title: Estimating mutant microsatellite allele frequencies in somatic cells by small-pool PCR.
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.
authors.affiliation: Department of Molecular Genetics, The University of Texas M.D. Anderson Cancer Center, Houston, TX 77030, USA.
authors.forename: Mary
authors.initials: M
authors.lastname: Coolbaugh-Murphy
authors.forename: Atousa
authors.initials: A
authors.lastname: Maleki
authors.forename: Lou
authors.initials: L
authors.lastname: Ramagli
authors.forename: Marsha
authors.initials: M
authors.lastname: Frazier
authors.forename: Benjamin
authors.initials: B
authors.lastname: Lichtiger
authors.forename: Darren G
authors.initials: DG
authors.lastname: Monckton
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Siciliano
authors.forename: Barry W
authors.initials: BW
authors.lastname: Brown
mesh_terms: D000328:Adult; D000368:Aged; D016477:Artifacts; D004252:DNA Mutational Analysis / Q000379:methods*; D005787:Gene Frequency / Q000235:genetics*; D042822:Genomic Instability / Q000235:genetics*; D006801:Humans; D008297:Male; D018895:Microsatellite Repeats / Q000235:genetics*; D009154:Mutation / Q000235:genetics*; D016133:Polymerase Chain Reaction; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity