Multiplex detection of KRAS mutations by a matrix-assisted laser desorption/ionization-time of flight mass spectrometry assay.

OBJECTIVES: Mutations of the Kirsten rat sarcoma viral oncogene (KRAS) gene are known to be important in the pathogenesis of a variety of cancers. Patients with mutant KRAS tumors do not respond to epidermal growth factor receptor (EGFR) inhibitors and fail to benefit from adjuvant chemotherapy. Testing for KRAS mutations is now being recommended as a tailored therapeutic strategy prior to anti-EGFR treatment; however, the low sensitivity of direct sequencing frequently leads to failure of detection of KRAS mutations in clinical samples. DESIGN AND METHODS: We developed restriction fragment mass polymorphism (RFMP) assays, to detect KRAS mutations in codons 12, 13, and 61. We performed RFMP analysis for KRAS on DNA isolated from eight different KRAS mutant cell lines and 34 formalin-fixed paraffin-embedded (FFPE) lung cancer tissues. RESULTS: Overall, the RFMP assay was in good concordance with direct sequencing analysis in the detection of KRAS mutations. By using dilutions of KRAS mutant DNA in wild type DNA from mutation cell lines with a known KRAS status, we confirmed that the RFMP assay has a higher analytical sensitivity, requiring only 3% of cells in a sample to have mutant alleles, compared to direct sequencing, which had a detection threshold of ~25%. In the FFPE samples, RFMP successfully detected KRAS genotypes in codons 12, 13, and 61. CONCLUSION: The RFMP might be efficiently applicable for the detection of KRAS mutations in a clinical setting, particularly for tumor samples containing abundant non-neoplastic cells.

Analysis of TPOX short tandem repeat locus with matrix-associated laser desorption/ionization time-of-flight-based restriction fragment mass polymorphism assay.

Short tandem repeat (STR) loci are routinely analyzed by capillary electrophoresis. However, this method has several disadvantages, including long operational time, low throughput, and inaccuracy. As a result of the introduction of matrix-associated laser desorption/ionization time-of-flight (MALDI-TOF) and electrospray ionization (ESI), mass spectrometry has become an alternative method for genotyping polymorphic STR loci. Here we established a restriction fragment mass polymorphism (RFMP) assay for genotyping STR locus, TPOX, by typeIIS restriction endonuclease cleavage of polymerase chain reaction (PCR) amplicon followed by MALDI-TOF mass spectrometry. The resulting TPOX genotypes from this assay were in good agreement with the results from direct DNA sequencing and GeneScan assays. Our results showed that the RFMP assay is an accurate and high-throughput method for analyzing long DNA fragments such as STR markers. Further research with multiple STR loci may allow this assay to be used for diverse applications such as forensics, paternity tests, and detection of genetic disorders.