Isothermal strand-displacement amplification applications for high-throughput genomics.
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.
Detter
John C
JC
United States Department of Energy Joint Genome Institute, Walnut Creek, California 94598, USA.
Jett
Jamie M
JM
Lucas
Susan M
SM
Dalin
Eileen
E
Arellano
Andre R
AR
Wang
Mei
M
Nelson
John R
JR
Chapman
Jarrod
J
Lou
Yunian
Y
Rokhsar
Dan
D
Hawkins
Trevor L
TL
Richardson
Paul M
PM
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
D004269:DNA, Bacterial