Synthesis in vitro and application of biotinylated DNA probes for human papilloma virus type 16 by utilizing the polymerase chain reaction

Abstract

The polymerase chain reaction (PCR) has been used to incorporate biotinylated deoxynucleotide triphosphate analogues into a 120 bp sequence from the E6 region of human papilloma virus type 16 (HPV 16). No loss of amplification efficiency is observed utilizing concentrations of up to 200 microM-biotin-11-dUTP, or 180 microM-biotin-7-dATP and -biotin-16-dUTP (where the numbers refer to the number of carbon atoms in the spacer arms). Internally biotinylated PCR products can be detected following slot-blot or vacuum transfer to mitrocellulose or nylon filters without prior electrophoretic separation of the reactants, since unincorporated biotinylated analogues pass through the filter. Internally biotinylated PCR products can also be applied as hybridization probes in Southern blot analysis or in situ hybridization. This system enables detection of PCR products or target sequences at levels below that for 5′-biotinylated probes and can be applied in an ‘open sandwich assay’ without the need for a separate labelled probe currently required in conventional sandwich assays. However, as hybridization probes, sensitivity may be limited by the steric hindrance of strand hybridization possibly due to the spacer arms linking the nucleotides to the biotin molecule.