Cargando…

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...

Descripción completa

Detalles Bibliográficos
Autores principales: Amiram, Miriam, Quiroz, Felipe Garcia, Callahan, Daniel J., Chilkoti, Ashutosh
Formato: Texto
Lenguaje:English
Publicado: 2011
Materias:
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
_version_ 1782201791477186560
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
work_keys_str_mv AT amirammiriam highlyparallelmethodforsynthesisofdnarepeatsenablesdiscoveryofsmartproteinpolymers
AT quirozfelipegarcia highlyparallelmethodforsynthesisofdnarepeatsenablesdiscoveryofsmartproteinpolymers
AT callahandanielj highlyparallelmethodforsynthesisofdnarepeatsenablesdiscoveryofsmartproteinpolymers
AT chilkotiashutosh highlyparallelmethodforsynthesisofdnarepeatsenablesdiscoveryofsmartproteinpolymers