Optimizing T4 DNA polymerase conditions enhances the efficiency of one-step sequence- and ligation-independent cloning SCIE SCOPUS

Cited 5 time in WEB OF SCIENCE Cited 4 time in Scopus
Title
Optimizing T4 DNA polymerase conditions enhances the efficiency of one-step sequence- and ligation-independent cloning
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)Jung, Hae Chang(정해창)Lee, Jung Hyun(이정현)
Publication Year
2017-09
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.
ISSN
0736-6205
URI
https://sciwatch.kiost.ac.kr/handle/2020.kiost/1162
DOI
10.2144/000114588
Bibliographic Citation
BIOTECHNIQUES, v.63, no.3, pp.125 - 130, 2017
Publisher
BIOTECHNIQUES OFFICE
Subject
PCR
Keywords
seamless cloning; SLIC; T4 DNA polymerase
Type
Article
Language
English
Document Type
Article
Publisher
BIOTECHNIQUES OFFICE
Related Researcher
Research Interests

marine biotechnology,molecular microbiology,해양생명공학,분자미생물학

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