Phosphorothioate-based ligase-independent gene cloning (PLICing): An enzyme-free and sequence-independent cloning method

Analytical Biochemistry
Milan BlanusaUlrich Schwaneberg

Abstract

Many ligase-independent cloning methods have been developed to overcome problems of standard restriction cloning such as low transformation efficiency and high background of vector with no insert. Most of these methods are still enzyme based, require time-consuming incubation and multiple purification steps, and/or might have a low robustness in handling. Thus, with the aim to establish a robust enzyme/ligase-free method, we developed the phosphorothioate-based ligase-independent gene cloning (PLICing) method, which is based on a chemical cleavage reaction of phosphorothioate bonds in an iodine/ethanol solution. After optimization of polymerase chain reaction (PCR) and DNA cleavage conditions, PLICing performs competitively with all commercialized methods in terms of handling and transformation efficiency. In addition, PLICing is absolutely sequence independent and surpasses other concepts regarding cloning efficiency given that none of the 240 analyzed clones showed any religation event for three different model genes. A developed fast PLICing protocol does not require any purification step and can be completed within 10 min. Due to its robustness, reliability, and simplicity, PLICing should prove to be a true alternative to o...Continue Reading

References

Aug 25, 1992·Nucleic Acids Research·S KaluzK B Reid
Nov 1, 1991·PCR Methods and Applications·P E NissonP C Watkins
Jan 1, 1991·Methods in Enzymology·D HanahanF R Bloom
Oct 25, 1990·Nucleic Acids Research·C Aslanidis, P J de Jong
Mar 25, 1995·Nucleic Acids Research·M Zhou, Z Hatahet
Jun 7, 1994·Proceedings of the National Academy of Sciences of the United States of America·S ChengR Higuchi
Jun 1, 1997·Trends in Biochemical Sciences·P N Hengen
Oct 10, 1997·Nucleic Acids Research·C Li, R M Evans

❮ Previous
Next ❯

Citations

Dec 14, 2011·Applied Microbiology and Biotechnology·Amol V ShivangeUlrich Schwaneberg
Oct 15, 2013·Molecular Biotechnology·Tsvetan KardashlievUlrich Schwaneberg
May 28, 2013·BioMed Research International·Philippe JeandetEric Courot
Apr 4, 2012·Expert Opinion on Drug Discovery·Billyana TsvetanovaFederico Katzen
Dec 12, 2012·Current Opinion in Biotechnology·Lars M Blank, Birgitta E Ebert
Sep 26, 2015·Applied Microbiology and Biotechnology·Amol V ShivangeUlrich Schwaneberg
Dec 20, 2014·Chemistry & Biology·Christian PitzlerUlrich Schwaneberg
May 8, 2013·The FEBS Journal·Anna J RuffUlrich Schwaneberg
Aug 12, 2015·ACS Synthetic Biology·Ana Mafalda CavaleiroMorten H H Nørholm
Sep 22, 2015·Journal of Biological Engineering·Pawel Jajesniak, Tuck Seng Wong
Jun 7, 2014·Biotechnology and Bioengineering·Jing ZhaoUlrich Schwaneberg
Sep 10, 2013·Biotechnology Advances·Kang Lan Tee, Tuck Seng Wong
Jun 14, 2014·Journal of Biotechnology·Christina A MüllerUlrich Schwaneberg
Jun 13, 2015·Microbial Cell Factories·Philana V van Summeren-WesenhagenJan Marienhagen
May 8, 2015·Applied Microbiology and Biotechnology·Nina LülsdorfUlrich Schwaneberg
Jul 16, 2013·Bioengineered·Philana V van Summeren-Wesenhagen, Jan Marienhagen
Jun 13, 2012·Journal of Biotechnology·Jan Marienhagen, Michael Bott
Aug 12, 2014·Applied Microbiology and Biotechnology·Feng ChengUlrich Schwaneberg
Oct 19, 2016·Biotechnology Journal·Jianhua YangUlrich Schwaneberg
Dec 23, 2016·Protein Engineering, Design & Selection : PEDS·Ketaki D BelsareUlrich Schwaneberg
Jan 18, 2017·Journal of the American Chemical Society·Arun ShivalingamTom Brown
May 18, 2016·Scientific Reports·Georgette KörferUlrich Schwaneberg
Apr 14, 2016·PloS One·Roger M BenoitManfred Auer
Jun 7, 2014·ACS Synthetic Biology·Morten T NielsenMorten H H Nørholm
Apr 25, 2015·Chemical Communications : Chem Comm·Nina LülsdorfUlrich Schwaneberg
Dec 31, 2017·Biotechnology and Bioengineering·Shohana IslamUlrich Schwaneberg
Apr 1, 2018·Chembiochem : a European Journal of Chemical Biology·John MandaweUlrich Schwaneberg
Oct 11, 2017·Chemistry : a European Journal·Alexander DennigUlrich Schwaneberg
Mar 13, 2012·Chembiochem : a European Journal of Chemical Biology·Zhenwei LiUlrich Schwaneberg
Sep 1, 2018·FEMS Microbiology Letters·Kenji OkanoAkihiko Kondo
Dec 13, 2016·Applied and Environmental Microbiology·Christina Andrea MüllerGabriele Berg
Dec 12, 2018·Chembiochem : a European Journal of Chemical Biology·Jennifer AschenbrennerJan Marienhagen
Apr 16, 2019·Biotechnology and Bioengineering·Lina ApitiusUlrich Schwaneberg
Sep 25, 2019·Biotechnology and Bioengineering·Sarah DedischUlrich Schwaneberg
Mar 17, 2012·Beilstein Journal of Organic Chemistry·Svenja StaudtHarald Gröger
Oct 25, 2017·Biotechnology and Bioengineering·Kristin RübsamUlrich Schwaneberg
Mar 24, 2018·Journal of Biological Engineering·José-Aníbal Mora-Villalobos, An-Ping Zeng

❮ Previous
Next ❯

Related Concepts

Related Feeds

Aminoglycosides

Aminoglycoside is a medicinal and bacteriologic category of traditional Gram-negative antibacterial medications that inhibit protein synthesis and contain as a portion of the molecule an amino-modified glycoside. Discover the latest research on aminoglycoside here.

Aminoglycosides (ASM)

Aminoglycoside is a medicinal and bacteriologic category of traditional Gram-negative antibacterial medications that inhibit protein synthesis and contain as a portion of the molecule an amino-modified glycoside. Discover the latest research on aminoglycoside here.