alexa Estimating mutant microsatellite allele frequencies in somatic cells by small-pool PCR.
Reproductive Medicine

Reproductive Medicine

Journal of Fertilization: In Vitro - IVF-Worldwide, Reproductive Medicine, Genetics & Stem Cell Biology

Author(s): CoolbaughMurphy M, Maleki A, Ramagli L, Frazier M, Lichtiger B,

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Abstract Identifying microsatellite instability (MSI) by partitioning DNA into multiple small pools containing only single genome amounts of DNA results in trapping both progenitor and low-frequency mutant alleles into pools where they can be identified and counted following PCR. Statistical approaches determining both the frequencies and the significant differences between frequencies of these Poisson-distributed alleles are presented. Results indicate a level of sensitivity and quantification not possible by standard PCR methods. Using material from colon cancer patients with high levels of MSI in their tumors, we also present the molecular and robotic methods for carrying out such studies. Validation experiments indicated mutants detectable at frequencies >0.03 above background. Frequencies obtained in tumor tissue (>0.25) met the expectations of the approach. Significant levels of MSI were detected in the constitutive tissue of the patient carrying a germ-line mutation for mismatch repair, suggesting both mechanistic and clinical applications of the procedure. This article was published in Genomics and referenced in Journal of Fertilization: In Vitro - IVF-Worldwide, Reproductive Medicine, Genetics & Stem Cell Biology

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