Detection of K-ras oncogene mutations by polymerase chain reaction-based ligase chain reaction.
Detection of K-ras oncogene mutations by polymerase chain reaction-based ligase chain reaction.
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通过基于聚合酶链式反应的连接酶链式反应检测 K-ras 癌基因突变。
DOI：
10.1006/abio.1996.0310
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发表时间：
1996
期刊：
影响因子：
--
通讯作者：
Modali,R
中科院分区：
文献类型：
--
作者：
Lehman,TA;Scott,F;Seddon,M;Kelly,K;Dempsey,EC;Wilson,VL;Mulshine,JL;Modali,R
为了评价检测三种常见K-rascodon 12突变的快速多重测定，开发了与野生型和突变基因座互补的引物对，并使用具有先前鉴定的突变状态的肺癌细胞系进行了测试。使用含有K-rascodon 12突变的加标样品，突变检测的灵敏度确定为至少1%。然后，通过分析既往接受过肺癌治疗的患者的支气管肺泡灌洗（BAL）标本，使用该测定法前瞻性评估肺癌高危个体气道中的K-ras状态。从BAL标本细胞团中提取DNA，并进行基于PCR的连接酶链反应，用于K-ras第12位密码子的第一位突变，并具有阳性和阴性对照。10例BAL标本中，4例有1个突变（GGT → TGT），1例有2个突变（GGT → TGT和GGT → AGT），其余均为野生型。BAL突变通过克隆和用mu-特异性探针筛选，然后进行确认测序来验证。
To evaluate a rapid multiplexed assay to detect three common K-rascodon 12 mutations, primer pairs complementary to the wild-type and mutant loci were developed and tested with lung cancer cell lines with previously identified mutation status. The sensitivity of detection of mutations was determined to be at least 1% using spiked samples containing K-rascodon 12 mutations. This assay was then used to evaluate prospectively K-rasstatus in airways of individuals at high risk of lung cancer by analysis of bronchoalveolar lavage (BAL) specimens from patients who have been previously treated for lung cancer. DNA was extracted from BAL specimen cell pellets, and PCR-based ligase chain reaction was performed for mutations in the first position of codon 12 of K-ras,with positive and negative controls. Of 10 BAL samples, 4 contained 1 mutation (GGT → TGT), 1 contained 2 mutations (GGT → TGT and GGT → AGT), and the rest were wild-type. The BAL mutations were validated by cloning and screening with mutant-specific probes followed by confirmation sequencing.