The online version of this article (doi:10.1186/bcr2722) contains supplementary material, which is available to authorized users.
MJL, HPO, and GJF are current employees of AmberGen, Inc., a developer of commercial diagnostic assays. SG is a former employee (within the last 5 years), and KJR is a co-founder of the company. This project was financed by AmberGen, in part using Small Business Innovation Research grant funds (from the National Institutes of Health) awarded to AmberGen (see Acknowledgements). AmberGen is the assignee on issued patents related to the commercial use of the HTS-PTT technologies described in this article. However, these patents do not preclude the use of the HTS-PTT by the general research community. In the article, we disclose in full detail the methodology required for researchers to perform HTS-PTT, which needs no specialized reagents or instrumentation (and requires nothing to be purchased from AmberGen).
MJL and KJR helped conceive of the HTS-PTT assay and its application to BRCA analysis, participated in the design and coordination of all studies in this article, and contributed significantly to the drafting of the manuscript. SG helped conceive of the HTS-PTT assay and its application to BRCA analysis and participated in the design and coordination of all studies in this article. SN supervised and coordinated the isolation and gel-PTT analysis of all clinical genomic DNA samples used in this article and contributed significantly to the drafting of the manuscript. GJF was responsible for performing and designing PCR amplifications, cell-free protein expressions, and HTS-PTT assays for optimization purposes and for the final analyses of the clinical samples. HPO performed advanced PCR assays that contributed to a better understanding of the effects of primer dimers and non-specific PCR extension products on the HTS-PTT assay for BRCA analysis and contributed significantly to the drafting of the manuscript. All authors read and approved the final manuscript.
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.
Additional file 1: Supplemental table S1 - BRCA1/2 Mutations Covered by the HTS-PTT. This table lists the mutation designations for the 50 patient genomic DNA samples tested which were positive for BRCA1/2 truncation mutations. HTS-PTT segments containing the mutation and the measured % C/N ratios are also listed. (DOC 76 KB)13058_2010_2709_MOESM1_ESM.DOC
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Breast Cancer Information Core (BIC) Database. [ http://research.nhgri.nih.gov/bic/]
Luce MC, Marra G, Chauhan DP, Laghi L, Carethers JM, Cherian SP, Hawn M, Binnie CG, Kam-Morgan LN, Cayouette MC, Koi M, Boland CR: In vitro transcription/translation assay for the screening of hMLH1 and hMSH2 mutations in familial colon cancer. Gastroenterology. 1995, 109: 1368-1374. 10.1016/0016-5085(95)90600-2. CrossRefPubMed
Andrulis IL, Anton-Culver H, Beck J, Bove B, Boyd J, Buys S, Godwin AK, Hopper JL, Li F, Neuhausen SL, Ozcelik H, Peel D, Santella RM, Southey MC, van Orsouw NJ, Venter DJ, Vijg J, Whittemore AS: Comparison of DNA- and RNA-based methods for detection of truncating BRCA1 mutations. Hum Mutat. 2002, 20: 65-73. 10.1002/humu.10097. CrossRefPubMed
- An ELISA-based high throughput protein truncation test for inherited breast cancer
Mark J Lim
Gabriel J Foster
Heather P Ostendorff
Kenneth J Rothschild
- BioMed Central
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