Synthetic DNA pp 193-203

Part of the Methods in Molecular Biology book series (MIMB, volume 1472) | Cite as

Simultaneous Removal of Multiple DNA Segments by Polymerase Chain Reactions



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.

Key words

Restriction-free cloning Polymerase chain reaction Synthetic DNA assembly and manipulation Multiplex gene removal Synthetic single-stranded bridging oligos 


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Copyright information

© Springer Science+Business Media New York 2017

Authors and Affiliations

  1. 1.Department of Biochemistry, School of Molecular and Cellular BiologyUniversity of Illinois at Urbana-ChampaignUrbanaUSA

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