Specific versus nonspecific isothermal DNA amplification through thermophilic polymerase and nicking enzyme activities

The isothermal exponential amplification reaction (EXPAR) combined with upstream generation of trigger oligonucleotides from a genomic target sequence and with downstream visual detection using DNA-functionalized gold nanospheres were employed to determine the cause of nonspecific DNA amplification observed in EXPAR. The results showed that early phase background amplification could be eliminated by physically separating the template and polymerase until the final reaction temperature is reached to enable detection of attomolar starting trigger concentrations.