建立經濟簡便、特異、靈敏的 ARMS-PCR 快速檢測方法，實現對 p53 基因外顯子突變熱點的快速鑒定。針對7個覆蓋 p53 基因90%突變類型以上的突變熱點，擴增健康人基因組中突變對應所在外顯子，并將擴增片段插入 T 載體后點突變引入突變熱點。通過調整 PCR 體系與引物特異性建立 ARMS-PCR 優化反應體系。應用優化的 ARMS-PCR 反應體系對臨床癌癥患者石蠟包埋組織與正常人樣本進行篩選、鑒定。實驗中成功擴增獲得了三個目的外顯子，并引入突變熱點，建立優化的 PCR 體系。對10個臨床癌癥患者石蠟包埋組織和10個健康人基因組 DNA 樣品篩選：10個臨床癌癥患者石蠟包埋組織共檢測出22個突變，10個健康人基因組 DNA 樣品沒有突變檢出。該法簡便快捷、特異性強，能為臨床治療與預后提供重要信息，是具有廣闊臨床應用前景的突變檢測方法。
關鍵詞: p53 基因 突變熱點 ARMS-PCR
Establishment and improvement of rapid detection of p53 gene mutation hot spot with ARMS-PCR
ABSTRACT
To establish an economic, simple, specific, sensitive and rapid detection method of ARMS-PCR for identification of mutation hot spots on p53 exons. Seven regions of mutation hot spots representing 90% of p53 exon mutations were amplified from the genome of healthy individuals, and cloned into T-vector followed by directed mutagenesis. ARMS-PCR system was optimized by adjusting the PCR conditions and primer specificity. The optimized ARMS-PCR reaction system was applied in the comparison analysis of paraffin-embedded tissues from clinical cancer patients and normal individuals. In this study, three target exons were amplified, and mutations at hot spots were introduced. An ARMS-PCR system was further established and optimized. In the screening of paraffin-embedded tissues from 10 clinical cancer patients and 10 genomic DNA samples of healthy individuals, 22 mutations were detected from clinical cancer patients. However, no mutations were detected from the genomes of healthy individuals. A simple, quick, and specific method was established to provide important information for clinic treatment and prognosis, with broad application potential in clinical for mutation detection.
Key words: p53 gene Mutation hot spot ARMS-PCR
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