| Autor: |
Stefanska, Aleksandra, Gaffke, Lidia, Kaczorowska, Anna-Karina, Plotka, Magdalena, Dabrowski, Slawomir, Kaczorowski, Tadeusz |
| Zdroj: |
Journal of Applied Genetics; May 2016, Vol. 57 Issue: 2 p239-249, 11p |
| Abstrakt: |
The radAgene 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 Mrof 38,397 which shows 100 % positional amino acid identity to Pyrococcus furiosusRadA and 27.1 % to the E. coliRecA protein. Recombinant RadA was overproduced in Escherichia colias 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 HADHAgene 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. |
| Databáze: |
Supplemental Index |
| Externí odkaz: |
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