Nucleic acid capture assay, a new method for direct quantitation of nucleic acids

Abstract

Technologies allowing direct detection of specic RNA/DNA sequences occasionally serve as an alternative to amplication methods for gene expression studies. In these direct methods the hybridization of probes takes place in complex mixtures, thus specicity and sensitivity still limit the use of current technologies. To address these challenges, we developed a new technique called the nucleic acid capture assay, involving a direct multi-capture system. This approach combines a 3-ethylene glycol scaffolding with the incorporation of 2-methoxy deoxyribonucleotides in the capture sequences. In our design, all nucleotides other than those complementary to the target mRNA have been replaced by an inert linker, resulting in signicant reductions in non-specic binding. We also provide a versatile method to detect the presence of captured targets by using specic labeled probes with alkaline phosphatase-conjugated anti-label antibodies. This direct, exible and reliable technique for gene expression analysis is well suited for highthroughput screening and has potential for DNA microarray applications.