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Detection of Mononucleotide Repeat Sequence Alterations in a Large Background of Normal DNA for Screening High-Frequency Microsatellite Instability Cancers

Authors' Affiliations: ¹ Department of Chemistry, Cleveland State University; ² Department of Medicine and Ireland Cancer Center, Case Western Reserve University and University Hospitals of Cleveland; and ³ Howard Hughes Medical Institute, Cleveland, Ohio

Requests for reprints: Baochuan Guo, Department of Chemistry, 2121 Euclid Avenue, Cleveland State University, SR 359, Cleveland, OH 44115. Phone: 216-687-2471; Fax: 216-687-9298; E-mail: B.GUO@CSUOHIO.EDU.

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-ß-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.