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Highly Parallel Method for Synthesis of DNA Repeats Enables Discovery of “Smart” Protein Polymers
Robust high-throughput synthesis methods are needed to expand the repertoire of repetitive protein-polymers for different applications. To address this need, we developed a new method, overlap-extension rolling circle amplification (OERCA), for the highly parallel synthesis of genes encoding repetit...
Autores principales: | , , , |
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Formato: | Texto |
Lenguaje: | English |
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2011
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Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3075872/ https://www.ncbi.nlm.nih.gov/pubmed/21258353 http://dx.doi.org/10.1038/nmat2942 |
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author | Amiram, Miriam Quiroz, Felipe Garcia Callahan, Daniel J. Chilkoti, Ashutosh |
author_facet | Amiram, Miriam Quiroz, Felipe Garcia Callahan, Daniel J. Chilkoti, Ashutosh |
author_sort | Amiram, Miriam |
collection | PubMed |
description | Robust high-throughput synthesis methods are needed to expand the repertoire of repetitive protein-polymers for different applications. To address this need, we developed a new method, overlap-extension rolling circle amplification (OERCA), for the highly parallel synthesis of genes encoding repetitive protein-polymers. OERCA involves a single PCR-type reaction for the rolling circle amplification of a circular DNA template and simultaneous overlap extension by thermal cycling. We characterized the variables that control OERCA and demonstrated its superiority over existing methods, its robustness, throughput and versatility by synthesizing variants of elastin-like polypeptides (ELPs) and protease-responsive polymers of a glucagon-like peptide-1 analog. Despite the GC-rich, highly repetitive sequences of ELPs, we synthesized remarkably large genes without recursive ligation. OERCA also enabled us to discover “smart” biopolymers that exhibit fully reversible thermally responsive behavior. This powerful strategy generates libraries of repetitive genes over a wide and tunable range of molecular weights in a “one-pot” parallel format. |
format | Text |
id | pubmed-3075872 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2011 |
record_format | MEDLINE/PubMed |
spelling | pubmed-30758722011-08-01 Highly Parallel Method for Synthesis of DNA Repeats Enables Discovery of “Smart” Protein Polymers Amiram, Miriam Quiroz, Felipe Garcia Callahan, Daniel J. Chilkoti, Ashutosh Nat Mater Article Robust high-throughput synthesis methods are needed to expand the repertoire of repetitive protein-polymers for different applications. To address this need, we developed a new method, overlap-extension rolling circle amplification (OERCA), for the highly parallel synthesis of genes encoding repetitive protein-polymers. OERCA involves a single PCR-type reaction for the rolling circle amplification of a circular DNA template and simultaneous overlap extension by thermal cycling. We characterized the variables that control OERCA and demonstrated its superiority over existing methods, its robustness, throughput and versatility by synthesizing variants of elastin-like polypeptides (ELPs) and protease-responsive polymers of a glucagon-like peptide-1 analog. Despite the GC-rich, highly repetitive sequences of ELPs, we synthesized remarkably large genes without recursive ligation. OERCA also enabled us to discover “smart” biopolymers that exhibit fully reversible thermally responsive behavior. This powerful strategy generates libraries of repetitive genes over a wide and tunable range of molecular weights in a “one-pot” parallel format. 2011-01-23 2011-02 /pmc/articles/PMC3075872/ /pubmed/21258353 http://dx.doi.org/10.1038/nmat2942 Text en Users may view, print, copy, download and text and data- mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use: http://www.nature.com/authors/editorial_policies/license.html#terms |
spellingShingle | Article Amiram, Miriam Quiroz, Felipe Garcia Callahan, Daniel J. Chilkoti, Ashutosh Highly Parallel Method for Synthesis of DNA Repeats Enables Discovery of “Smart” Protein Polymers |
title | Highly Parallel Method for Synthesis of DNA Repeats Enables Discovery of “Smart” Protein Polymers |
title_full | Highly Parallel Method for Synthesis of DNA Repeats Enables Discovery of “Smart” Protein Polymers |
title_fullStr | Highly Parallel Method for Synthesis of DNA Repeats Enables Discovery of “Smart” Protein Polymers |
title_full_unstemmed | Highly Parallel Method for Synthesis of DNA Repeats Enables Discovery of “Smart” Protein Polymers |
title_short | Highly Parallel Method for Synthesis of DNA Repeats Enables Discovery of “Smart” Protein Polymers |
title_sort | highly parallel method for synthesis of dna repeats enables discovery of “smart” protein polymers |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3075872/ https://www.ncbi.nlm.nih.gov/pubmed/21258353 http://dx.doi.org/10.1038/nmat2942 |
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