Exponential amplification of DNA with very low background using graphene oxide and single-stranded binding protein to suppress non-specific amplification

Abstract

The high background signal caused by non-specific amplification (NSA) is a serious issue when using the conventional exponential amplification reaction (EXPAR). We describe a novel method of suppressing NSA using 10 mg L−1 graphene oxide (GO) to prevent non-specific binding of DNA polymerase to the template, resulting in ultra-low background. A side effect of the use of GO is the slow release of ssDNA from the GO surface, with the positive signal decreasing accordingly. The problem of low signal intensity is addressed by applying a 2 μM solution of single-stranded binding protein (SSB) to accelerate the release of template for EXPAR. The use of GO and SSB in EXPAR can substantially suppress NSA, but it does not compromise the performance of the quantification step. The improved EXPAR presented here can detect concentration as low as 5 aM of trigger, which is approximately four orders of magnitude lower than that of conventional EXPAR under the same experimental conditions.