ArticleFrontiers in bioengineering and biotechnology2023
TLTC, a T5 exonuclease-mediated low-temperature DNA cloning method.
Article in Frontiers in bioengineering and biotechnology, 2023. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 14 papers.
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14 citing papers in PubMed, 25 citations in OpenAlex.
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- DAPE cloning with modified primers for producing designated lengths of 3' single-stranded ends in PCR products.PloS one · 2025Article
- Development of a starch-fermenting Zymomonas mobilis strain for bioethanol production.Microbial cell factories · 2024Article
- Heterologous Expression of KetoreductaseMolecules (Basel, Switzerland) · 2024Article
- A Whole-Process Visible Strategy for the Preparation of Rhizomucor miehei Lipase with Escherichia coli Secretion Expression System and the Immobilization.Microbial cell factories · 2024Article
- Overexpression of salusin‑β downregulates adipoR1 expression to prevent fatty acid oxidation in HepG2 cells.Molecular medicine reports · 2024Article
- Circular PCR as an efficient and precise umbrella of methods for the generation of circular dsDNA with staggered nicks: Mechanism and types.Biology methods & protocols · 2024Article
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Authors and funding
8 authors at 2 institutions in 1 country.
Funding
No grant is acknowledged in the PubMed record.
Abstract
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
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