Optimizing T4 DNA polymerase conditions enhances the efficiency of one-step sequence- and ligation-independent cloning
Previously, we developed a one-step sequence- and ligation-independent cloning (SLIC) method that is simple, fast, and cost-effective. However, although one-step SLIC generally works well, its cloning efficiency is occasionally poor, potentially due to formation of stable secondary structures within...
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| Format: | Article |
| Language: | English |
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Taylor & Francis Group
2017-09-01
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| Series: | BioTechniques |
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| Online Access: | https://www.future-science.com/doi/10.2144/000114588 |
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| author | Mohammad Nazrul Islam Kyeong Won Lee Hyung-Soon Yim Seong Hyuk Lee Hae Chang Jung Jung-Hyun Lee Jae-Yeon Jeong |
| author_facet | Mohammad Nazrul Islam Kyeong Won Lee Hyung-Soon Yim Seong Hyuk Lee Hae Chang Jung Jung-Hyun Lee Jae-Yeon Jeong |
| author_sort | Mohammad Nazrul Islam |
| collection | DOAJ |
| description | Previously, we developed a one-step sequence- and ligation-independent cloning (SLIC) method that is simple, fast, and cost-effective. However, although one-step SLIC generally works well, its cloning efficiency is occasionally poor, potentially due to formation of stable secondary structures within the single-stranded DNA (ssDNA) region generated by T4 DNA polymerase during the 2.5 min treatment at room temperature. To overcome this problem, we developed a modified thermo-regulated one-step SLIC approach by testing shorter T4 DNA polymerase treatment durations (5 s–2.5 min) over a wide range of temperatures (25–75°C). The highest cloning efficiency resulted when inserts with homology lengths <20 bases were treated with T4 DNA polymerase for 30 s at 50°C. This briefer T4 polymerase treatment at a higher temperature helps increase cloning efficiency for inserts with strong secondary structures at their ends, increasing the utility of one-step SLIC for the cloning of short fragments. |
| format | Article |
| id | doaj-art-89f2f455697d4d41b6f270c1b1741fa4 |
| institution | OA Journals |
| issn | 0736-6205 1940-9818 |
| language | English |
| publishDate | 2017-09-01 |
| publisher | Taylor & Francis Group |
| record_format | Article |
| series | BioTechniques |
| spelling | doaj-art-89f2f455697d4d41b6f270c1b1741fa42025-08-20T02:25:58ZengTaylor & Francis GroupBioTechniques0736-62051940-98182017-09-0163312513010.2144/000114588Optimizing T4 DNA polymerase conditions enhances the efficiency of one-step sequence- and ligation-independent cloningMohammad Nazrul Islam0Kyeong Won Lee1Hyung-Soon Yim2Seong Hyuk Lee3Hae Chang Jung4Jung-Hyun Lee5Jae-Yeon Jeong61Marine Biotechnology Research Center, Korea Institute of Ocean Science and Technology, Ansan, Republic of Korea1Marine Biotechnology Research Center, Korea Institute of Ocean Science and Technology, Ansan, Republic of Korea1Marine Biotechnology Research Center, Korea Institute of Ocean Science and Technology, Ansan, Republic of Korea1Marine Biotechnology Research Center, Korea Institute of Ocean Science and Technology, Ansan, Republic of Korea1Marine Biotechnology Research Center, Korea Institute of Ocean Science and Technology, Ansan, Republic of Korea1Marine Biotechnology Research Center, Korea Institute of Ocean Science and Technology, Ansan, Republic of Korea1Marine Biotechnology Research Center, Korea Institute of Ocean Science and Technology, Ansan, Republic of KoreaPreviously, we developed a one-step sequence- and ligation-independent cloning (SLIC) method that is simple, fast, and cost-effective. However, although one-step SLIC generally works well, its cloning efficiency is occasionally poor, potentially due to formation of stable secondary structures within the single-stranded DNA (ssDNA) region generated by T4 DNA polymerase during the 2.5 min treatment at room temperature. To overcome this problem, we developed a modified thermo-regulated one-step SLIC approach by testing shorter T4 DNA polymerase treatment durations (5 s–2.5 min) over a wide range of temperatures (25–75°C). The highest cloning efficiency resulted when inserts with homology lengths <20 bases were treated with T4 DNA polymerase for 30 s at 50°C. This briefer T4 polymerase treatment at a higher temperature helps increase cloning efficiency for inserts with strong secondary structures at their ends, increasing the utility of one-step SLIC for the cloning of short fragments.https://www.future-science.com/doi/10.2144/000114588seamless cloningSLICT4 DNA polymerase |
| spellingShingle | Mohammad Nazrul Islam Kyeong Won Lee Hyung-Soon Yim Seong Hyuk Lee Hae Chang Jung Jung-Hyun Lee Jae-Yeon Jeong Optimizing T4 DNA polymerase conditions enhances the efficiency of one-step sequence- and ligation-independent cloning BioTechniques seamless cloning SLIC T4 DNA polymerase |
| title | Optimizing T4 DNA polymerase conditions enhances the efficiency of one-step sequence- and ligation-independent cloning |
| title_full | Optimizing T4 DNA polymerase conditions enhances the efficiency of one-step sequence- and ligation-independent cloning |
| title_fullStr | Optimizing T4 DNA polymerase conditions enhances the efficiency of one-step sequence- and ligation-independent cloning |
| title_full_unstemmed | Optimizing T4 DNA polymerase conditions enhances the efficiency of one-step sequence- and ligation-independent cloning |
| title_short | Optimizing T4 DNA polymerase conditions enhances the efficiency of one-step sequence- and ligation-independent cloning |
| title_sort | optimizing t4 dna polymerase conditions enhances the efficiency of one step sequence and ligation independent cloning |
| topic | seamless cloning SLIC T4 DNA polymerase |
| url | https://www.future-science.com/doi/10.2144/000114588 |
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