PMID: 7580902Dec 1, 1994Paper

Minimal length requirement of the single-stranded tails for ligation-independent cloning (LIC) of PCR products

PCR Methods and Applications
C AslanidisG Schmitz

Abstract

The ligation-independent cloning of PCR products (LIC-PCR) is a versatile and highly efficient cloning procedure resulting in recombinant clones only. Recombinants are generated between PCR products and a PCR-amplified vector through defined complementary single-stranded (ss) ends artificially generated with T4 DNA polymerase. This procedure does not require restriction enzymes, alkaline phosphatase, or DNA ligase. The primers used for amplification contain an additional 12-nucleotide sequence at their 5' ends that is complementary in the vector- and insert-specific primers. The (3'-->5') exonuclease activity of T4 DNA polymerase is used in combination with a predetermined dNTP (dGTP for the inserts and dCTP for the vector) to specifically remove 12 nucleotides from each 3' end of the PCR fragments. Because of the complementarity of the ends that are generated, circularization can occur between vector and insert. The recombinant molecules do not require in vitro ligation for efficient bacterial transformation. To make this technique widely applicable, we have simplified the handling of the PCR fragments prior to LIC. The PCR products do not need further purification following the T4 DNA polymerase treatment. Incubation of vecto...Continue Reading

Citations

Feb 9, 2012·Applied Microbiology and Biotechnology·Tianwen WangJian Chen
Mar 10, 2010·Journal of Structural and Functional Genomics·William H EschenfeldtAndrzej Joachimiak
Jun 5, 2013·Molecular Biotechnology·Ren-Bin YangXian-En Zhang
Mar 17, 2012·Nucleic Acids Research·Jonathan Leo Schmid-BurgkYaakov Benenson
Sep 18, 2002·Nucleic Acids Research·William F DonahueKevin A Jarrell
Mar 29, 2008·BMC Biotechnology·Viola BardóczyTamás Mészáros
Sep 26, 2014·Mikrochimica Acta·Lena DanckertMarkus von Nickisch-Rosenegk
Jun 26, 2012·Protein Expression and Purification·Venuka DuraniThomas J Magliery
May 19, 2009·Journal of Bioscience and Bioengineering·Akira TachibanaToshizumi Tanabe
Dec 30, 2008·Trends in Biotechnology·Michael J CzarJean Peccoud
Jan 26, 2007·Protein Expression and Purification·Stephen D WeeksPatrick J Loll
Aug 24, 2013·Protein Expression and Purification·Tiana D WarrenDavid W Wood
Apr 14, 2016·PloS One·Roger M BenoitManfred Auer
Apr 27, 2017·Protein Engineering, Design & Selection : PEDS·Maxim RossmannMarko Hyvönen
Feb 13, 2007·Nature Methods·Mamie Z Li, Stephen J Elledge
Jan 24, 2019·PloS One·Ruhu Qi, Gottfried Otting
Mar 14, 2003·Nature·Eric PhizickyStanley Fields
Mar 1, 2019·European Journal of Psychotraumatology·Jurriaan JacobsMichel L A Dückers
Jul 30, 2017·Scientific Reports·Franziska M HeydenreichDmitry B Veprintsev
Aug 17, 2006·Critical Reviews in Biochemistry and Molecular Biology·Eric M Phizicky, Elizabeth J Grayhack
Jan 5, 1999·Applied and Environmental Microbiology·F von WintzingerodeU B Göbel
Jun 7, 2021·Experimental Cell Research·Khadijeh BarzamanLeila Farahmand
Nov 26, 2010·BioTechniques·Jie YangQingming Luo

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