Monitoring hybridization during polymerase chain reaction

The polymerase chain reaction (PCR) is usually analyzed by gel electrophore sis for size separation of PCR products. Additional separation techniques, such as single-stranded conformational polymorphism (SSCP), denaturing grad ient gel electrophoresis (DGGE), temperature gradient gel electrophoresis ( TGGE) and denaturing high-performance liquid chromatography (DHPLC), can al so be used to scan for sequence alterations. These techniques are all based on the effect of PCR product hybridization on mobility. Hybridization can also be monitored with fluorescence during PCR without chromatographic or e lectrophoretic separation. Continuous monitoring of PCR allows the detectio n, quantification and sequence specificity of PCR products to be assessed, often without any need for further analysis. In such a closed system, PCR q uantification with sensitivity to the single copy level can be achieved usi ng either double-stranded DNA binding dyes or fluorescently labeled allele- specific oligonucleotide (ASO) probes. Melting curve analysis with ASO prob es can be used to genotype various alleles, including single base alteratio ns. The integration of rapid cycle PCR and ASO probes in an automated syste m greatly facilitates research and clinical applications of nucleic acid an alysis in genetics, oncology, and infectious disease. (C) 2000 Elsevier Sci ence B.V. All rights reserved.