Optimizing T4 DNA polymerase conditions enhances the efficiency of one-step sequence- and ligation-independent cloning SCIE SCOPUS
DC Field | Value | Language |
---|---|---|
dc.contributor.author | Islam, Mohammad Nazrul | - |
dc.contributor.author | Lee, Kyeong Won | - |
dc.contributor.author | Yim, Hyung-Soon | - |
dc.contributor.author | Lee, Seong Hyuk | - |
dc.contributor.author | Jung, Hae Chang | - |
dc.contributor.author | Lee, Jung-Hyun | - |
dc.contributor.author | Jeong, Jae-Yeon | - |
dc.date.accessioned | 2020-04-16T10:25:05Z | - |
dc.date.available | 2020-04-16T10:25:05Z | - |
dc.date.created | 2020-01-28 | - |
dc.date.issued | 2017-09 | - |
dc.identifier.issn | 0736-6205 | - |
dc.identifier.uri | https://sciwatch.kiost.ac.kr/handle/2020.kiost/1162 | - |
dc.description.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. | - |
dc.description.uri | 1 | - |
dc.language | English | - |
dc.publisher | BIOTECHNIQUES OFFICE | - |
dc.subject | PCR | - |
dc.title | Optimizing T4 DNA polymerase conditions enhances the efficiency of one-step sequence- and ligation-independent cloning | - |
dc.type | Article | - |
dc.citation.endPage | 130 | - |
dc.citation.startPage | 125 | - |
dc.citation.title | BIOTECHNIQUES | - |
dc.citation.volume | 63 | - |
dc.citation.number | 3 | - |
dc.contributor.alternativeName | Mohammad Nazrul Islam | - |
dc.contributor.alternativeName | 이경원 | - |
dc.contributor.alternativeName | 임형순 | - |
dc.contributor.alternativeName | 이성혁 | - |
dc.contributor.alternativeName | 정해창 | - |
dc.contributor.alternativeName | 이정현 | - |
dc.identifier.bibliographicCitation | BIOTECHNIQUES, v.63, no.3, pp.125 - 130 | - |
dc.identifier.doi | 10.2144/000114588 | - |
dc.identifier.scopusid | 2-s2.0-85029772488 | - |
dc.identifier.wosid | 000410642400007 | - |
dc.type.docType | Article | - |
dc.description.journalClass | 1 | - |
dc.subject.keywordPlus | PCR | - |
dc.subject.keywordAuthor | seamless cloning | - |
dc.subject.keywordAuthor | SLIC | - |
dc.subject.keywordAuthor | T4 DNA polymerase | - |
dc.relation.journalWebOfScienceCategory | Biochemical Research Methods | - |
dc.relation.journalWebOfScienceCategory | Biochemistry & Molecular Biology | - |
dc.description.journalRegisteredClass | scie | - |
dc.description.journalRegisteredClass | scopus | - |
dc.relation.journalResearchArea | Biochemistry & Molecular Biology | - |