Tetraalkylammonium derivatives as real-time PCR enhancers and stabilizers of the qPCR mixtures containing SYBR Green I

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.

[1]  Y. Itoyama,et al.  A reproducible assay of polymerase chain reaction to detect trinucleotide repeat expansion of Huntington's disease and senile chorea. , 1996, Neurological Research.

[2]  S. Weissman,et al.  Uniform amplification of a mixture of deoxyribonucleic acids with varying GC content. , 1996, Genome research.

[3]  M K Sidhu,et al.  Dimethyl sulfoxide improves RNA amplification. , 1996, BioTechniques.

[4]  K. Fong,et al.  A specificity enhancer for polymerase chain reaction. , 1990, Nucleic acids research.

[5]  M. Hauser,et al.  Evaluation of a homemade SYBR green I reaction mixture for real-time PCR quantification of gene expression. , 2002, BioTechniques.

[6]  W. Lüke,et al.  Improvement of PCR amplified DNA sequencing with the aid of detergents. , 1990, Nucleic acids research.

[7]  Eric D. Green,et al.  Genome analysis : a laboratory manual , 1997 .

[8]  D. J. Sharkey,et al.  Monoclonal antibodies prepared against the DNA polymerase from Thermus aquaticus are potent inhibitors of enzyme activity. , 1994, Journal of immunological methods.

[9]  G. Sarkar,et al.  Formamide can dramatically improve the specificity of PCR. , 1990, Nucleic acids research.

[10]  J. Lanchbury,et al.  Strategy for controlling preferential amplification and avoiding false negatives in PCR typing. , 1996, BioTechniques.

[11]  C. Dieffenbach,et al.  PCR primer: a laboratory manual. , 2003 .

[12]  Frank Vitzthum,et al.  Investigations on DNA intercalation and surface binding by SYBR Green I, its structure determination and methodological implications. , 2004, Nucleic acids research.

[13]  M. Salimans,et al.  Rapid and simple method for purification of nucleic acids , 1990, Journal of clinical microbiology.

[14]  J. Zikán,et al.  ESTABLISHMENT AND CHARACTERIZATION OF PERMANENT MURINE HYBRIDOMAS SECRETING MONOCLONAL ANTI‐THY‐1 ANTIBODIES * , 1980, Journal of immunogenetics.

[15]  S. Loening,et al.  Dimethyl sulfoxide as additive in ready-to-use reaction mixtures for real-time polymerase chain reaction analysis with SYBR Green I dye. , 2001, Analytical biochemistry.

[16]  Stephen A. Bustin,et al.  A-Z of Quantitative PCR , 2004 .

[17]  C. Schutt,et al.  Novel sulfoxides facilitate GC-rich template amplification. , 2002, BioTechniques.

[18]  C. Schutt,et al.  The enhancement of PCR amplification by low molecular-weight sulfones. , 2001, Gene.

[19]  C. Schutt,et al.  The enhancement of PCR amplification by low molecular weight amides. , 2001, Nucleic acids research.

[20]  P. Hengen.  Optimizing multiplex and LA-PCR with betaine. , 1997, Trends in biochemical sciences.

[21]  J F Medrano,et al.  Organic solvents as facilitators of polymerase chain reaction. , 1991, BioTechniques.

[22]  E. Chevet,et al.  Low concentrations of tetramethylammonium chloride increase yield and specificity of PCR. , 1995, Nucleic acids research.

[23]  N Sasaki,et al.  Thermostabilization and thermoactivation of thermolabile enzymes by trehalose and its application for the synthesis of full length cDNA. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[24]  C. J. Di Como,et al.  Effects of ethidium bromide and SYBR Green I on different polymerase chain reaction systems. , 2000, Journal of biochemical and biophysical methods.

[25]  Improved stability and electrophoretic properties of preformed fluorescent cationic dye-DNA complexes in a taps-tetrapentylammonium buffer in agarose slab gels. , 1997, Analytical biochemistry.

[26]  J. Dougherty,et al.  The thermal stability of oligonucleotide duplexes is sequence independent in tetraalkylammonium salt solutions: application to identifying recombinant DNA clones. , 1988, Nucleic acids research.

[27]  P. Dráber,et al.  Functional expression of the endogenous Thy‐1 gene and the transfected murine Thy‐1.2 gene in rat basophilic leukemia cells , 1991, European journal of immunology.

[28]  Bruce Budowle,et al.  Use of formamide to improve amplification of HLA DQα sequences , 1991 .

[29]  M. Ashburner.  A Laboratory manual , 1989 .

[30]  K Varadaraj,et al.  Denaturants or cosolvents improve the specificity of PCR amplification of a G + C-rich DNA using genetically engineered DNA polymerases. , 1994, Gene.

[31]  P. V. von Hippel,et al.  Alteration of the relative stability of dA-dT and dG-dC base pairs in DNA. , 1973, Proceedings of the National Academy of Sciences of the United States of America.

[32]  C. Saint,et al.  Comparison of SYTO9 and SYBR Green I for real-time polymerase chain reaction and investigation of the effect of dye concentration on amplification and DNA melting curve analysis. , 2005, Analytical biochemistry.

[33]  Andrej-Nikolai Spiess,et al.  Trehalose is a potent PCR enhancer: lowering of DNA melting temperature and thermal stabilization of taq polymerase by the disaccharide trehalose. , 2004, Clinical chemistry.

[34]  Petr Dráber,et al.  New specificity and yield enhancer of polymerase chain reactions , 2000, Nucleic Acids Res..