Quantitative polymerase chain reaction by monitoring enzymatic activity of DNA polymerase.
Nucleic acid amplification by polymerase chain reaction (PCR) is a very powerful technique in terms of sensitivity but is limited in terms of ability to perform accurate quantitation. While there is a theoretical correlation between copies of input target sequence and those of PCR product, the quantitative nature of this relationship is obscured by unpredictable variations in reaction conditions and by inhibitory and/or stimulatory substances which might be present in sample preparations, especially those derived from biological fluids. To reliably estimate copies of input DNA target from PCR product, we designed a combination of internal and external control systems coupled to DNA/RNA hybridization and enzymatic immunodetection techniques. The internal control system served to monitor amplification efficiency and to correct for the effects of inhibitors or stimuli on the efficiency of the DNA amplification. The assay is quantitative, nonisotopic, and can be widely applied to assessment of the quantity of DNA present in a wide range of preparations.
Yang
B
B
Eudowood Division of Infectious Diseases, Department of Pediatrics, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205.
Yolken
R
R
Viscidi
R
R
D001483:Base Sequence; D004279:DNA, Viral / Q000235:genetics; D004259:DNA-Directed DNA Polymerase; D005069:Evaluation Studies as Topic; D005784:Gene Amplification; D006678:HIV / Q000235:genetics; D006801:Humans; D007124:Immunoenzyme Techniques; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D015347:RNA Probes; D012015:Reference Standards
D004279:DNA, Viral; D015347:RNA Probes; D004259:DNA-Directed DNA Polymerase