HOMOGENEOUS PHASE PYROPHOSPHATE (PPI) MEASUREMENT (H3PIM) - A NONRADIOACTIVE, QUANTITATIVE DETECTION SYSTEM FOR NUCLEIC-ACID SPECIFIC HYBRIDIZATION METHODOLOGIES INCLUDING GENE AMPLIFICATION

A one-step, non-radioactive, homogeneous phase revelation system designed to detect and quantify nucleic acid hybridization is described. The principle of. the procedure, termed homogeneous phase pyrophosphate (PPi) measurement (H3PIM), is to detect and quantify the release of PPi from nucleotides, which occurs stoichiometrically when nucleic acids are synthesized. The method does not require any special reagents before the H3PIM revelation step. H3PIM is particularly adapted to quantitative measurement of gene amplification or cDNA gene expression using PCR, but can also be used following random priming or simultaneous multi-step nucleic acid amplification. This rapid, sensitive, liquid phase procedure permits the design of low-cost, fully automated devices for gene detection and quantification.