Linear-after-the-exponential (LATE)-PCR: an advanced method of asymmetric PCR and its uses in quantitative real-time analysis.
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 CFDelta508 allele and the Tay-Sachs disease TSD 1278 allele from single heterozygous cells.
Sanchez
J Aquiles
JA
Department of Biology, MS 008, Brandeis University, 415 South Street, Waltham, MA 02454-9110, USA.
Pierce
Kenneth E
KE
Rice
John E
JE
Wangh
Lawrence J
LJ
D000483:Alleles; D002460:Cell Line; D003550:Cystic Fibrosis / Q000235:genetics; D017931:DNA Primers / Q000235:genetics; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results; D012107:Research Design; D012680:Sensitivity and Specificity; D013661:Tay-Sachs Disease / Q000235:genetics; D013696:Temperature; D013997:Time Factors
D017931:DNA Primers