Unexpected Mechanism and Inhibition Effect for Nonspecific Amplification Involving Dynamic Binding of Primers with Background DNA

Nonspecific amplification is a serious issue in DNA detection as it can lead to false-positive results and reduce specificity. It is very important to well understand its mechanism through sequencing nonspecific products. Here, an approach is developed using a nanopore sequencing technique after acquiring the long repetitive sequence of DNA products from nonspecific amplification. Based on the sequencing results, a new mechanism of nonspecific amplification designated as dynamic mismatched primer binding (DMPB) with the background DNA (bgDNA) is proposed. Unexpectedly, our findings show that the primers (∼20 nt) can bind to bgDNA for primer extension when only 6–11 fully matched (9–14 mismatched) base pairs are formed. After the single-stranded DNAs (ssDNAs) attached to the first primer are produced, more interestingly, with the aid of DNA polymerase, the other primer can bind to these ssDNAs in the case that the fully matched base pairs formed between them are even shorter than 6 bp. As a result, perfect "seeds" for polymerase chain reaction with information on both primers are produced so that exponential nonspecific amplification can occur. The DMPB mechanism can explain nonspecific amplification in other approaches as well. Finally, a mini-hairpin DNA is used to effectively inhibit nonspecific amplification by preventing the formation of an unexpected primer–bgDNA complex.