Quadruplex priming amplification (QPA) is a straightforward assay that allows isothermal amplification of DNA and
possesses an intrinsic real-time detection mechanism. The key point of QPA is that a monomolecular DNA oligomer,
GGGTGGGTGGGTGGG (G3T), is capable of forming a quadruplex structure with unusually high thermal stability. In contrast,
the starting primer, which is a truncated version of G3T, is missing guanine residues critical for quadruplex formation. As a
result, the truncated sequence primes to primer binding site (PBS) without complication. When the polymerase adds the missing
guanines, the extended primer spontaneously folds into a DNA quadruplex and the PBS is ready for the next priming event. In
addition, primers containing fluorescent nucleotides, such as 3-methylisoxanthopterin, demonstrate a strong increase in
fluorescence upon quadruplex formation, which permits simple and effective quantification without extra probe molecules.
The intrinsic detection mechanism of QPA allows for further simplification of the assay by complete separation of the
recognition step from the signal amplification. In contrast, all current detection mechanisms require the presence of probe
molecules in the amplification reaction, since fluorescence signal is created upon hybridization of probes to amplicons. Earlier
studies demonstrated that QPA can be successfully adopted for molecular diagnostics, which requires amplification of short
(<30 nucleotides (nt)) DNA segments. For instance, QPA can achieve 1010-fold amplification in less than 40 min. QPA reaction
can be widely used in biomedical, molecular biology, genetics and forensic examination, genetic diseases and detection of
pathogenic microorganisms. Also, in scientific research. The RNA quadruplex demonstrates even higher thermal stability, than
DNA quadruplex. The QRA reaction that employs the RNA-containing primers enables the expansion of reaction temperature.
That will increase the use of QPA.