Detection of mononucleotide repeat sequence alterations in a large background of normal DNA for screening high-frequency microsatellite instability cancers

Clin Cancer Res. 2006 Jan 15;12(2):454-9. doi: 10.1158/1078-0432.CCR-05-0919.


Purpose: 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.

Experimental design: 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.

Conclusion: 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.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Acetyltransferases / genetics
  • Bacterial Proteins / genetics
  • Base Pair Mismatch / genetics
  • Biomarkers, Tumor / genetics*
  • Colorectal Neoplasms / diagnosis
  • Colorectal Neoplasms / genetics*
  • DNA, Neoplasm / analysis*
  • Endometrial Neoplasms / diagnosis
  • Endometrial Neoplasms / genetics*
  • Feces / chemistry
  • Female
  • Genetic Variation
  • Genomic Instability*
  • Humans
  • Microsatellite Repeats / genetics*
  • Mutation
  • Polymerase Chain Reaction / methods
  • Protein Serine-Threonine Kinases
  • Receptor, Transforming Growth Factor-beta Type II
  • Receptors, Transforming Growth Factor beta / genetics
  • Tumor Cells, Cultured


  • Bacterial Proteins
  • Biomarkers, Tumor
  • DNA, Neoplasm
  • Receptors, Transforming Growth Factor beta
  • bleomycin N-acetyltransferase
  • Acetyltransferases
  • Protein Serine-Threonine Kinases
  • Receptor, Transforming Growth Factor-beta Type II