Abstract
PCR is a common method to produce desired DNA fragments from templates. The oligonucleotide primers used for PCR must contain annealing sequences that are usually 20–30 nucleotides long and identical to a part of template DNA. However, primers often contain additional sequences at their 5′ ends, which are restriction enzyme sites, recombination targeting sequences, or overlap sequences for fusion PCR. When these additional sequences are attached to their annealing sequences, the annealing sequences can be shortened. Here, we describe universal GC-rich annealing sequences useful for overlap extension PCR and simple in-frame addition of desired sequences.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Dieffenbach CW, Lowe TMJ, Dveksler GS (1995) General concepts for PCR primer design. In: Dieffenbach CW, Dveksler GS (eds) PCR primer: a laboratory manual. Cold Spring Harbor Laboratory Press, New York, pp 133–142
Suggs SV, Hirose T, Miyake T et al (1981) Use of synthetic oligodeoxyribonucleotides for the isolation of specific cloned DNA sequences. ICN-UCLA Symp Dev Biol 23:683–693
Cha-aim K, Fukunaga T, Hoshida H et al (2009) Reliable fusion PCR mediated by GC-rich overlap sequences. Gene 434:43–49
Heckman KL, Pease LR (2007) Gene splicing and mutagenesis by PCR-driven overlap extension. Nat Protoc 2:924–932
Lu Q (2005) Seamless cloning and gene fusion. Trends Biotechnol 23:199–207
Vallejo AN, Pogulis RJ, Pease LR (1994) In vitro synthesis of novel genes: mutagenesis and recombination by PCR. Genome Res 4:s123–s130
Levis R (1995) Strategies for cloning PCR products. In: Dieffenbach CW, Dveksler GS (eds) PCR primer: a laboratory manual. Cold Spring Harbor Laboratory Press, New York, pp 539–554
Baudin A, Ozier-Kalogeropoulos O, Denouel A et al (1993) A simple and efficient method for direct gene deletion in Saccharomyces cerevisiae. Nucleic Acids Res 21:3329–3330
Kakihara Y, Matsuura Y, Hoshida H et al (2005) Cost-saving design of PCR primers containing additional sequences. ITE Lett 6:135–139
Benedikt W, Walter N (2000) Mitochondria-targeted green fluorescent proteins: convenient tools for the study of organelle biogenesis in Saccharomyces cerevisiae. Yeast 16:1421–1427
Keppler-Ross S, Douglas L, Konopka JB et al (2010) Recognition of yeast by murine macrophages requires mannan but not glucan. Eukaryot Cell 9:1776–1787
Amberg DC, Burke D, Strathern JN (2005) Method in yeast genetics: a Cold Spring Harbor Laboratory course manual. Cold Spring Harbor Laboratory Press, New York
Nonklang S, Abdel-Banat BM, Cha-aim K et al (2008) High-temperature ethanol fermentation and transformation with linear DNA in the thermotolerant yeast Kluyveromyces marxianus DMKU3-1042. Appl Environ Microbiol 74:7514–7521
Abdel-Banat BM, Nonklang S, Hoshida H et al (2010) Random and targeted gene integrations through the control of non-homologous end joining in the yeast Kluyveromyces marxianus. Yeast 27:29–39
Zha H, Fisk HA, Yaffe MP et al (1996) Structure-function comparisons of the proapoptotic protein Bax in yeast and mammalian cells. Mol Cell Biol 16:6494–6508
Monosov EZ, Wenzel TJ, Lüers GH et al (1996) Labeling of peroxisomes with green fluorescent protein in living P. pastoris cells. J Histochem Cytochem 44:581–589
Cha-Aim K, Hoshida H, Fukunaga T et al (2012) Fusion PCR via novel overlap sequences. Methods Mol Biol 852:97–110
Winzeler EA, Shoemaker DD, Astromoff A et al (1999) Functional characterization of the S. cerevisiae genome by gene deletion and parallel analysis. Science 285:901–906
Acknowledgments
The authors thank Yukie Misumi for her technical assistance. This work was supported in part by the Adaptable and Seamless Technology Transfer Program through Target-Driven R & D and the Advanced Low Carbon Technology Research and Development Program (JST, Japan).
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2014 Springer Science+Business Media, New York
About this protocol
Cite this protocol
Nakamura, M., Suzuki, A., Hoshida, H., Akada, R. (2014). Minimum GC-Rich Sequences for Overlap Extension PCR and Primer Annealing. In: Valla, S., Lale, R. (eds) DNA Cloning and Assembly Methods. Methods in Molecular Biology, vol 1116. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-764-8_12
Download citation
DOI: https://doi.org/10.1007/978-1-62703-764-8_12
Published:
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-62703-763-1
Online ISBN: 978-1-62703-764-8
eBook Packages: Springer Protocols