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 involving hybridization, for the enzyme-linked immunosorbent assay (ELISA)-based detection, quantitation, and validation of polymerase chain reaction (PCR)-amplified DNA. Both procedures are applicable to any PCR reaction, and do not require specially synthesized or enzyme-tagged oligonucleotides. We obtained accurate quantitation of PCR-amplified human cc10kDa cDNA with a sensitivity of about 0.6 fmoles. This cDNA was also used to detect single-base insertions, deletions, and substitutions specifically. Additionally, we could readily distinguish zinc-finger Y chromosome-specific genomic sequences in mixtures of male and female cells and normal and mutant cystic fibrosis transmembrane conductance regulator (CFTR) gene sequences in crude fibroblast lysates. To our knowledge, this is the first report of point mutation detection in solution by ELISA without allele-specific amplification or ligation. These novel procedures have vast potential for basic and clinical applications, including gene expression studies, rapid screening of genetic diseases, detection of oncogene and anti-oncogene mutations, and identification of pathogens (e.g., HIV-1) in clinical specimens.
Miele
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Section on Development Genetics, NICHD, NIH, Bethesda, MD 20892.
Peri
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Cordella-Miele
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Mukherjee
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D001483:Base Sequence; D002478:Cells, Cultured; D003550:Cystic Fibrosis; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D018076:DNA, Complementary / Q000032:analysis* / Q000097:blood; D004268:DNA-Binding Proteins / Q000235:genetics; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D005260:Female; D005347:Fibroblasts / Q000737:chemistry; D006801:Humans; D051741:Kruppel-Like Transcription Factors; D008214:Lymphocytes / Q000737:chemistry; D008297:Male; D008565:Membrane Proteins / Q000235:genetics; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D015345:Oligonucleotide Probes; D017354:Point Mutation; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D014157:Transcription Factors; D016335:Zinc Fingers / Q000235:genetics
C505032:CFTR protein, human; D018076:DNA, Complementary; D004268:DNA-Binding Proteins; D051741:Kruppel-Like Transcription Factors; D008565:Membrane Proteins; D015345:Oligonucleotide Probes; D014157:Transcription Factors; C054430:ZFY protein, human; D019005:Cystic Fibrosis Transmembrane Conductance Regulator