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Nucleic Acids Research 2006 34(9):e69; doi:10.1093/nar/gkl350
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Published online 17 May 2006

© The Author 2006. Published by Oxford University Press. All rights reserved
The online version of this article has been published under an open access model. Users are entitled to use, reproduce, disseminate, or display the open access version of this article for non-commercial purposes provided that: the original authorship is properly and fully attributed; the Journal and Oxford University Press are attributed as the original place of publication with the correct citation details given; if an article is subsequently reproduced or disseminated not in its entirety but only in part or as a derivative work this must be clearly indicated. For commercial re-use, please contact journals.permissions@oxfordjournals.org

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Improvements of rolling circle amplification (RCA) efficiency and accuracy using Thermus thermophilus SSB mutant protein

Jin Inoue3, Yasushi Shigemori and Tsutomu Mikawa1,2,3,*

Aisin Cosmos R&D Co. Ltd. 2-1-5 Kazusa-kamatari, Kisarazu, Chiba 292-0818 Japan 1 RIKEN Harima Institute/SPring-8 Mikazuki cho, Hyogo 679-5148, Japan 2 RIKEN Discovery Research Institute 2-1 Hirosawa, Wako, Saitama 351-0198, Japan 3 International Graduate School of Arts and Science, Yokohama City University 1-7-29, Suehiro-cho, Tsurumi-ku, Yokohama 230-0045, Japan

*To whom correspondence should be addressed. Tel: +81 45 508 7224; Fax: +81 45 508 7364; Email: mikawa{at}riken.jp

Received December 13, 2005. Revised February 6, 2006. Accepted April 19, 2006.

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.

The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors


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