METHOD FOR ENHANCING DETECTION ABILITY OF NUCLEIC ACID ASSAYS EMPLOYING POLYMERASE CHAIN REACTION

摘要

This invention relates to a new and novel method for significantly enhancing the sensitivity of assays aimed at detecting the presence of target deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) sequences in a sample. Specifically, this method applies to techniques that employ polymerase chain reactions (PCR?) or other nucleic amplificatio techniques to amplify copies of the target DNA or RNA (via reverse-transclil,tase followed by PCR, Murakawa et ah 1988 DNA 7-287) to allow for detection. The method of thepresent invention combines the following four steps for the first time: DNA amplicon synthesis and amplification in a modified PCR using a target nucleic acid sequence as a template; amplicon transcription into RNA and amplification; RNA:DNA hybrid formation; and immunochemical detection of heteroduplex nucleic acid sequences. This method extends the standard PCR techniques to accommodate amplification of copies of the PCR product and transcription into RNA, which, following binding to DNA sequences facilitates immunochemical detection of the RNA:DNA hybrids thereby formed. This combination of steps results in a new assay with increased simplicity, speed and sensitivity, including a greatly improved signal-to-noise ratio, over standard nucleic acid detection methods. This improved method will be extremely useful for performing analyses in the foiensic, clinical, agricultural and biological fields.