title: [Expression and purification of a novel thermophilic bacterial single-stranded DNA-binding protein and enhancement the synthesis of DNA and cDNA].
abstract: OBJECTIVE
  Express a novel species of single-stranded DNA-binding protein (SSB) derived from Thermococcus kodakarensis KOD1, abbreviated kod-ssb. And evaluate the effect of kod-ssb on PCR-based DNA amplification and reverse transcription.
  
  
  METHODS
  We express kod-ssb with the Transrtta (DE3), and kod-ssb was purified by affinity chromatography on a Ni2+ Sepharose column, detected by SDS-PAGE. To evaluate the effect of kod-ssb on PCR-based DNA amplification, the human beta globin gene was used as template to amplify a 5-kb, 9-kb and 13-kb. And to detect the effect of kod-ssb on reverse transcription, we used RNA from flu cell culture supernatant extraction as templates to implement qRT-PCR reaction.
  
  
  RESULTS
  The plasmid pET11a-kod was transformed into Transetta (DE3) and the recombinant strain Transetta (pET11 a-kod) was obtained. The kod-ssb was highly expressed when the recombinant strain Transetta(pET11a-kod) was induced by IPTG. The specific protein was detected by SDS-PAGE. To confirm that kod-ssb can enhance target DNA synthesis and reduce PCR by-products, 5-, 9-, and 13-kb human beta globin gene fragments were used as templates for PCR. When PCR reactions did not include SSB proteins, the specific PCR product was contaminated with non-specific products. When kod -ssb was added, kod-ssb significantly enhanced amplification of the 5-, 9-and 13-kb target product and minimised the non-specific PCR products. To confirm that kod-ssb can enhance target cDNA synthesis, RNA from flu cell culture supernatant extraction was used as templates for qRT-PCR reaction. The results was that when kod-ssb was added, kod-ssb significantly enhanced the synthesis of cDNA, average Ct value is 19.42, and the average Ct value without kod-ssb is 22.15.
  
  
  CONCLUSIONS
  kod-ssb may in future be used to enhance DNA and cDNA amplification.
authors.affiliation: Department of Hepatobiliary Surgery Tianjin Fifth Central Hospital, China.
authors.forename: Xiao-Wei
authors.initials: XW
authors.lastname: Jia
authors.forename: Guo-Hui
authors.initials: GH
authors.lastname: Zhang
authors.forename: Hai-Yan
authors.initials: HY
authors.lastname: Shi
mesh_terms: D019843:Archaeal Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D002846:Chromatography, Affinity; D004269:DNA, Bacterial / Q000235:genetics* / Q000378:metabolism; D018076:DNA, Complementary / Q000235:genetics* / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D015870:Gene Expression; D019712:Thermococcus / Q000235:genetics* / Q000378:metabolism
chemical_list: D019843:Archaeal Proteins; D004269:DNA, Bacterial; D018076:DNA, Complementary; D004268:DNA-Binding Proteins