
Y
r;B
_-
__== =L -dMlMM:MlMo.-,.;w _
_
_
Prim
5
Atag
Figure 1. In the first step, two or even more mutagenic primers (0, 0) and Primer A are annealed to the template. Primer A has a suitable restriction site (y) and a 5'add-on sequence which is not complementary to the template. After primer extension and ligation reaction the mutated DNA strand is selectively amplified by PCR in the 2nd step by using the two outer Primers Atag and B*. Primer Atag is identical to the 5' add-on sequence of Primer A, whereas primer B* is complementary to the other end of the wild-type DNA sequence also containing a suitable restriction site (x) to reclone the mutated fragment. Primer Incorporated
Template ss.DNA
i
SO dsDNA50
II 92 66
and II | 75 r40
Table I. The percentage of clones which were positive for Primer I and/or Primer II as assayed by looking at size and number of fragments in restriction digests. A total of 100 clones each from ds or ss templates were tested for incorporation of each Primer.
