title: Detection of mononucleotide repeat sequence alterations in a large background of normal DNA for screening high-frequency microsatellite instability cancers.
abstract: OBJECTIVE
  Mutations in mononucleotide repeat sequence (MRS) are good indicators of high-frequency microsatellite instability (MSI-H) cancers, but it has been a challenge to detect such mutations in a large background of wild-type DNA; as in this setting, PCR errors often generate false positive mutant alleles. In this study, we developed a general strategy, referred to as probe clamping primer extension-PCR (PCPE-PCR), to detect MRS alterations in a large background of wild-type DNA.
  
  
  METHODS
  In PCPE-PCR, genomic DNA is first subjected to PCPE, in which mutant single-strand DNA molecules are preferentially produced. Next, genomic DNA is removed to enrich for the mutant DNA fraction. Thereafter, PCR is carried out using the remaining single-strand DNA molecules as templates. Finally, the PCR products are analyzed to reveal the MSI-H status. In this study, the sensitivity of this new method was first examined by spiking mutant DNA into wild-type DNA at specific ratios followed by studying whether this method is applicable to fecal DNA testing.
  
  
  RESULTS
  We showed that PCPE-PCR could detect both mutated BAT26 and transforming growth factor-beta-RII (A)10 markers in the presence of 500-fold excess of normal DNA and that as few as three copies of mutated DNA could be detected. In addition, we showed that this technology could detect MSI-H colorectal cancer by fecal DNA analysis.
  
  
  CONCLUSIONS
  PCPE-PCR is sensitive. In addition, PCPE-PCR is simple and amendable to a cost-effective and high-throughput screening operation. This technology may be applicable to noninvasive screening of MSI-H cancer.
authors.affiliation: Department of Chemistry, Cleveland State University, 2121 Euclid Avenue, Cleveland, OH 44115, USA.
authors.forename: Xiyuan
authors.initials: X
authors.lastname: Sun
authors.forename: Yiding
authors.initials: Y
authors.lastname: Liu
authors.forename: Jim
authors.initials: J
authors.lastname: Lutterbaugh
authors.forename: Wei-Dong
authors.initials: WD
authors.lastname: Chen
authors.forename: Sanford D
authors.initials: SD
authors.lastname: Markowitz
authors.forename: Baochuan
authors.initials: B
authors.lastname: Guo
mesh_terms: D000123:Acetyltransferases / Q000235:genetics; D001426:Bacterial Proteins / Q000235:genetics; D020137:Base Pair Mismatch / Q000235:genetics; D014408:Biomarkers, Tumor / Q000235:genetics*; D015179:Colorectal Neoplasms / Q000175:diagnosis / Q000235:genetics*; D004273:DNA, Neoplasm / Q000032:analysis*; D016889:Endometrial Neoplasms / Q000175:diagnosis / Q000235:genetics*; D005243:Feces / Q000737:chemistry; D005260:Female; D014644:Genetic Variation; D042822:Genomic Instability; D006801:Humans; D018895:Microsatellite Repeats / Q000235:genetics*; D009154:Mutation; D016133:Polymerase Chain Reaction / Q000379:methods; D017346:Protein Serine-Threonine Kinases; D000077294:Receptor, Transforming Growth Factor-beta Type II; D018125:Receptors, Transforming Growth Factor beta / Q000235:genetics; D014407:Tumor Cells, Cultured
chemical_list: D001426:Bacterial Proteins; D014408:Biomarkers, Tumor; D004273:DNA, Neoplasm; D018125:Receptors, Transforming Growth Factor beta; C087954:bleomycin N-acetyltransferase; D000123:Acetyltransferases; D017346:Protein Serine-Threonine Kinases; D000077294:Receptor, Transforming Growth Factor-beta Type II