Isothermal Strand-Displacement Amplification Applications for High-Throughput Genomics

• Published in: Genomics (San Diego, Calif.) Vol. 80; no. 6; pp. 691 - 698
• Main Authors: Detter, John C, Jett, Jamie M, Lucas, Susan M, Dalin, Eileen, Arellano, Andre R, Wang, Mei, Nelson, John R, Chapman, Jarrod, Lou, Yunian, Rokhsar, Dan, Hawkins, Trevor L, Richardson, Paul M
• Format: Journal Article
• Language: English
• Published: San Diego, CA Elsevier Inc 01.12.2002; Elsevier
• Subjects: Analytical biochemistry: general aspects, technics, instrumentation; Analytical, structural and metabolic biochemistry; Biological and medical sciences; Carcinogenesis, carcinogens and anticarcinogens; DNA amplification; DNA isolation; DNA, Bacterial - chemistry; DNA, Bacterial - genetics; Foods and miscellaneous; Fundamental and applied biological sciences. Psychology; Genome, Bacterial; Genomics - methods; Medical sciences; Molecular and cellular biology; Molecular genetics; Mutagenesis. Repair; Nucleic Acid Amplification Techniques - methods; RCA; rolling circle amplification; Sequence Analysis, DNA - statistics & numerical data; Tumors; whole genome amplification; Xylella; RCA; DNA amplification; rolling circle amplification; Xylella; DNA isolation; whole genome amplification; Insulation; Genomics; Gene amplification; DNA; Technique; Molecular biology; Genome
• ISSN: 0888-7543; 1089-8646
• DOI: 10.1006/geno.2002.7020

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 Φ29 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.