The simplification of methods for the analysis of amplified nucleic acid sequences has largely focused on the development of nonradioisotopic detection reagents, and on the development of instrument systems to perform PCR in microwell plates. Sample preparation methods remain largely unchanged from techniques described a decade or more ago. While DNA extraction is relatively simple, most methods have concentrated on single tube techniques, which remain labor-intensive and whose application in screening tests is still problematic. The labile nature of RNA extracts makes reproducible, robust RNA extraction for analysis even more difficult. We propose to apply affinity based hybrid collection techniques developed in house for PCR product detection and quantitation to the problem of sample preparation for PCR in a screening assay. We further propose to develop these methods for specific application to the quantitation of HIV-I titers in genital secretions, a problem of immediate relevance in the study of HIV- I sexual transmission.Awardee's statement of the potential commercial applications of the research: In contrast to developments in PCR instrument systems and product detection schemes, little assay simplification has been attained in the area of sample preparation. We propose to adapt our proprietary capture plate assay for PCR products to the capture of specific nucleic acid sequences prior to amplification. The abihty to perform sample preparation in the 96-well screening format has obvious advantages, particularly for wide-scale application of PCR in the clinical laboratory.National Institute of Allergy and Infectious Diseases (NIAID)
