Optimizing T4 DNA polymerase conditions enhances the efficiency of one-step sequence- and ligation-independent cloning
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Title
- Optimizing T4 DNA polymerase conditions enhances the efficiency of one-step sequence- and ligation-independent cloning
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Author(s)
- Islam, Mohammad Nazrul; Lee, Kyeong Won; Yim, Hyung-Soon; Lee, Seong Hyuk; Jung, Hae Chang; Lee, Jung-Hyun; Jeong, Jae-Yeon
- KIOST Author(s)
- Lee, Kyeong Won(이경원); Yim, Hyung Soon(임형순); Lee, Seong Hyuk(이성혁); Lee, Jung Hyun(이정현)
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Alternative Author(s)
- Mohammad Nazrul Islam; 이경원; 임형순; 이성혁; 정해창; 이정현
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Publication Year
- 2017-09
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Abstract
- 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 degrees C). The highest cloning efficiency resulted when inserts with homology lengths <20 bases were treated with T4 DNA polymerase for 30 s at 50 degrees 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.
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ISSN
- 0736-6205
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URI
- https://sciwatch.kiost.ac.kr/handle/2020.kiost/1162
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DOI
- 10.2144/000114588
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Bibliographic Citation
- BIOTECHNIQUES, v.63, no.3, pp.125 - 130, 2017
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Publisher
- BIOTECHNIQUES OFFICE
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Subject
- PCR
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Keywords
- seamless cloning; SLIC; T4 DNA polymerase
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Type
- Article
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Language
- English
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Document Type
- Article
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