Single-stranded DNA binding protein facilitates specific enrichment of circular DNA molecules using rolling circle amplification

Tsutomu Mikawa, Jin Inoue, Yasushi Shigemori

Research output: Contribution to journalArticlepeer-review

2 Citations (Scopus)

Abstract

Many techniques in molecular biology require the use of pure nucleic acids in general and circular DNA (plasmid or mitochondrial) in particular. We have developed a method to separate these circular molecules from a mixture containing different species of nucleic acids using rolling circle amplification (RCA). RCA of plasmid or genomic DNA using random hexamers and bacteriophage Phi29 DNA polymerase has become increasingly popular for the amplification of template DNA in DNA sequencing protocols. Recently, we reported that the mutant single-stranded DNA binding protein (SSB) from Thermus thermophilus (TthSSB) HB8 eliminates nonspecific DNA products in RCA reactions. We developed this method for separating circular nucleic acids from a mixture having different species of nucleic acids. Use of the mutant TthSSB resulted in an enhancement of plasmid or mitochondrial DNA content in the amplified product by approximately 500×. The use of mutant TthSSB not only promoted the amplification of circular target DNA over the background but also could be used to enhance the amplification of circular targets over linear targets. Crown

Original languageEnglish
Pages (from-to)81-84
Number of pages4
JournalAnalytical Biochemistry
Volume391
Issue number2
DOIs
Publication statusPublished - 2009 Aug 15
Externally publishedYes

Keywords

  • Mitochondrial DNA
  • Rolling circle amplification
  • Single-stranded DNA binding protein
  • Thermus thermophilus HB8

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

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