ARMS-PCR快速检测p53基因突变热点方法的建立及优化

论文编号:SW233  论文字数:15427,页数:36

ARMS-PCR 快速检测 p53 基因突变热点的建立及优化

摘　　要

 建立经济简便、特异、灵敏的 ARMS-PCR 快速检测方法，实现对 p53 基因外显子突变热点的快速鉴定。针对7个覆盖 p53 基因90%突变类型以上的突变热点，扩增健康人基因组中突变对应所在外显子，并将扩增片段插入 T 载体后点突变引入突变热点。通过调整 PCR 体系与引物特异性建立 ARMS-PCR 优化反应体系。应用优化的 ARMS-PCR 反应体系对临床癌症患者石蜡包埋组织与正常人样本进行筛选、鉴定。实验中成功扩增获得了三个目的外显子，并引入突变热点，建立优化的 PCR 体系。对10个临床癌症患者石蜡包埋组织和10个健康人基因组 DNA 样品筛选：10个临床癌症患者石蜡包埋组织共检测出22个突变，10个健康人基因组 DNA 样品没有突变检出。该法简便快捷、特异性强，能为临床治疗与预后提供重要信息，是具有广阔临床应用前景的突变检测方法。
 
关键词: p53 基因　突变热点　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
目　　录

 摘要 I
 ABSTRACT II
 1前言 1
 1.1研究背景 1
 1.2 p53 基因 1
 1.3突变检测的方法 2
 1.4研究趋势及其展望 5
 2材料与方法 6
 2.1实验材料 6
 2.1.1实验样品 6
 2.1.2菌种和质粒 6
 2.1.3实验试剂 6
 2.1.4实验仪器 7
 2.2实验方法 8
 2.2.1实验流程示意图 8
 2.2.2基因组DNA的提取 8
 2.2.3引物的设计 10
 2.2.4突变热点所在外显子的 PCR 扩增 12
 2.2.5野生型质粒的点突变 14
 2.2.6 ARMS-PCR模型的建立与优化 15
 2.2.7 ARMS-PCR对样品的检测 16
 3结果与分析 18
 3.1实验结果与分析 18
 3.1.1基因组DNA电泳图 18
 3.1.2外显子扩增及菌液PCR鉴定电泳图 18
 3.1.3质粒提取电泳图 19
 3.1.4点突变测序结果分析图 19
 3.1.5 ARMS电泳图 21
 3.1.6样品ARMS-PCR检测结果 24
 3.2小结 26
 4总结 28
 参考文献 29
 谢辞 31