an elisa-based high throughput protein truncation test for inherited breast cancer一种elisa-based高通量蛋白质截断检测遗传性乳腺癌.pdfVIP

Lim et al. Breast Cancer Research 2010, 12:R78 /content/12/5/R78 RESEARCH ARTICLE Open Access An ELISA-based high throughput protein truncation test for inherited breast cancer 1* 1 2 1 3 1,4 Mark J Lim , Gabriel J Foster , Sadanand Gite , Heather P Ostendorff , Steven Narod , Kenneth J Rothschild Abstract Introduction: 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) 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, SDSand 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. Methods: 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 w