ARMS-PCR快速檢測p53基因突變熱點(diǎn)方法的建立及優(yōu)化

論文編號:SW233  論文字?jǐn)?shù):15427,頁數(shù):36

ARMS-PCR 快速檢測 p53 基因突變熱點(diǎn)的建立及優(yōu)化

摘　　要

 建立經(jīng)濟(jì)簡便、特異、靈敏的 ARMS-PCR 快速檢測方法，實(shí)現(xiàn)對 p53 基因外顯子突變熱點(diǎn)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(diǎn)，擴(kuò)增健康人基因組中突變對應(yīng)所在外顯子，并將擴(kuò)增片段插入 T 載體后點(diǎn)突變引入突變熱點(diǎn)。通過調(diào)整 PCR 體系與引物特異性建立 ARMS-PCR 優(yōu)化反應(yīng)體系。應(yīng)用優(yōu)化的 ARMS-PCR 反應(yīng)體系對臨床癌癥患者石蠟包埋組織與正常人樣本進(jìn)行篩選、鑒定。實(shí)驗(yàn)中成功擴(kuò)增獲得了三個目的外顯子，并引入突變熱點(diǎn)，建立優(yōu)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(qiáng)，能為臨床治療與預(yù)后提供重要信息，是具有廣闊臨床應(yīng)用前景的突變檢測方法。
 
關(guān)鍵詞: p53 基因　突變熱點(diǎn)　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　　錄

 摘要 I
 ABSTRACT II
 1前言 1
 1.1研究背景 1
 1.2 p53 基因 1
 1.3突變檢測的方法 2
 1.4研究趨勢及其展望 5
 2材料與方法 6
 2.1實(shí)驗(yàn)材料 6
 2.1.1實(shí)驗(yàn)樣品 6
 2.1.2菌種和質(zhì)粒 6
 2.1.3實(shí)驗(yàn)試劑 6
 2.1.4實(shí)驗(yàn)儀器 7
 2.2實(shí)驗(yàn)方法 8
 2.2.1實(shí)驗(yàn)流程示意圖 8
 2.2.2基因組DNA的提取 8
 2.2.3引物的設(shè)計 10
 2.2.4突變熱點(diǎn)所在外顯子的 PCR 擴(kuò)增 12
 2.2.5野生型質(zhì)粒的點(diǎn)突變 14
 2.2.6 ARMS-PCR模型的建立與優(yōu)化 15
 2.2.7 ARMS-PCR對樣品的檢測 16
 3結(jié)果與分析 18
 3.1實(shí)驗(yàn)結(jié)果與分析 18
 3.1.1基因組DNA電泳圖 18
 3.1.2外顯子擴(kuò)增及菌液PCR鑒定電泳圖 18
 3.1.3質(zhì)粒提取電泳圖 19
 3.1.4點(diǎn)突變測序結(jié)果分析圖 19
 3.1.5 ARMS電泳圖 21
 3.1.6樣品ARMS-PCR檢測結(jié)果 24
 3.2小結(jié) 26
 4總結(jié) 28
 參考文獻(xiàn) 29
 謝辭 31