Abstract
Precise DNA manipulation is a key enabling technology for synthetic biology. Approaches based on restriction digestion are often limited by the presence of certain restriction enzyme recognition sites. Recent development of restriction-free cloning approaches has greatly enhanced the flexibility and speed of molecular cloning. Most restriction-free cloning methods focus on DNA assembly. Much less work has been dedicated towards DNA removal. Here we introduce a protocol that allows simultaneous removal of multiple DNA segments from a plasmid using polymerase chain reactions (PCR). Our approach will be beneficial to applications in multiple sites mutagenesis, DNA library construction, genetic and protein engineering, and synthetic biology.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Purnick PEM, Weiss R (2009) The second wave of synthetic biology: from modules to systems. Nat Rev Mol Cell Biol 10(6):410–422. doi:10.1038/nrm2698
Khalil AS, Collins JJ (2010) Synthetic biology: applications come of age. Nat Rev Genet 11(5):367–379. doi:10.1038/nrg2775
Lale R, Valla S (2014) DNA cloning and assembly methods. Springer, New York, NY. doi:10.1007/978-1-62703-764-8
Bryksin AV, Matsumura I (2010) Overlap extension PCR cloning: a simple and reliable way to create recombinant plasmids. Biotechniques 48(6):463–465. doi:10.2144/000113418
Erijman A, Dantes A, Bernheim R, Shifman JM, Peleg Y (2011) Transfer-PCR (TPCR): a highway for DNA cloning and protein engineering. J Struct Biol 175(2):171–177. doi:10.1016/j.jsb.2011.04.005
Zuo PJ, Rabie ABM (2010) One-step DNA fragment assembly and circularization for gene cloning. Curr Issues Mol Biol 12:11–16
van den Ent F, Lowe J (2006) RF cloning: a restriction-free method for inserting target genes into plasmids. J Biochem Biophys Methods 67(1):67–74. doi:10.1016/j.jbbm.2005.12.008
Quan JY, Tian JD (2009) Circular polymerase extension cloning of complex gene libraries and pathways. PLoS One 4(7):6441. doi:10.1371/Journal.Pone.0006441
Zhang Y, Werling U, Edelmann W (2012) SLiCE: a novel bacterial cell extract-based DNA cloning method. Nucleic Acids Res 40(8), e55. doi:10.1093/nar/gkr1288
You C, Zhang XZ, Zhang YH (2012) Simple cloning via direct transformation of PCR product (DNA Multimer) to Escherichia coli and Bacillus subtilis. Appl Environ Microbiol 78(5):1593–1595. doi:10.1128/AEM.07105-11
Aslanidis C, Dejong PJ (1990) Ligation-independent cloning of PCR products (LIC-PCR). Nucleic Acids Res 18(20):6069–6074. doi:10.1093/nar/18.20.6069
Raman M, Martin K (2014) One solution for cloning and mutagenesis: in-fusion (R) HD cloning plus. Nat Methods 11(9):Iii–V
Thieme F, Engler C, Kandzia R, Marillonnet S (2011) Quick and clean cloning: a ligation-independent cloning strategy for selective cloning of specific PCR products from non-specific mixes. PLoS One 6(6):12. doi:10.1371/journal.pone.0020556
Li MZ, Elledge SJ (2007) Harnessing homologous recombination in vitro to generate recombinant DNA via SLIC. Nat Methods 4(3):251–256. doi:10.1038/nmeth1010
Gibson DG, Young L, Chuang RY, Venter JC, Hutchison CA, Smith HO (2009) Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat Methods 6(5):343–345. doi:10.1038/Nmeth.1318
de Kok S, Stanton LH, Slaby T, Durot M, Holmes VF, Patel KG, Platt D, Shapland EB, Serber Z, Dean J, Newman JD, Chandran SS (2014) Rapid and reliable DNA assembly via ligase cycling reaction. ACS Synth Biol 3(2):97–106. doi:10.1021/sb4001992
Paetzold B, Carolis C, Ferrar T, Serrano L, Lluch-Senar M (2013) In situ overlap and sequence synthesis during DNA assembly. ACS Synth Biol 2(12):750–755. doi:10.1021/sb400067v
Trubitsyna M, Michlewski G, Cai Y, Elfick A, French CE (2014) PaperClip: rapid multi-part DNA assembly from existing libraries. Nucleic Acids Res. doi:10.1093/nar/gku829
Pfirrmann T, Lokapally A, Andreasson C, Ljungdahl P, Hollemann T (2013) SOMA: a single oligonucleotide mutagenesis and cloning approach. PLoS One 8(6), e64870. doi:10.1371/journal.pone.0064870
Krishnakumar R, Grose C, Haft DH, Zaveri J, Alperovich N, Gibson DG, Merryman C, Glass JI (2014) Simultaneous non-contiguous deletions using large synthetic DNA and site-specific recombinases. Nucleic Acids Res 42(14), e111. doi:10.1093/nar/gku509
Cornils K, Thielecke L, Huser S, Forgber M, Thomaschewski M, Kleist N, Hussein K, Riecken K, Volz T, Gerdes S, Glauche I, Dahl A, Dandri M, Roeder I, Fehse B (2014) Multiplexing clonality: combining RGB marking and genetic barcoding. Nucleic Acids Res 42(7), e56. doi:10.1093/nar/gku081
Krishnamurthy VV, Khamo JS, Cho E, Schornak C, Zhang K (2015) Multiplex gene removal by two-step polymerase chain reactions. Anal Biochem 481:7–9. doi:10.1016/j.ab.2015.03.033
Krishnamurthy VV, Khamo JS, Cho E, Schornak C, Zhang K (2015) Polymerase chain reaction-based gene removal from plasmids. Data in Brief 4:75–82. doi:10.1016/j.dib.2015.04.024
Acknowledgements
K.Z. thanks the funding support from the University of Illinois at Urbana-Champaign (UIUC).
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2017 Springer Science+Business Media New York
About this protocol
Cite this protocol
Krishnamurthy, V., Zhang, K. (2017). Simultaneous Removal of Multiple DNA Segments by Polymerase Chain Reactions. In: Hughes, R. (eds) Synthetic DNA. Methods in Molecular Biology, vol 1472. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6343-0_15
Download citation
DOI: https://doi.org/10.1007/978-1-4939-6343-0_15
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-6341-6
Online ISBN: 978-1-4939-6343-0
eBook Packages: Springer Protocols