Homogeneous amplification and mutation scanning of the p53 gene using fluorescent melting curves.
Homogeneous amplification and mutation scanning of the p53 gene using fluorescent melting curves.
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DOI：
10.1093/clinchem/48.8.1321
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发表时间：
2002-08
影响因子：
9.3
通讯作者：
Haleigh Millward;W. Samowitz;C. Wittwer;P. Bernard
中科院分区：
文献类型：
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作者：
Haleigh Millward;W. Samowitz;C. Wittwer;P. Bernard
在恶性肿瘤中，基因突变经常发生在肿瘤抑制基因如p53中，并且是零星分布的。我们描述了一种均匀的扩增和突变扫描方法，并将该方法应用于p53基因。方法使用一系列重叠荧光素标记的寡核苷酸与野生型p53序列互补，我们通过异常探针：靶熔化温度（T(m)）检测结直肠癌的体细胞突变。由于脱氧鸟苷猝灭，探针在退火过程中荧光减弱。探针沿着要扫描的序列移动，每个试管使用两到三个探针，并将重叠的探针放置在不同的反应中。扩增后，将反应冷却至退火探针，然后缓慢加热（0.1℃/s），同时连续监测荧光。体细胞突变在肿瘤组织检测的变化，从一个典型的野生型融化曲线轮廓利用白细胞DNA。结果：从基因组白细胞DNA开始，一次扫描（约30分钟）完成了p53基因DNA结合域（外显子5-8）的完整扫描。为了证明原理，对63例结肠癌的p53外显子6-8进行了探针扫描，结果显示与直接测序检测野生型DNA变化100%一致。结论单标记杂交探针扫描p53突变是一种均匀、快速、灵敏的方法，可用于研究和临床诊断。
BACKGROUND In malignancy, gene mutations frequently occur in tumor suppressor genes such as p53 and are sporadically located. We describe a homogeneous method for amplification and mutation scanning, and apply the method to the p53 gene. METHODS Using a series of overlapping fluorescein-labeled oligonucleotides complementary to a wild-type p53 sequence, we detected somatic mutations in colorectal cancers by aberrant probe:target melting temperatures (T(m)). The probes were designed so that fluorescence decreased on target annealing as a result of deoxyguanosine quenching. Probes were walked along the sequence to be scanned, using two to three probes per cuvette and placing overlapping probes in separate reactions. After amplification, the reaction was cooled to anneal probes and then slowly heated (0.1 degrees C/s) while fluorescence was continuously monitored. Somatic mutations in tumor tissue were detected by changes from a characteristic wild-type melting curve profile using leukocyte DNA. RESULTS A complete scanning of the DNA binding domain (exons 5-8) of the p53 gene was completed in a single run ( approximately 30 min) starting from genomic leukocyte DNA. To show proof-of-principle, p53 exons 6-8 from 63 colon cancers were probe-scanned and showed 100% agreement with direct sequencing for detecting alterations from wild-type DNA. CONCLUSIONS p53 mutation scanning by single-labeled hybridization probes is a homogeneous, rapid, and sensitive method with application in both research and clinical diagnostics.