title: 1,2-propanediol-trehalose mixture as a potent quantitative real-time PCR enhancer.
abstract: BACKGROUND
  Quantitative real-time PCR (qPCR) is becoming increasingly important for DNA genotyping and gene expression analysis. For continuous monitoring of the production of PCR amplicons DNA-intercalating dyes are widely used. Recently, we have introduced a new qPCR mix which showed improved amplification of medium-size genomic DNA fragments in the presence of DNA dye SYBR green I (SGI). In this study we tested whether the new PCR mix is also suitable for other DNA dyes used for qPCR and whether it can be applied for amplification of DNA fragments which are difficult to amplify.
  
  
  RESULTS
  We found that several DNA dyes (SGI, SYTO-9, SYTO-13, SYTO-82, EvaGreen, LCGreen or ResoLight) exhibited optimum qPCR performance in buffers of different salt composition. Fidelity assays demonstrated that the observed differences were not caused by changes in Taq DNA polymerase induced mutation frequencies in PCR mixes of different salt composition or containing different DNA dyes. In search for a PCR mix compatible with all the DNA dyes, and suitable for efficient amplification of difficult-to-amplify DNA templates, such as those in whole blood, of medium size and/or GC-rich, we found excellent performance of a PCR mix supplemented with 1 M 1,2-propanediol and 0.2 M trehalose (PT enhancer). These two additives together decreased DNA melting temperature and efficiently neutralized PCR inhibitors present in blood samples. They also made possible more efficient amplification of GC-rich templates than betaine and other previously described additives. Furthermore, amplification in the presence of PT enhancer increased the robustness and performance of routinely used qPCRs with short amplicons.
  
  
  CONCLUSIONS
  The combined data indicate that PCR mixes supplemented with PT enhancer are suitable for DNA amplification in the presence of various DNA dyes and for a variety of templates which otherwise can be amplified with difficulty.
authors.affiliation: Department of Signal Transduction, Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, Vídeňská 1083, 142 20 Prague 4, Czech Republic.
authors.forename: Helena
authors.initials: H
authors.lastname: Horáková
authors.forename: Iva
authors.initials: I
authors.lastname: Polakovičová
authors.forename: Gouse M
authors.initials: GM
authors.lastname: Shaik
authors.forename: Jiří
authors.initials: J
authors.lastname: Eitler
authors.forename: Viktor
authors.initials: V
authors.lastname: Bugajev
authors.forename: Lubica
authors.initials: L
authors.lastname: Dráberová
authors.forename: Petr
authors.initials: P
authors.lastname: Dráber
mesh_terms: D000818:Animals; D004247:DNA / Q000737:chemistry / Q000235:genetics; D005456:Fluorescent Dyes / Q000737:chemistry; D006801:Humans; D051379:Mice; D008810:Mice, Inbred C57BL; D016133:Polymerase Chain Reaction / Q000295:instrumentation*; D019946:Propylene Glycol / Q000737:chemistry*; D014199:Trehalose / Q000737:chemistry*
chemical_list: D005456:Fluorescent Dyes; D019946:Propylene Glycol; D004247:DNA; D014199:Trehalose