Estimating mutant microsatellite allele frequencies in somatic cells by small-pool PCR

Genomics
Mary Coolbaugh-MurphyBarry W Brown

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.

Citations

Oct 31, 2006·Radiation and Environmental Biophysics·Wael Abdel MegidJeff W Bacher
Feb 9, 2006·Nucleic Acids Research·Elizabeth C Chao, Steven M Lipkin
Nov 29, 2007·European Journal of Human Genetics : EJHG·Rui GaoXi Lin
Jun 16, 2010·International Journal of Radiation Biology·Jackie HainesSimon Bouffler
Jan 24, 2008·British Journal of Cancer·R J C SlebosW G Yarbrough
Mar 21, 2009·Pain Practice : the Official Journal of World Institute of Pain·Cary A Brown
Jan 7, 2010·Human Mutation·Mary I Coolbaugh-MurphyMichael J Siciliano
Aug 13, 2004·Environmental Microbiology·Edward F DeLong
Jul 31, 2013·Journal of Medical Genetics·Alicia SemakaMichael R Hayden
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Sep 13, 2005·Mechanisms of Ageing and Development·Darren G Monckton
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