Methods / Real-time PCR applications

Linear-After-The-Exponential (LATE)–PCR: An advanced method of asymmetric PCR and its uses in quan


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Conventional asymmetric PCR is inefficient and difficult to optimize because limiting the concentration of one primer lowers its melting temperature below the reaction annealing temperature. Linear-After-The-Exponential (LATE)–PCR describes a new paradigm for primer design that renders assays as efficient as symmetric PCR assays, regardless of primer ratio. LATE-PCR generates single-stranded products with predictable kinetics for many cycles beyond the exponential phase. LATE-PCR also introduces new probe design criteria that uncouple hybridization probe detection from primer annealing and extension, increase probe reliability, improve allele discrimination, and increase signal strength by 80�50% relative to symmetric PCR. These improvements in PCR are particularly useful for real-time quantitative analysis of target numbers in small samples. LATE-PCR is adaptable to high throughput applications in fields such as clinical diagnostics, biodefense, forensics, and DNA sequencing. We showcase LATE-PCR via amplification of the cystic fibrosis Δ allele and the Tay-Sachs disease allele from single heterozygous cells.

Proceedings of the National Academy of Sciences of the United States of America.2003:101(7):1933

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