NONRADIOMETRIC ELISA-BASED QUANTITATION AND VALIDATION OF POLYMERASE CHAIN REACTION-AMPLIFIED DNA, INCLUDING DETECTION OF POINT MUTATIONS, WITHOUT ALLELE-SPECIFIC AMPLIFICATION, OR LIGATION

We describe two simple novel procedures, one direct and the other invo lving hybridization, for the enzyme-linked immunosorbent assay (ELISA) -based detection, quantitation, and validation of polymerase chain rea ction (PCR)-amplified DNA, Both procedures are applicable to any PCR r eaction, and do not require specially synthesized or enzyme-tagged oli gonucleotides, We obtained accurate quantitation of PCR-amplified huma n cc10kDa cDNA with a sensitivity of about 0.6 fmoles, This cDNA was a lso used to detect single-base insertions, deletions, and substitution s specifically, Additionally, we could readily distinguish zinc-finger Y chromosome-specific genomic sequences in mixtures of male and femal e cells and normal and mutant cystic fibrosis transmembrane conductanc e regulator (CFTR) gene sequences in crude fibroblast lysates, To our knowledge, this is the first report of point mutation detection in sol ution by ELISA without allele-specific amplification or ligation, Thes e novel procedures have vast potential for basic and clinical applicat ions, including gene expression studies, rapid screening of genetic di seases, detection of oncogene and anti-oncogene mutations, and identif ication of pathogens (e,g,, HIV-1) in clinical specimens,