Highly thermostable RadA protein from the archaeon Pyrococcus woesei enhances specificity of simplex and multiplex PCR assays.
The radA gene of the hyperthermophilic archaeon Pyrococcus woesei (Thermococcales) was cloned and overexpressed in Escherichia coli. The 1050-bp gene codes for a 349-amino-acid polypeptide with an M r of 38,397 which shows 100 % positional amino acid identity to Pyrococcus furiosus RadA and 27.1 % to the E. coli RecA protein. Recombinant RadA was overproduced in Escherichia coli as a His-tagged fusion protein and purified to electrophoretic homogeneity using a simple procedure consisting of ammonium sulfate precipitation and metal-affinity chromatography. In solution RadA exists as an undecamer (11-mer). The protein binds both to ssDNA and dsDNA. RadA has been found to be highly thermostable, it remains almost unaffected by a 4-h incubation at 94 °C. The addition of the RadA protein to either simplex or multiplex PCR assays, significantly improves the specificity of DNA amplification by eliminating non-specific products. Among applications tested the RadA protein proved to be useful in allelic discrimination assay of HADHA gene associated with long-chain 3-hydroxylacyl-CoA dehydrogenase deficiency that in infancy may lead to hypotonia, serious heart and liver problems and even sudden death.
Department of Microbiology, University of Gdansk, Wita Stwosza 59, 80-308, Gdansk, Poland.
Aleksandra
A
Stefanska
Department of Microbiology, University of Gdansk, Wita Stwosza 59, 80-308, Gdansk, Poland.
Lidia
L
Gaffke
Collection of Plasmids and Microorganisms, University of Gdansk, Wita Stwosza 59, 80-308, Gdansk, Poland.
Anna-Karina
AK
Kaczorowska
Department of Microbiology, University of Gdansk, Wita Stwosza 59, 80-308, Gdansk, Poland.
Magdalena
M
Plotka
A&A Biotechnology, Aleja Zwyciestwa 96/98, 81-451, Gdynia, Poland.
Slawomir
S
Dabrowski
Department of Microbiology, University of Gdansk, Wita Stwosza 59, 80-308, Gdansk, Poland. tadeusz.kaczorowski@biol.ug.edu.pl.
Tadeusz
T
Kaczorowski
D019843:Archaeal Proteins / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D019641:DNA, Archaeal / Q000235:genetics; D004277:DNA, Single-Stranded / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D006358:Hot Temperature; D008969:Molecular Sequence Data; D060885:Multiplex Polymerase Chain Reaction; D055550:Protein Stability; D019711:Pyrococcus / Q000201:enzymology / Q000235:genetics*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
D019843:Archaeal Proteins; D019641:DNA, Archaeal; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; C117252:RadA protein, archaeal; D011994:Recombinant Proteins
Hyperthermophiles; RecA; Thermal stability; Thermococcales