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.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Chalfie M, Tu Y, Euskirchen G et al (1994) Green fluorescent protein as a marker for gene expression. Science 263:802–805
Crocker J, Tamori Y, Erives A (2008) Evolution acts on enhancer organization to fine-tune gradient threshold readouts. PLoS Biol 6:e263
Hamamatsu N, Aita T, Nomiya Y et al (2005) Biased mutation-assembling: an efficient method for rapid directed evolution through simultaneous mutation accumulation. Protein Eng Des Sel 18:265–271
Mullis K, Faloona F, Scharf S et al (1986) Specific enzymatic amplification of DNA in vitro: the polymerase chain reaction. Cold Spring Harb Symp Quant Biol 51:263–273
Horton RM, Hunt HD, Ho SN et al (1989) Engineering hybrid genes without the use of restriction enzymes: gene splicing by overlap extension. Gene 77:61–68
Yon J, Fried M (1989) Precise gene fusion by PCR. Nucleic Acids Res 17:4895
Yolov AA, Shabarova ZA (1990) Constructing DNA by polymerase recombination. Nucleic Acids Res 18:3983–3986
Lundberg KS, Shoemaker DD, Adams MW et al (1991) High-fidelity amplification using a thermostable DNA polymerase isolated from Pyrococcus furiosus. Gene 108:1–6
Shevchuk NA, Bryksin AV, Nusinovich YA et al (2004) Construction of long DNA molecules using long PCR-based fusion of several fragments simultaneously. Nucleic Acids Res 32:e19
Heckman KL, Pease LR (2007) Gene splicing and mutagenesis by PCR-driven overlap extension. Nat Protoc 2:924–932
Ho SN, Hunt HD, Horton RM et al (1989) Site-directed mutagenesis by overlap extension using the polymerase chain reaction. Gene 77:51–59
Fire A, Harrison SW, Dixon D (1990) A modular set of lacZ fusion vectors for studying gene expression in Caenorhabditis elegans. Gene 93:189–198
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2012 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Nelson, M.D., Fitch, D.H.A. (2012). Overlap Extension PCR: An Efficient Method for Transgene Construction. In: Orgogozo, V., Rockman, M. (eds) Molecular Methods for Evolutionary Genetics. Methods in Molecular Biology, vol 772. Humana Press. https://doi.org/10.1007/978-1-61779-228-1_27
Download citation
DOI: https://doi.org/10.1007/978-1-61779-228-1_27
Published:
Publisher Name: Humana Press
Print ISBN: 978-1-61779-227-4
Online ISBN: 978-1-61779-228-1
eBook Packages: Springer Protocols