O'Connell Catherine D, Tully Lois A, Devaney Joseph M, Marino Michael A, Jakupciak John P, Atha Donald H
Biotechnology Division, National Institute of Standards and Technology, Gaithersburg, Maryland 20899, USA.
Mol Diagn. 2003;7(2):85-97. doi: 10.1007/BF03260024.
Numerous DNA-based tests are currently in use or under development for the detection of mutations associated with disease. Most of the current methods use PCR amplification technologies and detection after separation or chromatography of the products. We have developed a panel of standard reference materials consisting of 12 plasmid clones containing a 2.0 kb region of the TP53 gene, including exons 5-9. Eleven of these clones contain a single mutation within the mutational hot spots of the TP53 gene, the twelfth is wild-type in this region of the gene. The mutations are amino acid (aa) 128: C to T; aa 175: G to A; aa 237: T to C; aa 245: G to A; aa 248: C to T; aa 248: G to A; aa 249: G to T; aa 273: C to T; aa 273: G to A; aa 282: C to T; and aa 328: T to C. These standard reference materials (SRMs), created by site-directed mutagenesis of wild-type TP53 from a human cell line, include the specific mutations most commonly found to be associated with cancer. Their use will improve disease detection by serving as validation materials to monitor errors in measurement methods, including PCR amplification, amplicon separation, and data analysis from different technology platforms.
The single point mutations of the panel were validated by capillary electrophoresis single-strand conformational polymorphism analysis, denaturing gradient gel electrophoresis, and denaturing high-performance liquid chromatography, as well as full sequence analysis of both DNA strands of the cloned material. For both heteroduplex analysis methods, the presence of the mutations was resolved for each SRM.
The generation of a standard TP53 reference panel and demonstration that the panel can successfully validate mutation detection across different mutation scanning technology platforms. Hence, this panel functions as an SRM to normalize results obtained from different laboratories using different techniques.
目前有许多基于DNA的检测方法正在使用或处于研发阶段，用于检测与疾病相关的突变。当前大多数方法都使用PCR扩增技术，并在产物分离或色谱分析后进行检测。我们开发了一组标准参考物质，由12个质粒克隆组成，包含TP53基因的一个2.0 kb区域，包括外显子5至9。其中11个克隆在TP53基因的突变热点区域内含有一个单一突变，第12个在该基因区域为野生型。这些突变分别是氨基酸（aa）128：C突变为T；aa 175：G突变为A；aa 237：T突变为C；aa 245：G突变为A；aa 248：C突变为T；aa 248：G突变为A；aa 249：G突变为T；aa 273：C突变为T；aa 273：G突变为A；aa 282：C突变为T；以及aa 328：T突变为C。这些标准参考物质（SRM）是通过对人细胞系野生型TP53进行定点诱变产生的，包括最常发现的与癌症相关的特定突变。它们的使用将通过作为验证材料来监测测量方法中的误差，包括PCR扩增、扩增子分离以及来自不同技术平台的数据分析，从而改善疾病检测。
通过毛细管电泳单链构象多态性分析、变性梯度凝胶电泳、变性高效液相色谱以及对克隆材料两条DNA链的全序列分析，对该组单点突变进行了验证。对于两种异源双链分析方法，均确定了每个SRM中突变的存在情况。
生成了标准的TP53参考组，并证明该组能够成功验证不同突变扫描技术平台的突变检测。因此，该组可作为SRM来标准化使用不同技术的不同实验室获得的结果。
