Isothermal amplification of long DNA segments by quadruplex priming amplification

Abstract

Amplification of long DNA segments with the highest possible specificity and lowest bias is one of the main goals of modern genomics. Quadruplex priming amplification (QPA) is a single-primer isothermal method, which employs the free energy of quadruplex structures as the driving force for DNA amplification without any extra reaction components. As a result, QPA represents one of the simplest isothermal assays and was previously shown to be suitable for amplification of relatively short DNA sequences. The current study reveals that single-primer QPA can be used for both exponential and linear amplification of relatively long DNA segments (>100 nt), and switching between these modes can be accomplished by simple re-design of the primer used. While exponential amplification resulted in production of some undesired higher molecular weight species, linear QPA demonstrated highly specific amplification of the target molecules without any side products.