Single Cell Diagnostics

Volume 132 of the series Methods in Molecular Medicine™ pp 65-85

Linear-After-The-Exponential Polymerase Chain Reaction and Allied Technologies

Real-Time Detection Strategies for Rapid, Reliable Diagnosis from Single Cells
  • Kenneth E. PierceAffiliated withDepartment of Biology, Brandeis University
  • , Lawrence J. WanghAffiliated withDepartment of Biology, Brandeis University

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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 LATEPCR signals provides a means for improving the accuracy of single cell genetic diagnosis.

Key Words

Asymmetric PCR cell lysis fluorescent probes molecular beacons proteinase K real-time PCR