Abstract
A polymerase chain reaction (PCR) in water droplets with water-in-oil emulsion (emulsion PCR) facilitates parallel amplification of a single-molecule template. The amplified DNA can be immobilized onto microbeads bound to primer DNA. The product, termed a “bead library”, has various applications such as next-generation sequencing (NGS) and the directed evolution of various functional biomolecules. Here, we describe a method for genomic library construction on microbeads using emulsion PCR.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Tawfik DS, Griffiths AD (1998) Man-made cell-like compartments for molecular evolution. Nat Biotechnol 16:652–656
Williams R, Peisajovich SG, Miller OJ, Magdassi S, Tawfik DS, Griffiths AD (2006) Amplification of complex gene libraries by emulsion PCR. Nat Methods 3:545–550
Dressman D, Yan H, Traverso G, Kinzler KW, Vogelstein B (2003) Transforming single DNA molecules into fluorescent magnetic particles for detection and enumeration of genetic variations. Proc Natl Acad Sci U S A 100:8817–8822
Kojima T, Takei Y, Ohtsuka M, Kawarasaki Y, Yamane T, Nakano H (2005) PCR amplification from single DNA molecules on magnetic beads in emulsion: application for high-throughput screening of transcription factor targets. Nucleic Acids Res 33, e150
Gan R, Yamanaka Y, Kojima T, Nakano H (2008) Microbeads display of proteins using emulsion PCR and cell-free protein synthesis. Biotechnol Prog 24:1107–1114
Gan R, Furuzawa S, Kojima T, Kanie K, Kato R, Okochi M, Honda H, Nakano H (2010) Directed evolution of angiotensin II-inhibiting peptides using a microbead display. J Biosci Bioeng 109:411–417
Stapleton JA, Swartz JR (2010) Development of an in vitro compartmentalization screen for high-throughput directed evolution of [FeFe] hydrogenases. PLoS One 5, e15275
Kojima T, Hashimoto Y, Kato M, Kobayashi T, Nakano H (2010) High-throughput screening of DNA binding sites for transcription factor AmyR from Aspergillus nidulans using DNA beads display system. J Biosci Bioeng 109:519–525
Kojima T, Ohuchi S, Ito Y, Nakano H (2012) High-throughput screening method for promoter activity using bead display and a ligase ribozyme. J Biosci Bioeng 114:671–676
Margulies M, Egholm M, Altman WE, Attiya S, Bader JS, Bemben LA, Berka J, Braverman MS, Chen YJ, Chen ZT et al (2005) Genome sequencing in microfabricated high-density picolitre reactors. Nature 437:376–380
Shendure J, Porreca GJ, Reppas NB, Lin X, McCutcheon JP, Rosenbaum AM, Wang MD, Zhang K, Mitra RD, Church GM (2005) Accurate multiplex polony sequencing of an evolved bacterial genome. Science 309:1728–1732
Valouev A, Ichikawa J, Tonthat T, Stuart J, Ranade S, Peckham H, Zeng K, Malek JA, Costa G, McKernan K et al (2008) A high-resolution, nucleosome position map of C. elegans reveals a lack of universal sequence-dictated positioning. Genome Res 18:1051–1063
Smith AM, Heisler LE, St Onge RP, Farias-Hesson E, Wallace IM, Bodeau J, Harris AN, Perry KM, Giaever G, Pourmand N, Nislow C (2010) Highly-multiplexed barcode sequencing: an efficient method for parallel analysis of pooled samples. Nucleic Acids Res 38:e142
Rothberg JM, Hinz W, Rearick TM, Schultz J, Mileski W, Davey M, Leamon JH, Johnson K, Milgrew MJ, Edwards M et al (2011) An integrated semiconductor device enabling non-optical genome sequencing. Nature 475:348–352
Acknowledgments
This work was supported by Grant-in-Aid awards (22760605 and 23360367) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (MEXT).
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2015 Springer Science+Business Media New York
About this protocol
Cite this protocol
Kojima, T., Zhu, B., Nakano, H. (2015). Construction of a DNA Library on Microbeads Using Whole Genome Amplification. In: Kroneis, T. (eds) Whole Genome Amplification. Methods in Molecular Biology, vol 1347. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2990-0_6
Download citation
DOI: https://doi.org/10.1007/978-1-4939-2990-0_6
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-2989-4
Online ISBN: 978-1-4939-2990-0
eBook Packages: Springer Protocols