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Updated: Jul 6, 2026

Rapid Assembly of Multi-Gene Constructs using Modular Golden Gate Cloning
Published on: February 5, 2021
A method for generating sticky-end PCR products which facilitates unidirectional cloning and the one-step assembly of
Andrew Walker1, James Taylor, Duncan Rowe
1Geneservice Ltd., 2 Cambridge Science Park, Milton Road, Cambridge CB4 0FE, UK.
Abstract:
We have developed and tested a method for the restriction enzyme-independent generation of sticky-end PCR products. The method is suitable for use with a proof-reading polymerase such as pfu, or any other heat-stable polymerase which produces a blunt-end product. The technique can be used to achieve unidirectional cloning of PCR products with an efficiency greater than 90%. Because the sequences of the sticky ends are defined by the user and potentially can be of any length, the method can also be exploited for the one-step construction of recombinant plasmids from multiple functional cassettes, without the use of restriction enzymes.
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