PCR is a formidable and potent technology that serves as an indispensable tool in a wide range of biological disciplines. However, due to the ease of use and often lack of rigorous standards many PCR applications can lead to highly variable, inaccurate, and ultimately meaningless results. Thus, rigorous method validation must precede its broad adoption to any new application. Multi-template samples possess particular features, which make their PCR analysis prone to artifacts and biases: multiple homologous templates present in copy numbers that vary within several orders of magnitude. Such conditions are a breeding ground for chimeras and heteroduplexes. Differences in template amplification efficiencies and template competition for reaction compounds undermine correct preservation of the original template ratio. In addition, the presence of inhibitors aggravates all of the above-mentioned problems. Inhibitors might also have ambivalent effects on the different templates within the same sample. Yet, no standard approaches exist for monitoring inhibitory effects in multitemplate PCR, which is crucial for establishing compatibility between samples.

• Klíčová slova
• CDCE, constant denaturing capillary electrophoresis, Chimera, DGGE, denaturing gradient gel electrophoresis, DHPLC, denaturing high-performance liquid chromatography, HPLC, high-performance liquid chromatography, Multi-template PCR, PAAG, polyacrylamide gel, SSCA, single strand conformation analysis, T-RFLP, terminal restriction fragment length polymorphism, TGGE, temperature gradient gel electrophoresis,
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH

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.

• MeSH
• DNA chemie   genetika   MeSH
• fluorescenční barviva chemie   MeSH
• lidé MeSH
• myši inbrední C57BL MeSH
• myši MeSH
• polymerázová řetězová reakce přístrojové vybavení   MeSH
• propylenglykol chemie   MeSH
• trehalosa chemie   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• DNA MeSH
• fluorescenční barviva MeSH
• propylenglykol MeSH
• trehalosa MeSH

Tetraalkylammonium (TAA) derivatives have been reported to serve as stabilizers of asymmetrical cyanine dyes in aqueous solutions and to increase the yield and efficiency of polymerase chain reaction (PCR) detected by end-point analysis. In this study, we compared the ability of various TAA derivatives (with alkyl chain ranging from 1 to 5 carbons) and some other compounds to serve as enhancers of real-time PCR based on fluorescence detection from intercalating dye SYBR Green I (SGI). Our data indicate that TAA chlorides and some other TAA derivatives serve as potent enhancers of SGI-monitored real-time PCR. Optimal results were obtained with 10-16 mM tetrapropylammonium chloride. The effect of TAA compounds was dependent on the nature of counter ions present and composition of the reaction mixtures used. Based on measurements of SGI-generated fluorescence signal in the presence of PCR-amplified DNA fragments, oligonucleotide primers and/or various additives, we propose that TAA-derivatives reduce the binding of SGI to oligonucleotide primers and thus enhance primer-template interactions during annealing phase. Furthermore, these compounds serve as stabilizers of SGI-containing PCR mixtures. The combined data indicate that TAA derivatives might be a new class of additives contributing to robustness of real-time PCR monitored by asymmetrical cyanine dye SGI.

• MeSH
• benzothiazoly MeSH
• chinoliny MeSH
• diaminy MeSH
• fluorescenční barviva analýza   MeSH
• kvartérní amoniové sloučeniny chemie   MeSH
• myši inbrední C57BL MeSH
• myši MeSH
• organické látky analýza   MeSH
• polymerázová řetězová reakce metody   MeSH
• teplota MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• srovnávací studie MeSH
• Názvy látek
• benzothiazoly MeSH
• chinoliny MeSH
• diaminy MeSH
• fluorescenční barviva MeSH
• kvartérní amoniové sloučeniny MeSH
• organické látky MeSH
• SYBR Green I MeSH Prohlížeč