Quantification of polymerase chain reaction products by affinity-based hybrid collection

Abstract

We have used oligonucleotides modified with biotin in the 5'-end as primers in the polymerase chain reaction (PCR)-amplification. This results in the synthesis of 5'-biotinylated DNA molecules, which are detected by hybridization to a labelled probe in solution. The formed hybrids are collected on an avidin-matrix by mediation of the biotin residue of the target molecules. The affinity-based hybrid collection method is quantitative and makes it possible to measure the amount of DNA produced in the PCR-amplification. At low concentrations of template the efficiency of the process is close to 100%, making it possible to detect the presence of a few molecules of target DNA in 25 cycles. With high template concentrations the efficiency of the process is low.