Lleonart Matilde E, Ramón y Cajal Santiago, Groopman John D, Friesen Marlin D
Unit of Gene-Environment Interactions, International Agency for Research on Cancer, 69372 Lyon, France.
Nucleic Acids Res. 2004 Mar 22;32(5):e53. doi: 10.1093/nar/gnh051.
Short oligonucleotide mass analysis (SOMA) is a technique by which small sequences of mutated and wild-type DNA, produced by PCR amplification and restriction digestion, are characterized by HPLC-electrospray ionization tandem mass spectrometry. We have adapted the method to specifically detect two common point mutations at codon 12 of the c-K-ras gene. Mutations in DNA from 121 colon tumor samples were identified by SOMA and validated by comparison with sequencing. SOMA correctly identified 26 samples containing the 12GAT mutation and four samples containing the 12AGT mutation. Sequencing did not reveal mutant DNA in three samples out of the 26 samples shown by SOMA to contain the 12GAT mutation. In these three samples, the presence of mutant DNA was confirmed by SOMA analysis after selective PCR amplification in the presence of BstN1 restriction enzyme. Additional mutations in codons 12 and 13 were revealed by sequencing in 24 additional samples, and their presence did not interfere with the correct identification of G to A or G to T mutations in codon 12. These results provide the basis for a sensitive and specific method to detect c-K-ras codon 12-mutated DNA at levels below 10-12% of wild-type DNA.
短寡核苷酸质量分析（SOMA）是一种技术，通过该技术，经聚合酶链反应（PCR）扩增和限制性酶切产生的突变型和野生型DNA小序列，可通过高效液相色谱-电喷雾电离串联质谱进行表征。我们已对该方法进行改良，以特异性检测c-K-ras基因第12密码子处的两种常见点突变。通过SOMA鉴定了121份结肠肿瘤样本DNA中的突变，并与测序结果进行比较以验证。SOMA正确鉴定出26份含有12GAT突变的样本和4份含有12AGT突变的样本。在SOMA显示含有12GAT突变的26份样本中，有3份样本测序未发现突变型DNA。在这3份样本中，在存在BstN1限制性酶的情况下进行选择性PCR扩增后，通过SOMA分析证实了突变型DNA的存在。另外24份样本经测序发现第12和13密码子存在其他突变，且这些突变的存在并不干扰对第12密码子处G到A或G到T突变的正确鉴定。这些结果为在野生型DNA水平低于10-12%时检测c-K-ras第12密码子突变型DNA的灵敏且特异方法提供了依据。
