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PLoS One. 2014 Dec 31;9(12):e115318. doi: 10.1371/journal.pone.0115318. eCollection 2014.

Hot Fusion: an efficient method to clone multiple DNA fragments as well as inverted repeats without ligase.

Author information

1
Biotechnology, Monsanto Company, St. Louis, Missouri, United States of America.

Abstract

Molecular cloning is utilized in nearly every facet of biological and medical research. We have developed a method, termed Hot Fusion, to efficiently clone one or multiple DNA fragments into plasmid vectors without the use of ligase. The method is directional, produces seamless junctions and is not dependent on the availability of restriction sites for inserts. Fragments are assembled based on shared homology regions of 17-30 bp at the junctions, which greatly simplifies the construct design. Hot Fusion is carried out in a one-step, single tube reaction at 50 °C for one hour followed by cooling to room temperature. In addition to its utility for multi-fragment assembly Hot Fusion provides a highly efficient method for cloning DNA fragments containing inverted repeats for applications such as RNAi. The overall cloning efficiency is in the order of 90-95%.

PMID:
25551825
PMCID:
PMC4281135
DOI:
10.1371/journal.pone.0115318
[Indexed for MEDLINE]
Free PMC Article

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