Quantitative Analysis of DNA Sequences by PCR and Solid-Phase Minisequencing
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The PCR technique provides a highly specific and sensitive means for analyzing nucleic acids, but it does not allow their direct quantification. This limitation is because the efficiency of PCR depends on the amount of template sequence present in the sample, and the amplification is exponential only at low template concentrations (1). Owing to this plateau effect of PCR, the amount of amplification product does not directly reflect the original amount of template. Moreover, subtle differences in reaction conditions, such as material from biological samples, may cause significant sample-to-sample variation in the final yield of the PCR product.
The problem of performing accurate quantitative PCR analyses has been addressed by two principal approaches. A quantitative PCR result can be obtained by “kinetic PCR,” in which the amplification process is monitored at numerous times or concentration points (2,3). Most conveniently, the amplification process can be monitored in...
KeywordsMolecular Genetic Laboratory Molecular Beacon Probe Determine Allele Frequency Minisequencing Reaction Scintillate Polystyrene
- 2.Murphy LD, Herzog CE, Rudick JB, Fojo AT, Bates SE (1990) Use of the polymer-ase chain reaction in the quantitation of mdr-1 gene expression. Biochemistry 29:10,351–10,356Google Scholar