Detection of K-ras mutations in azoxymethane-induced aberrant crypt foci in mice using LNA-mediated real-time PCR clamping and mutant-specific probes

Abstract

Azoxymethane, a rodent colon-specific carcinogen, induce DNA damage, and causes proto-oncogene K-ras point mutations and subsequent tumor formation if DNA damage is not repaired or removed. The present study was designed to detect and characterize K-ras mutations in azoxymethane (AOM)-induced aberrant crypt foci (ACF) in mice, and determine whether dietary supplementation of selenium influences K-ras mutations frequency in ACF using a new PCR technique of locked nucleic acid-mediated real-time PCR clamping combined with mutant-specific probes. K-ras mutations were identified in 33% of AOM-induced ACF. In addition to G to A transition mutation, specific G to T transversion mutation was also identified for the first time in mouse ACF. Furthermore, selenium intake was associated with reduced ACF formation and reduced K-ras mutations rate, respectively, from 112 and 37% in mice fed control diet to 65 and 14% in mice fed selenium-containing diet ( p < 0.05). This is the first report of the use of one-step LNA-mediated real-time PCR clamping to detect K-ras mutations in AOM-induced colon cancer model. It is highly sensitive and can be applied to the detection of early genetic alterations in carcinogen-based animal models.