Linear-after-the-exponential polymerase chain reaction and allied technologies. Real-time detection strategies for rapid, reliable diagnosis from single cells

Methods Mol Med. 2007:132:65-85.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cells / chemistry
  • Cystic Fibrosis / diagnosis
  • DNA / analysis
  • DNA / genetics
  • DNA Primers / chemistry
  • Genetic Diseases, Inborn / diagnosis*
  • Heterozygote
  • Humans
  • Mutation
  • Polymerase Chain Reaction / methods*
  • Polymerase Chain Reaction / standards*
  • Polymorphism, Single Nucleotide
  • Sensitivity and Specificity
  • Sequence Analysis, DNA / methods*
  • Time Factors

Substances

  • DNA Primers
  • DNA