Overlap extension PCR: an efficient method for transgene construction.

Matthew D. Nelson, David H A Fitch

Research output: Contribution to journalArticle

Abstract

Combining genes or regulatory elements to make hybrid genes is a widely used methodology throughout the biological sciences. Here, we describe an optimized approach for hybrid gene construction called overlap extension PCR. In this method, the polymerase chain reaction (PCR) is employed for efficient and reliable construction of hybrid genes. A PCR-based approach does not rely on available restriction sites or other specific sequences, an advantage over more conventional cloning or recombineering methods. With the use of high-fidelity DNA polymerase, this method can be used for making even very large constructs (>20 kb) with minimal unwanted mutations. Finally, overlap extension-PCR can be used as a means for site-directed mutagenesis, introducing desired mutations to the final hybrid gene.

Original languageEnglish (US)
Pages (from-to)459-470
Number of pages12
JournalMethods in Molecular Biology
Volume772
StatePublished - 2011

Fingerprint

Transgenes
Polymerase Chain Reaction
Genes
Mutation
Biological Science Disciplines
DNA-Directed DNA Polymerase
Regulator Genes
Site-Directed Mutagenesis
Organism Cloning

ASJC Scopus subject areas

  • Medicine(all)

Cite this

Overlap extension PCR : an efficient method for transgene construction. / Nelson, Matthew D.; Fitch, David H A.

In: Methods in Molecular Biology, Vol. 772, 2011, p. 459-470.

Research output: Contribution to journalArticle

@article{ccd5103a245e40139b62054d8c6a6697,
title = "Overlap extension PCR: an efficient method for transgene construction.",
abstract = "Combining genes or regulatory elements to make hybrid genes is a widely used methodology throughout the biological sciences. Here, we describe an optimized approach for hybrid gene construction called overlap extension PCR. In this method, the polymerase chain reaction (PCR) is employed for efficient and reliable construction of hybrid genes. A PCR-based approach does not rely on available restriction sites or other specific sequences, an advantage over more conventional cloning or recombineering methods. With the use of high-fidelity DNA polymerase, this method can be used for making even very large constructs (>20 kb) with minimal unwanted mutations. Finally, overlap extension-PCR can be used as a means for site-directed mutagenesis, introducing desired mutations to the final hybrid gene.",
author = "Nelson, {Matthew D.} and Fitch, {David H A}",
year = "2011",
language = "English (US)",
volume = "772",
pages = "459--470",
journal = "Methods in Molecular Biology",
issn = "1064-3745",
publisher = "Humana Press",

}

TY - JOUR

T1 - Overlap extension PCR

T2 - an efficient method for transgene construction.

AU - Nelson, Matthew D.

AU - Fitch, David H A

PY - 2011

Y1 - 2011

N2 - Combining genes or regulatory elements to make hybrid genes is a widely used methodology throughout the biological sciences. Here, we describe an optimized approach for hybrid gene construction called overlap extension PCR. In this method, the polymerase chain reaction (PCR) is employed for efficient and reliable construction of hybrid genes. A PCR-based approach does not rely on available restriction sites or other specific sequences, an advantage over more conventional cloning or recombineering methods. With the use of high-fidelity DNA polymerase, this method can be used for making even very large constructs (>20 kb) with minimal unwanted mutations. Finally, overlap extension-PCR can be used as a means for site-directed mutagenesis, introducing desired mutations to the final hybrid gene.

AB - Combining genes or regulatory elements to make hybrid genes is a widely used methodology throughout the biological sciences. Here, we describe an optimized approach for hybrid gene construction called overlap extension PCR. In this method, the polymerase chain reaction (PCR) is employed for efficient and reliable construction of hybrid genes. A PCR-based approach does not rely on available restriction sites or other specific sequences, an advantage over more conventional cloning or recombineering methods. With the use of high-fidelity DNA polymerase, this method can be used for making even very large constructs (>20 kb) with minimal unwanted mutations. Finally, overlap extension-PCR can be used as a means for site-directed mutagenesis, introducing desired mutations to the final hybrid gene.

UR - http://www.scopus.com/inward/record.url?scp=84857206161&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=84857206161&partnerID=8YFLogxK

M3 - Article

C2 - 22065455

AN - SCOPUS:84857206161

VL - 772

SP - 459

EP - 470

JO - Methods in Molecular Biology

JF - Methods in Molecular Biology

SN - 1064-3745

ER -