title: Isothermal strand-displacement amplification applications for high-throughput genomics.
abstract: Amplification of source DNA is a nearly universal requirement for molecular biology applications. The primary methods currently available to researchers are limited to in vivo amplification in Escherichia coli hosts and the polymerase chain reaction. Rolling-circle DNA replication is a well-known method for synthesis of phage genomes and recently has been applied as rolling circle amplification (RCA) of specific target sequences as well as circular vectors used in cloning. Here, we demonstrate that RCA using random hexamer primers with 29 DNA polymerase can be used for strand-displacement amplification of different vector constructs containing a variety of insert sizes to produce consistently uniform template for end-sequencing reactions. We show this procedure to be especially effective in a high-throughput plasmid production sequencing process. In addition, we demonstrate that whole bacterial genomes can be effectively amplified from cells or small amounts of purified genomic DNA without apparent bias for use in downstream applications, including whole genome shotgun sequencing.
authors.affiliation: United States Department of Energy Joint Genome Institute, Walnut Creek, California 94598, USA.
authors.forename: John C
authors.initials: JC
authors.lastname: Detter
authors.forename: Jamie M
authors.initials: JM
authors.lastname: Jett
authors.forename: Susan M
authors.initials: SM
authors.lastname: Lucas
authors.forename: Eileen
authors.initials: E
authors.lastname: Dalin
authors.forename: Andre R
authors.initials: AR
authors.lastname: Arellano
authors.forename: Mei
authors.initials: M
authors.lastname: Wang
authors.forename: John R
authors.initials: JR
authors.lastname: Nelson
authors.forename: Jarrod
authors.initials: J
authors.lastname: Chapman
authors.forename: Yunian
authors.initials: Y
authors.lastname: Lou
authors.forename: Dan
authors.initials: D
authors.lastname: Rokhsar
authors.forename: Trevor L
authors.initials: TL
authors.lastname: Hawkins
authors.forename: Paul M
authors.initials: PM
authors.lastname: Richardson
mesh_terms: D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D016680:Genome, Bacterial; D023281:Genomics / Q000379:methods*; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D017422:Sequence Analysis, DNA / Q000706:statistics & numerical data
chemical_list: D004269:DNA, Bacterial