Abstract
Accurate detection of gene sequences in single cells is the ultimate challenge of 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 insure high efficiency and specificity. 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.
This chapter is revised from an earlier version published in “Methods Mol Med” (2007) vol. 132 pp. 65–85
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Acknowledgments
Aquiles Sanchez, John Rice, Christina Hartshorn, Kevin Soares, and Jesse Salk have made contributions to the development and testing of LATE-PCR. This work was funded by Brandeis University.
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Pierce, K.E., Wangh, L.J. (2011). LATE-PCR and Allied Technologies: Real-Time Detection Strategies for Rapid, Reliable Diagnosis from Single Cells. In: Theophilus, B., Rapley, R. (eds) PCR Mutation Detection Protocols. Methods in Molecular Biology, vol 688. Humana Press. https://doi.org/10.1007/978-1-60761-947-5_5
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DOI: https://doi.org/10.1007/978-1-60761-947-5_5
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