Thermal stability of quadruplex primers for highly versatile isothermal DNA amplification.
Biophys Chem
; 185: 14-8, 2014 Jan.
Article
em En
| MEDLINE
| ID: mdl-24317195
ABSTRACT
Quadruplex priming amplification (QPA) allows isothermal amplification of nucleic acids with improved yield and simplified detection. This assay is based on a DNA quadruplex, GGGTGGGTGGGTGGG (G3T), which in the presence of specific cations possesses unusually high thermal stability. QPA employs truncated G3T sequences as primers, which upon polymerase elongation, self-dissociate from the binding site and allow the next round of priming without thermal unfolding of amplicons. The rate of amplification strongly depends on the thermal stability of the primer/primer binding site (PBS) complex and to date QPA has been demonstrated to work over a narrow temperature range. To expand the capabilities of QPA, in the present study, we studied the fold and thermodynamic properties of the wild-type G3T and variants containing sequence modifications or extensions at the 5'-end. Circular dichroism studies demonstrate that the substitution of thymidines by other nucleotides or GC addition at the 5'-end does not change the parallel fold of G3T. Thermal unfolding experiments revealed that purine bases incorporated at loop positions and 5'-end dinucleotide extension significantly destabilize the quadruplex, while loop pyrimidines have almost no effect. Overall, the results of these studies suggest that linear isothermal QPA can be performed over a wide temperature range to accommodate both thermophilic and mesophilic DNA polymerases.
Palavras-chave
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
DNA
/
Primers do DNA
/
Técnicas de Amplificação de Ácido Nucleico
/
Quadruplex G
Idioma:
En
Ano de publicação:
2014
Tipo de documento:
Article