title: Linear-after-the-exponential polymerase chain reaction and allied technologies. Real-time detection strategies for rapid, reliable diagnosis from single cells.
abstract: Accurate detection of gene sequences in single cells is the ultimate challenge to polymerase chain reaction (PCR) sensitivity. Unfortunately, commonly used conventional and real-time PCR techniques are often too unreliable at that level to provide the accuracy needed for clinical diagnosis. Here we provide details of linear-after-the-exponential-PCR (LATE-PCR), a method similar to asymmetric PCR in the use of primers at different concentrations, but with novel design criteria to ensure high efficiency and specificity. Compared with conventional PCR, LATE-PCR increases the signal strength and allele discrimination capability of oligonucleotide probes such as molecular beacons and reduces variability among replicate samples. The analysis of real-time kinetics of LATE-PCR signals provides a means for improving the accuracy of single cell genetic diagnosis.
authors: Department of Biology, Brandeis University, Waltham, MA, USA.
authors: Kenneth E
authors: KE
authors: Pierce
authors: Lawrence J
authors: LJ
authors: Wangh
mesh_terms: D002477:Cells / Q000737:chemistry; D003550:Cystic Fibrosis / Q000175:diagnosis; D004247:DNA / Q000032:analysis / Q000235:genetics; D017931:DNA Primers / Q000737:chemistry; D030342:Genetic Diseases, Inborn / Q000175:diagnosis*; D006579:Heterozygote; D006801:Humans; D009154:Mutation; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000592:standards*; D020641:Polymorphism, Single Nucleotide; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA / Q000379:methods*; D013997:Time Factors
chemical_list: D017931:DNA Primers; D004247:DNA