K-RAS mutations are common in human cancers and play a very important role in various processes of cancer development, including cancer initiation, metastasis, prognosis and response for treatment [
1‐
7,
10,
11,
30‐
32]. Therefore, mutation detection of
K-RAS is of clinical importance in cancer studies. In this study, we present a simple assay to detect
K-RAS mutations in colorectal cancer. Several methods can be used to detect
K-RAS mutations, including PCR and direct sequencing, PCR-RFLP and direct sequencing, PCR-SSCP and direct sequencing, PCR and probe hybridization, and allele-specific competitive blocker PCR [
19‐
26,
33,
34]. These methods are laborious because the mutation hotspots in exons 2, 3 and 4 have to be screened separately. Furthermore, the common mutations seen at codons 12, 13, 61 and 146 are not the result of any single nucleotide change; instead, every base at these codons has 3 or 4 types of base changes. It is therefore difficult to use the above-mentioned methods to precisely detect all the base changes without using direct sequencing or hybridization. Although the probe hybridization method requires no direct sequencing, it requires several probes to accurately determine base changes. With our method, the detection of base changes in three key exons of
K-RAS can be combined into one assay, which allows a sample to be screened for all
K-RAS mutation hotspots simultaneously. Because the technique is a sequencing-based approach, additional sequencing is not necessary. Meanwhile, the advantage of using primer extension over hybridization with allele-specific oligonucleotide probes to distinguish sequence variants is based on the high accuracy of the nucleotide incorporation reaction catalyzed by a DNA polymerase compared with the caveats of hybridization. These caveats include differences in thermal stability between mismatched and perfectly matched hybrids formed with the allele-specific oligonucleotide probes. Current products of the thermostable enzymes used in primer extension reaction have very low error rates and have increased efficiency and specificity for ddNTPs [
35]. These characteristics provide negligible primer misincorporation and excellent discrimination between wild, heterozygous and homozygous genotypes. Additionally, the same reaction conditions can be used to detect any variable nucleotide regardless of the nucleotide sequence flanking the variable site. Another advantage of the primer extension reaction is its multiplexing capability, with several mutations being detected in a single reaction tube. Although multiplex PCR-SSCP- or PCR-ARMS- based methods can also simultaneously detect several mutations, PCR-SSCP require further confirmation by direct sequencing, and using PCR-ARMS to detect all the changes at codons 12, 13, 61 and 146 require more primers than are possible for one reaction tube. Our multiplex primer extension methods can detect more than 40 types of changes at these 4 codons. Compared with direct sequencing, primer extension reaction assays are faster because automated fluorescent capillary electrophoresis of the products requires only 25 minutes in comparison with more than one hour for capillary electrophoresis require for standard sequencing. However, we should point out that our proposed method can only detect
K-RAS mutations in a DNA mixture containing at least 5% mutant DNA. Moreover, our method can not detect
K-RAS mutations occurred outside the codons that we targeted although direct sequencing we performed did not find any either. In the past, primer extension-based methods were used for detecting a single base change across several conditions, and they have rarely been used to detect the three base changes at a given codon [
36‐
40]. In this study, we extend the application to detect all the base changes at a given codon of
K-RAS gene simultaneously and established it as a simple and fast way to screen
K-RAS gene. Our results indicated a strong association between
K-RAS mutation in exon 2 and clinical outcome in terms of survival, lymph node involvement, and tumor size. Mutated exon 2 of
K-RAS, thus represents a molecular lesion in the development of more aggressive disease. The effectiveness of cetuximab in colorectal cancer is strongly associated with
K-RAS mutation status [
12]. The method that we demonstrated in this report provides a simple, fast, and reliable way to identify
K-RAS mutation for the purpose of clinical evaluation in colorectal cancer. Meanwhile, the principle of the method can also be used to detect
H-RAS and
N-RAS mutations, or to detect the mutation at a given codon in other genes in colorectal and other cancers