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TLTC, a T5 exonuclease–mediated low-temperature DNA cloning method
Molecular cloning is used in a wide variety of biological and medical research. Here, we developed a rapid and efficient DNA-assembling method for routine laboratory work. We discovered that the cleavage speed of T5 exonuclease is approximately 3 nt/min at 0°C and hence developed a T5 exonuclease–me...
Autores principales: | , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Frontiers Media S.A.
2023
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10457141/ https://www.ncbi.nlm.nih.gov/pubmed/37635997 http://dx.doi.org/10.3389/fbioe.2023.1167534 |
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author | Yu, Fang Li, Xia Wang, Fei Liu, Yang Zhai, Chao Li, Wenqiang Ma, Lixin Chen, Wanping |
author_facet | Yu, Fang Li, Xia Wang, Fei Liu, Yang Zhai, Chao Li, Wenqiang Ma, Lixin Chen, Wanping |
author_sort | Yu, Fang |
collection | PubMed |
description | Molecular cloning is used in a wide variety of biological and medical research. Here, we developed a rapid and efficient DNA-assembling method for routine laboratory work. We discovered that the cleavage speed of T5 exonuclease is approximately 3 nt/min at 0°C and hence developed a T5 exonuclease–mediated low-temperature sequence- and ligation-independent cloning method (TLTC). Two homologous regions of 15 bp–25 bp compatible with the ends of the vector backbones were introduced into the inserts through PCR. Approximately 120 fmol of inserts and linear vectors was mixed at a molar ratio of approximately 3:1 and treated with 0.5 U of T5 exonuclease at 0°C for 5 min. Then, the mixture was transformed into Escherichia coli to generate recombinant plasmids. Single segment and multi-segments can be assembled efficiently using TLTC. For single segment, the overall cloning efficiency is above 95%. Moreover, extra nucleotides in the vectors can be removed during TLTC. In conclusion, an extremely simple and fast DNA cloning/assembling method was established in the present study. This method facilitates routine DNA cloning and synthesis of DNA fragments. |
format | Online Article Text |
id | pubmed-10457141 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2023 |
publisher | Frontiers Media S.A. |
record_format | MEDLINE/PubMed |
spelling | pubmed-104571412023-08-26 TLTC, a T5 exonuclease–mediated low-temperature DNA cloning method Yu, Fang Li, Xia Wang, Fei Liu, Yang Zhai, Chao Li, Wenqiang Ma, Lixin Chen, Wanping Front Bioeng Biotechnol Bioengineering and Biotechnology Molecular cloning is used in a wide variety of biological and medical research. Here, we developed a rapid and efficient DNA-assembling method for routine laboratory work. We discovered that the cleavage speed of T5 exonuclease is approximately 3 nt/min at 0°C and hence developed a T5 exonuclease–mediated low-temperature sequence- and ligation-independent cloning method (TLTC). Two homologous regions of 15 bp–25 bp compatible with the ends of the vector backbones were introduced into the inserts through PCR. Approximately 120 fmol of inserts and linear vectors was mixed at a molar ratio of approximately 3:1 and treated with 0.5 U of T5 exonuclease at 0°C for 5 min. Then, the mixture was transformed into Escherichia coli to generate recombinant plasmids. Single segment and multi-segments can be assembled efficiently using TLTC. For single segment, the overall cloning efficiency is above 95%. Moreover, extra nucleotides in the vectors can be removed during TLTC. In conclusion, an extremely simple and fast DNA cloning/assembling method was established in the present study. This method facilitates routine DNA cloning and synthesis of DNA fragments. Frontiers Media S.A. 2023-08-11 /pmc/articles/PMC10457141/ /pubmed/37635997 http://dx.doi.org/10.3389/fbioe.2023.1167534 Text en Copyright © 2023 Yu, Li, Wang, Liu, Zhai, Li, Ma and Chen. https://creativecommons.org/licenses/by/4.0/This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. |
spellingShingle | Bioengineering and Biotechnology Yu, Fang Li, Xia Wang, Fei Liu, Yang Zhai, Chao Li, Wenqiang Ma, Lixin Chen, Wanping TLTC, a T5 exonuclease–mediated low-temperature DNA cloning method |
title | TLTC, a T5 exonuclease–mediated low-temperature DNA cloning method |
title_full | TLTC, a T5 exonuclease–mediated low-temperature DNA cloning method |
title_fullStr | TLTC, a T5 exonuclease–mediated low-temperature DNA cloning method |
title_full_unstemmed | TLTC, a T5 exonuclease–mediated low-temperature DNA cloning method |
title_short | TLTC, a T5 exonuclease–mediated low-temperature DNA cloning method |
title_sort | tltc, a t5 exonuclease–mediated low-temperature dna cloning method |
topic | Bioengineering and Biotechnology |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10457141/ https://www.ncbi.nlm.nih.gov/pubmed/37635997 http://dx.doi.org/10.3389/fbioe.2023.1167534 |
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