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

DOI Web Site 被引用文献1件
  • J. Aquiles Sanchez
    Department of Biology, MS 008, Brandeis University, 415 South Street, Waltham, MA 02454-9110
  • Kenneth E. Pierce
    Department of Biology, MS 008, Brandeis University, 415 South Street, Waltham, MA 02454-9110
  • John E. Rice
    Department of Biology, MS 008, Brandeis University, 415 South Street, Waltham, MA 02454-9110
  • Lawrence J. Wangh
    Department of Biology, MS 008, Brandeis University, 415 South Street, Waltham, MA 02454-9110

書誌事項

公開日
2004-02-09
DOI
  • 10.1073/pnas.0305476101
公開者
Proceedings of the National Academy of Sciences

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<jats:p> 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 80–250% 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 <jats:italic>CF</jats:italic> Δ <jats:italic>508</jats:italic> allele and the Tay-Sachs disease <jats:italic>TSD 1278</jats:italic> allele from single heterozygous cells. </jats:p>

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