An ELISA-based high throughput protein truncation test for inherited breast cancer.
An ELISA-based high throughput protein truncation test for inherited breast cancer.
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DOI：
10.1186/bcr2722
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发表时间：
2010
期刊：
影响因子：
--
通讯作者：
Rothschild KJ
中科院分区：
文献类型：
--
作者：
Lim MJ;Foster GJ;Gite S;Ostendorff HP;Narod S;Rothschild KJ
乳腺癌是美国女性人口中诊断最多的癌症，也是癌症死亡的第二大原因。据估计，所有乳腺癌中有5%至10%是遗传性的，由乳腺癌易感基因（BRCA 1/2）突变引起。多达90%的突变是无义突变，导致截短的多肽产物。一种流行且低成本的突变检测方法是蛋白质截短试验（PTT），其中BRCA 1/2的靶区域被PCR扩增，在无细胞蛋白质合成系统中转录/翻译，并通过十二烷基硫酸钠聚丙烯酰胺凝胶电泳（SDS-PAGE）和放射自显影分析截短的多肽。我们以前报道了一种新的高重复性固相PTT（HTS-PTT）的基础上，酶联免疫吸附测定（ELISA）的格式，消除了需要放射性，SDS-PAGE和主观解释的结果。在这里，我们报告了下一代HTS-PTT使用三重表位标记的蛋白质，并首次证明了其对BRCA 1/2分析的临床基因组DNA样本的有效性。从血液来源的基因组DNA或细胞系mRNA中PCR扩增BRCA 1/2开放阅读框的外显子11的片段。PCR引物掺入用于无细胞转录/翻译和表位标记的元件。然后将无细胞表达的新生蛋白质抗体捕获到微量滴定板的威尔斯孔上，并使用针对N-和C-末端表位标签的抗体以ELISA形式测量截短多肽的相对量。在100例血液来源的临床基因组DNA样本中，BRCA 1/2外显子11截短突变的诊断灵敏度为100%，特异性为96%，这些样本先前使用传统的基于凝胶的PTT进行检测。还证明了使用mRNA源材料进行BRCA 1/2全基因覆盖的可行性。总体而言，HTS-PTT为分析截短突变提供了一种简单、定量、客观、低成本和高通量的方法，可替代基于凝胶的PTT用于BRCA分析。几乎任何实验室都可以使用该技术，唯一需要的主要仪器是PCR热循环仪和基本微孔板读数器。与传统的基于凝胶的PTT相比，HTS-PTT提供了优异的一致性。
Breast cancer is the most diagnosed and second leading cause of cancer deaths in the U.S. female population. An estimated 5 to 10 percent of all breast cancers are inherited, caused by mutations in the breast cancer susceptibility genes (BRCA1/2). As many as 90% of all mutations are nonsense mutations, causing a truncated polypeptide product. A popular and low cost method of mutation detection has been the protein truncation test (PTT), where target regions of BRCA1/2 are PCR amplified, transcribed/translated in a cell-free protein synthesis system and analyzed for truncated polypeptides by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and autoradiography. We previously reported a novel High Throughput Solid-Phase PTT (HTS-PTT) based on an enzyme-linked immunosorbent assay (ELISA) format that eliminates the need for radioactivity, SDS-PAGE and subjective interpretation of the results. Here, we report the next generation HTS-PTT using triple-epitope-tagged proteins and demonstrate, for the first time, its efficacy on clinical genomic DNA samples for BRCA1/2 analysis. Segments of exons 11 of BRCA1/2 open reading frames were PCR amplified from either blood derived genomic DNA or cell line mRNA. PCR primers incorporate elements for cell-free transcription/translation and epitope tagging. Cell-free expressed nascent proteins are then antibody-captured onto the wells of a microtiter plate and the relative amount of truncated polypeptide measured using antibodies against the N- and C-terminal epitope tags in an ELISA format. 100% diagnostic sensitivity and 96% specificity for truncating mutations in exons 11 of BRCA1/2 were achieved on one hundred blood-derived clinical genomic DNA samples which were previously assayed using the conventional gel based PTT. Feasibility of full gene coverage for BRCA1/2 using mRNA source material is also demonstrated. Overall, the HTS-PTT provides a simple, quantitative, objective, low cost and high throughput method for analysis of truncating mutations as an alternative to gel based PTT for BRCA analysis. The technology is readily accessible to virtually any laboratory, with the only major instrumentation required being a PCR thermocycler and a basic micro-well plate reader. When compared to conventional gel based PTT, the HTS-PTT provides excellent concordance.
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影响因子：
3.5
作者：
HEIM, RA;KAMMORGAN, LNW;LUCE, MC
通讯作者：
LUCE, MC
影响因子：
3.9
作者：
Andrulis, IL;Anton-Culver, H;Whittemore, AS
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Whittemore, AS
影响因子：
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作者：
EVANS, J;REUBEN, A;CRAFT, J
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CRAFT, J
影响因子：
3.9
作者：
Kahmann, S;Herter, P;Müller, O
通讯作者：
Müller, O
影响因子：
46.9
作者：
Gite, S;Lim, M;Rothschild, K
通讯作者：
Rothschild, K