A NOVEL RAPID HYBRIDIZATION TECHNIQUE - PAPER-CHROMATOGRAPHY HYBRIDIZATION ASSAY (PACHA)

A new DNA hybridization technique, based on chromatographic migration of DNA on a nitrocellulose strip passing through an immobilized probe area, is described. The new paper chromatography hybridization assay ( PACHA) is faster and simpler to use than the conventional dot hybridiz ation assay. In this assay, an aliquot of biotinylated, PCR-amplified target DNA is applied to one end of a nitrocellulose strip. The DNA mi grates to the opposite end of the strip by capillary forces and hybrid izes to a specific DNA probe immobilized in a reaction zone (RZ), loca ted in the middle of the strip. Unhybridized DNA migrates away from th e RZ. The biotinylated hybrid is visualized by a color reaction employ ing a streptavidin-alkaline phosphatase (SA-AP) conjugate and a specif ic chromogenic substrate. The new PACHA technique allows for detection of as little as 1-5 pg of specific human papilloma virus 16 (HPV16) D NA in 25 min of hybridization. In this system, the hybridization effic iency is controlled by the flow velocity of the hybridization solution (HS) and by the volume of the amplified labeled DNA migrating across the immobilized probe. Glycerol (30%) or polyvinyl pyrrolidone (PVP)(1 %) reduces the flow rate by a factor of 2.5-3 and increases the sensit ivity of the assay by a factor of 5.2 for glycerol and 2.6 for PVP. Th is novel method ensures efficient hybridization to multiple probes and appears to be superior to currently available solid-phase hybridizati on techniques.