Format

Send to

Choose Destination
See comment in PubMed Commons below
Nucleic Acids Res. 2006 May 17;34(9):e69.

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

Author information

  • 1Aisin Cosmos R&D Co. Ltd, 2-1-5 Kazusa-kamatari, Kisarazu, Chiba 292-0818 Japan.

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.

PMID:
16707659
PMCID:
PMC1463899
DOI:
10.1093/nar/gkl350
[PubMed - indexed for MEDLINE]
Free PMC Article
PubMed Commons home

PubMed Commons

0 comments
How to join PubMed Commons

    Supplemental Content

    Full text links

    Icon for Silverchair Information Systems Icon for PubMed Central
    Loading ...
    Support Center