癌関連遺伝子の変異検出のための新方法 : -限定延長法 (RSNE法)-の開発

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  • ガン カンレン イデンシ ノ ヘンイ ケンシュツ ノ タメ ノ シン ホウホウ ゲンテイ エンチョウホウ RSNEホウ ノ カイハツ
  • Development of the Restricted Single Nucleotide Extension (RSNE) Method, a New Method to Detect Somatic Gene Mutations of Low Abundance

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A new method to detect gene mutations in clinical tissue samples is developed. By this method, we can detect highly frequent 12 mutations at 9 positions in the hot spot region of p53 gene that are as low as 1% in abundance in the samples. The principle of this method lies on the very high nucleotide specificity in the single nucleotide primer extension reactions. This implies that the primer extension reaction is carried out in the absence of dNTPs and labeled terminator nucleotides, which are exclusively complementary to the mutant nucleotides, are included in each reaction mixtures. We developed a simple protocol which includes guanidine HCl washing of the template DNA to remove trace amount of residual dNTPs in the sample templates as well as addition of ddGTP in the extension reaction mixtures to suppress unfavorable extension reactions. The protocol was examined for sensitivity and multiplicity of the detection with mutant DNA preparations of either synthesized DNA or PCR amplified DNA of human cell lines, MiaPaCa2, TE-6, DLD1, RPMI8226 and PC3. The highly frequent 12 mutations in the synthesized DNA were selected according to the p53 mutation database of IARC (The International Agency for Research in Cancer). These mutant DNA preparations had been diluted with largely excess amount of native human DNA to emulate DNA preparations of clinical tissue samples. All the 12 mutations were detected with 5 separate reaction tubes, suggesting high potential of this method in detecting many mutations with limited number of reaction tubes. This method is used to analyze paraffin embedded tissue samples to investigate possible application to clinical samples.

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