High-throughput mutagenesis using a two-fragment PCR approach

Abstract Site-directed scanning mutagenesis is a powerful protein engineering technique which allows studies of protein functionality at single amino acid resolution and design of stabilized proteins for structural and biophysical work. However, creating libraries of hundreds of mutants remains a ch...

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Autores principales:	Franziska M. Heydenreich, Tamara Miljuš, Rolf Jaussi, Roger Benoit, Dalibor Milić, Dmitry B. Veprintsev
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2017
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/1b11ce312b1949debc1d11e568ec22af
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spelling	oai:doaj.org-article:1b11ce312b1949debc1d11e568ec22af2021-12-02T16:06:47ZHigh-throughput mutagenesis using a two-fragment PCR approach10.1038/s41598-017-07010-42045-2322https://doaj.org/article/1b11ce312b1949debc1d11e568ec22af2017-07-01T00:00:00Zhttps://doi.org/10.1038/s41598-017-07010-4https://doaj.org/toc/2045-2322Abstract Site-directed scanning mutagenesis is a powerful protein engineering technique which allows studies of protein functionality at single amino acid resolution and design of stabilized proteins for structural and biophysical work. However, creating libraries of hundreds of mutants remains a challenging, expensive and time-consuming process. The efficiency of the mutagenesis step is the key for fast and economical generation of such libraries. PCR artefacts such as misannealing and tandem primer repeats are often observed in mutagenesis cloning and reduce the efficiency of mutagenesis. Here we present a high-throughput mutagenesis pipeline based on established methods that significantly reduces PCR artefacts. We combined a two-fragment PCR approach, in which mutagenesis primers are used in two separate PCR reactions, with an in vitro assembly of resulting fragments. We show that this approach, despite being more laborious, is a very efficient pipeline for the creation of large libraries of mutants.Franziska M. HeydenreichTamara MiljušRolf JaussiRoger BenoitDalibor MilićDmitry B. VeprintsevNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 7, Iss 1, Pp 1-11 (2017)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Franziska M. Heydenreich Tamara Miljuš Rolf Jaussi Roger Benoit Dalibor Milić Dmitry B. Veprintsev High-throughput mutagenesis using a two-fragment PCR approach
description	Abstract Site-directed scanning mutagenesis is a powerful protein engineering technique which allows studies of protein functionality at single amino acid resolution and design of stabilized proteins for structural and biophysical work. However, creating libraries of hundreds of mutants remains a challenging, expensive and time-consuming process. The efficiency of the mutagenesis step is the key for fast and economical generation of such libraries. PCR artefacts such as misannealing and tandem primer repeats are often observed in mutagenesis cloning and reduce the efficiency of mutagenesis. Here we present a high-throughput mutagenesis pipeline based on established methods that significantly reduces PCR artefacts. We combined a two-fragment PCR approach, in which mutagenesis primers are used in two separate PCR reactions, with an in vitro assembly of resulting fragments. We show that this approach, despite being more laborious, is a very efficient pipeline for the creation of large libraries of mutants.
format	article
author	Franziska M. Heydenreich Tamara Miljuš Rolf Jaussi Roger Benoit Dalibor Milić Dmitry B. Veprintsev
author_facet	Franziska M. Heydenreich Tamara Miljuš Rolf Jaussi Roger Benoit Dalibor Milić Dmitry B. Veprintsev
author_sort	Franziska M. Heydenreich
title	High-throughput mutagenesis using a two-fragment PCR approach
title_short	High-throughput mutagenesis using a two-fragment PCR approach
title_full	High-throughput mutagenesis using a two-fragment PCR approach
title_fullStr	High-throughput mutagenesis using a two-fragment PCR approach
title_full_unstemmed	High-throughput mutagenesis using a two-fragment PCR approach
title_sort	high-throughput mutagenesis using a two-fragment pcr approach
publisher	Nature Portfolio
publishDate	2017
url	https://doaj.org/article/1b11ce312b1949debc1d11e568ec22af
work_keys_str_mv	AT franziskamheydenreich highthroughputmutagenesisusingatwofragmentpcrapproach AT tamaramiljus highthroughputmutagenesisusingatwofragmentpcrapproach AT rolfjaussi highthroughputmutagenesisusingatwofragmentpcrapproach AT rogerbenoit highthroughputmutagenesisusingatwofragmentpcrapproach AT dalibormilic highthroughputmutagenesisusingatwofragmentpcrapproach AT dmitrybveprintsev highthroughputmutagenesisusingatwofragmentpcrapproach
_version_	1718384827326005248

High-throughput mutagenesis using a two-fragment PCR approach

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