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De novo DNA synthesis using single molecule PCR
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 vit...
Autores principales: | , , , , , |
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Formato: | Texto |
Lenguaje: | English |
Publicado: |
Oxford University Press
2008
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2553596/ https://www.ncbi.nlm.nih.gov/pubmed/18667587 http://dx.doi.org/10.1093/nar/gkn457 |
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author | Yehezkel, Tuval Ben Linshiz, Gregory Buaron, Hen Kaplan, Shai Shabi, Uri Shapiro, Ehud |
author_facet | Yehezkel, Tuval Ben Linshiz, Gregory Buaron, Hen Kaplan, Shai Shabi, Uri Shapiro, Ehud |
author_sort | Yehezkel, Tuval Ben |
collection | PubMed |
description | 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. |
format | Text |
id | pubmed-2553596 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2008 |
publisher | Oxford University Press |
record_format | MEDLINE/PubMed |
spelling | pubmed-25535962008-10-01 De novo DNA synthesis using single molecule PCR Yehezkel, Tuval Ben Linshiz, Gregory Buaron, Hen Kaplan, Shai Shabi, Uri Shapiro, Ehud Nucleic Acids Res Methods Online 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. Oxford University Press 2008-10 2008-07-30 /pmc/articles/PMC2553596/ /pubmed/18667587 http://dx.doi.org/10.1093/nar/gkn457 Text en © 2008 The Author(s) http://creativecommons.org/licenses/by-nc/2.0/uk/ 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. |
spellingShingle | Methods Online Yehezkel, Tuval Ben Linshiz, Gregory Buaron, Hen Kaplan, Shai Shabi, Uri Shapiro, Ehud De novo DNA synthesis using single molecule PCR |
title | De novo DNA synthesis using single molecule PCR |
title_full | De novo DNA synthesis using single molecule PCR |
title_fullStr | De novo DNA synthesis using single molecule PCR |
title_full_unstemmed | De novo DNA synthesis using single molecule PCR |
title_short | De novo DNA synthesis using single molecule PCR |
title_sort | de novo dna synthesis using single molecule pcr |
topic | Methods Online |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2553596/ https://www.ncbi.nlm.nih.gov/pubmed/18667587 http://dx.doi.org/10.1093/nar/gkn457 |
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