Rapid hierarchical assembly of medium-size DNA cassettes

Synthetic biology applications call for efficient methods to generate large gene cassettes that encode complex gene circuits in order to avoid simultaneous delivery of multiple plasmids encoding individual genes. Multiple methods have been proposed to achieve this goal. Here, we describe a novel protocol that allows one-step cloning of up to four gene-size DNA fragments, followed by a second assembly of these concatenated sequences into large circular DNA. The protocols described here comprise a simple, cheap and fast solution for routine construction of cassettes with up to 10 gene-size components.

[1]  Daniel V Santi,et al.  Total synthesis of multi-kilobase DNA sequences from oligonucleotides , 2007, Nature Protocols.

[2]  Gert Schmitz,et al.  Minimal length requirement of the single-stranded tails for ligation-independent cloning (LIC) of PCR products. , 1994, PCR methods and applications.

[3]  H. Brenner,et al.  Gene transfer into individual muscle fibers and conditional gene expression in living animals , 2000, Cell and Tissue Research.

[4]  R. Stewart,et al.  Human Induced Pluripotent Stem Cells Free of Vector and Transgene Sequences , 2009, Science.

[5]  P. D. de Jong,et al.  Ligation-independent cloning of PCR products (LIC-PCR). , 1990, Nucleic acids research.

[6]  Fernando Geu-Flores,et al.  USER fusion: a rapid and efficient method for simultaneous fusion and cloning of multiple PCR products , 2007, Nucleic acids research.

[7]  Zhen Xie,et al.  Rationally-designed logic integration of regulatory signals in mammalian cells , 2010, Nature nanotechnology.

[8]  J. Yates,et al.  Stable replication of plasmids derived from Epstein–Barr virus in various mammalian cells , 1985, Nature.

[9]  Timothy B. Stockwell,et al.  Complete Chemical Synthesis, Assembly, and Cloning of a Mycoplasma genitalium Genome , 2008, Science.

[10]  J Craig Venter,et al.  One-step assembly in yeast of 25 overlapping DNA fragments to form a complete synthetic Mycoplasma genitalium genome , 2008, Proceedings of the National Academy of Sciences.

[11]  D. G. Gibson,et al.  Enzymatic assembly of DNA molecules up to several hundred kilobases , 2009, Nature Methods.

[12]  J. Rossant,et al.  Isolation of human iPS cells using EOS lentiviral vectors to select for pluripotency , 2009, Nature Methods.

[13]  A. Schepers,et al.  The Affinity of EBNA1 for Its Origin of DNA Synthesis Is a Determinant of the Origin's Replicative Efficiency , 2008, Journal of Virology.

[14]  W. Donahue,et al.  Rapid gene cloning using terminator primers and modular vectors. , 2002, Nucleic acids research.

[15]  S. Cole,et al.  Sequences Human Induced Pluripotent Stem Cells Free of Vector and Transgene , 2012 .

[16]  Sarah J Kodumal,et al.  Total synthesis of long DNA sequences: synthesis of a contiguous 32-kb polyketide synthase gene cluster. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[17]  T. Ichisaka,et al.  Induction of Pluripotent Stem Cells from Adult Human Fibroblasts by Defined Factors , 2007, Cell.

[18]  R. Weiss,et al.  A universal RNAi-based logic evaluator that operates in mammalian cells , 2007, Nature Biotechnology.

[19]  M. Kimmel,et al.  Conflict of interest statement. None declared. , 2010 .

[20]  Shulan Tian,et al.  Induced Pluripotent Stem Cell Lines Derived from Human Somatic Cells , 2007, Science.

[21]  K. Woltjen,et al.  Virus free induction of pluripotency and subsequent excision of reprogramming factors , 2009, Nature.