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Nucleic Acids Research Advance Access published online on July 30, 2008
Nucleic Acids Research, doi:10.1093/nar/gkn457
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© 2008 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Methods Online

De novo DNA synthesis using single molecule PCR

Tuval Ben Yehezkel1, Gregory Linshiz1,2, Hen Buaron1, Shai Kaplan2, Uri Shabi1 and Ehud Shapiro1,2,*

1Department of Biological Chemistry and 2Department of Computer Science and Applied Mathematics, Weizmann Institute of Science, Rehovot 76100, Israel

*To whom correspondence should be addressed. Tel: +972 8 934 4506; Email: Ehud.Shapiro{at}weizmann.ac.il

Received April 1, 2008. Revised July 1, 2008. Accepted July 2, 2008.

The throughput of DNA reading (sequencing) has dramatically increased recently due to the incorporation of in vitro clonal amplification. The throughput of DNA writing (synthesis) is trailing behind, with cloning and sequencing constituting the main bottleneck. To overcome this bottleneck, an in vitro alternative for in vivo DNA cloning must be integrated into DNA synthesis methods. Here we show how a new single molecule PCR (smPCR)-based procedure can be employed as a general substitute to in vivo cloning thereby allowing for the first time in vitro DNA synthesis. We integrated this rapid and high fidelity in vitro procedure into our earlier recursive DNA synthesis and error correction procedure and used it to efficiently construct and error-correct a 1.8-kb DNA molecule from synthetic unpurified oligos completely in vitro. Although we demonstrate incorporating smPCR in a particular method, the approach is general and can be used in principle in conjunction with other DNA synthesis methods as well.

The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors.


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