title: Improvements of rolling circle amplification (RCA) efficiency and accuracy using Thermus thermophilus SSB mutant protein.
abstract: Rolling circle amplification (RCA) of plasmid or genomic DNA using random hexamers and bacteriophage phi29 DNA polymerase has become increasingly popular in the amplification of template DNA in DNA sequencing. We have found that the mutant protein of single-stranded DNA binding protein (SSB) from Thermus thermophilus (Tth) HB8 enhances the efficiency of amplification of DNA templates. In addition, the TthSSB mutant protein increased the specificity of phi29 DNA polymerase. We have overexpressed the native and mutant forms of TthSSB protein in Escherichia coli and purified them to homogeneity. In vitro, these proteins were found to bind specifically to single-stranded DNA. Addition of TthSSB mutant protein to RCA halved the elongation time required for phi29 DNA polymerase to synthesize DNA fragments in RCA. Furthermore, the presence of the TthSSB mutant protein essentially eliminates nonspecific DNA products in RCA reactions.
authors.affiliation: Aisin Cosmos R&D Co. Ltd, 2-1-5 Kazusa-kamatari, Kisarazu, Chiba 292-0818 Japan.
authors.forename: Jin
authors.initials: J
authors.lastname: Inoue
authors.forename: Yasushi
authors.initials: Y
authors.lastname: Shigemori
authors.forename: Tsutomu
authors.initials: T
authors.lastname: Mikawa
mesh_terms: D017103:Bacillus Phages / Q000201:enzymology; D001426:Bacterial Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D004247:DNA / Q000096:biosynthesis*; D004261:DNA Replication; D004277:DNA, Single-Stranded / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D015894:Genome, Human; D006801:Humans; D009154:Mutation; D016133:Polymerase Chain Reaction / Q000379:methods; D013698:Templates, Genetic; D016963:Thermus thermophilus
chemical_list: D001426:Bacterial Proteins; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D004247:DNA; D004259:DNA-Directed DNA Polymerase