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Rapid and efficient site-specific mutagenesis without phenotypic selection.
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| Zeitschriftentitel: | Proceedings of the National Academy of Sciences |
|---|---|
| Personen und Körperschaften: | |
| In: | Proceedings of the National Academy of Sciences, 82, 1985, 2, S. 488-492 |
| Format: | E-Article |
| Sprache: | Englisch |
| veröffentlicht: |
Proceedings of the National Academy of Sciences
|
| Schlagwörter: |
| author_facet |
Kunkel, T A Kunkel, T A |
|---|---|
| author |
Kunkel, T A |
| spellingShingle |
Kunkel, T A Proceedings of the National Academy of Sciences Rapid and efficient site-specific mutagenesis without phenotypic selection. Multidisciplinary |
| author_sort |
kunkel, t a |
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Kunkel, T A 0027-8424 1091-6490 Proceedings of the National Academy of Sciences Multidisciplinary http://dx.doi.org/10.1073/pnas.82.2.488 <jats:p>Several single-base substitution mutations have been introduced into the lacZ alpha gene in cloning vector M13mp2, at 40-60% efficiency, in a rapid procedure requiring only transfection of the unfractionated products of standard in vitro mutagenesis reactions. Two simple additional treatments of the DNA, before transfection, produce a site-specific mutation frequency approaching 100%. The approach is applicable to phenotypically silent mutations in addition to those that can be selected. The high efficiency, approximately equal to 10-fold greater than that observed using current methods without enrichment procedures, is obtained by using a DNA template containing several uracil residues in place of thymine. This template has normal coding potential for the in vitro reactions typical of site-directed mutagenesis protocols but is not biologically active upon transfection into a wild-type (i.e., ung+) Escherichia coli host cell. Expression of the desired change, present in the newly synthesized non-uracil-containing covalently closed circular complementary strand, is thus strongly favored. The procedure has been applied to mutations introduced via both oligonucleotides and error-prone polymerization. In addition to its utility in changing DNA sequences, this approach can potentially be used to examine the biological consequences of specific lesions placed at defined positions within a gene.</jats:p> Rapid and efficient site-specific mutagenesis without phenotypic selection. Proceedings of the National Academy of Sciences |
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| title |
Rapid and efficient site-specific mutagenesis without phenotypic selection. |
| title_unstemmed |
Rapid and efficient site-specific mutagenesis without phenotypic selection. |
| title_full |
Rapid and efficient site-specific mutagenesis without phenotypic selection. |
| title_fullStr |
Rapid and efficient site-specific mutagenesis without phenotypic selection. |
| title_full_unstemmed |
Rapid and efficient site-specific mutagenesis without phenotypic selection. |
| title_short |
Rapid and efficient site-specific mutagenesis without phenotypic selection. |
| title_sort |
rapid and efficient site-specific mutagenesis without phenotypic selection. |
| topic |
Multidisciplinary |
| url |
http://dx.doi.org/10.1073/pnas.82.2.488 |
| publishDate |
1985 |
| physical |
488-492 |
| description |
<jats:p>Several single-base substitution mutations have been introduced into the lacZ alpha gene in cloning vector M13mp2, at 40-60% efficiency, in a rapid procedure requiring only transfection of the unfractionated products of standard in vitro mutagenesis reactions. Two simple additional treatments of the DNA, before transfection, produce a site-specific mutation frequency approaching 100%. The approach is applicable to phenotypically silent mutations in addition to those that can be selected. The high efficiency, approximately equal to 10-fold greater than that observed using current methods without enrichment procedures, is obtained by using a DNA template containing several uracil residues in place of thymine. This template has normal coding potential for the in vitro reactions typical of site-directed mutagenesis protocols but is not biologically active upon transfection into a wild-type (i.e., ung+) Escherichia coli host cell. Expression of the desired change, present in the newly synthesized non-uracil-containing covalently closed circular complementary strand, is thus strongly favored. The procedure has been applied to mutations introduced via both oligonucleotides and error-prone polymerization. In addition to its utility in changing DNA sequences, this approach can potentially be used to examine the biological consequences of specific lesions placed at defined positions within a gene.</jats:p> |
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| description | <jats:p>Several single-base substitution mutations have been introduced into the lacZ alpha gene in cloning vector M13mp2, at 40-60% efficiency, in a rapid procedure requiring only transfection of the unfractionated products of standard in vitro mutagenesis reactions. Two simple additional treatments of the DNA, before transfection, produce a site-specific mutation frequency approaching 100%. The approach is applicable to phenotypically silent mutations in addition to those that can be selected. The high efficiency, approximately equal to 10-fold greater than that observed using current methods without enrichment procedures, is obtained by using a DNA template containing several uracil residues in place of thymine. This template has normal coding potential for the in vitro reactions typical of site-directed mutagenesis protocols but is not biologically active upon transfection into a wild-type (i.e., ung+) Escherichia coli host cell. Expression of the desired change, present in the newly synthesized non-uracil-containing covalently closed circular complementary strand, is thus strongly favored. The procedure has been applied to mutations introduced via both oligonucleotides and error-prone polymerization. In addition to its utility in changing DNA sequences, this approach can potentially be used to examine the biological consequences of specific lesions placed at defined positions within a gene.</jats:p> |
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| series | Proceedings of the National Academy of Sciences |
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| spelling | Kunkel, T A 0027-8424 1091-6490 Proceedings of the National Academy of Sciences Multidisciplinary http://dx.doi.org/10.1073/pnas.82.2.488 <jats:p>Several single-base substitution mutations have been introduced into the lacZ alpha gene in cloning vector M13mp2, at 40-60% efficiency, in a rapid procedure requiring only transfection of the unfractionated products of standard in vitro mutagenesis reactions. Two simple additional treatments of the DNA, before transfection, produce a site-specific mutation frequency approaching 100%. The approach is applicable to phenotypically silent mutations in addition to those that can be selected. The high efficiency, approximately equal to 10-fold greater than that observed using current methods without enrichment procedures, is obtained by using a DNA template containing several uracil residues in place of thymine. This template has normal coding potential for the in vitro reactions typical of site-directed mutagenesis protocols but is not biologically active upon transfection into a wild-type (i.e., ung+) Escherichia coli host cell. Expression of the desired change, present in the newly synthesized non-uracil-containing covalently closed circular complementary strand, is thus strongly favored. The procedure has been applied to mutations introduced via both oligonucleotides and error-prone polymerization. In addition to its utility in changing DNA sequences, this approach can potentially be used to examine the biological consequences of specific lesions placed at defined positions within a gene.</jats:p> Rapid and efficient site-specific mutagenesis without phenotypic selection. Proceedings of the National Academy of Sciences |
| spellingShingle | Kunkel, T A, Proceedings of the National Academy of Sciences, Rapid and efficient site-specific mutagenesis without phenotypic selection., Multidisciplinary |
| title | Rapid and efficient site-specific mutagenesis without phenotypic selection. |
| title_full | Rapid and efficient site-specific mutagenesis without phenotypic selection. |
| title_fullStr | Rapid and efficient site-specific mutagenesis without phenotypic selection. |
| title_full_unstemmed | Rapid and efficient site-specific mutagenesis without phenotypic selection. |
| title_short | Rapid and efficient site-specific mutagenesis without phenotypic selection. |
| title_sort | rapid and efficient site-specific mutagenesis without phenotypic selection. |
| title_unstemmed | Rapid and efficient site-specific mutagenesis without phenotypic selection. |
| topic | Multidisciplinary |
| url | http://dx.doi.org/10.1073/pnas.82.2.488 |