A high-throughput nonisotopic protein truncation test

• Published in: Nature biotechnology Vol. 21; no. 2; pp. 194 - 197
• Main Authors: Gite, Sadanand, Lim, Mark, Carlson, Rick, Olejnik, Jerzy, Zehnbauer, Barbara, Rothschild, Kenneth
• Format: Journal Article
• Language: English
• Published: New York Nature Publishing Group US 01.02.2003; Nature; Nature Publishing Group
• Subjects: Adenomatous Polyposis Coli - genetics; Adenomatous Polyposis Coli Protein - genetics; Agriculture; Bioinformatics; Biological and medical sciences; Biomedical and Life Sciences; Biomedical Engineering/Biotechnology; Biomedicine; Biotechnology; Breast cancer; Chemiluminescence; Codon, Nonsense - genetics; Colorectal carcinoma; Deoxyribonucleic acid; DNA; DNA Mutational Analysis - methods; DNA, Neoplasm - genetics; Dystrophy; Feasibility Studies; Frameshift Mutation - genetics; Fundamental and applied biological sciences. Psychology; Gene Expression Profiling - methods; Gene Expression Regulation, Neoplastic; Genes, APC; Genetic technics applied to diagnosis; Health. Pharmaceutical industry; Humans; Industrial applications and implications. Economical aspects; Inspection; Kidneys; Life Sciences; Mutation; Ovarian cancer; Polymerase Chain Reaction - methods; Proteins - genetics; RNA, Transfer; Sequence Analysis, Protein - methods; technical-report; Human; Molecular form; Method; Polyposis; Protein; Colonic disease; Cell line; Truncated shape; Diagnosis; Large scale; Mutation; Solid phase; Detection; Tumor cell
• DOI: 10.1038/nbt779
• ISSN: 1087-0156

Nonsense or frameshift mutations, which result in a truncated gene product, are prevalent in a variety of disease-related genes 1 , including APC (implicated in colorectal cancer) 2 , 3 , 4 , 5 , 6 , BRCA1 and BRCA2 (breast and ovarian cancer) 7 , 8 , 9 , PKD1 (polycystic kidney disease) 10 , NF1 and NF2 (neurofibromatosis) 11 , 12 , and DMD (Duchenne muscular dystrophy) 13 . Such chain-truncating mutations can be detected using the protein truncation test (PTT). This test is based on cell-free transcription and translation of either PCR-amplified portions of the target gene or RT-PCR amplified target mRNA, followed by analysis of the product(s) for shortened polypeptide fragments. However, conventional PTT is not easily adapted to high-throughput applications because it involves SDS-PAGE followed by autoradiography or western blotting. It is also subject to human error, as it relies on visual inspection to detect the mobility of shifted bands. To overcome these limitations, we have developed a high-throughput solid-phase protein truncation test (HTS-PTT). HTS-PTT uses a combination of misaminoacylated tRNAs 14 , 15 , which incorporate affinity tags for surface capture of the cell-free expressed protein fragments, and specially designed PCR primers, which introduce N- and C-terminal markers for measuring the relative level of shortened polypeptides produced by the chain-truncation mutation. After cell-free translation of the protein fragments, capture and detection are accomplished in a single well using a standard 96-well microtiter plate enzyme-linked immunosorbent assay (ELISA) format and chemiluminescence readout. We demonstrate the use of the technique to detect chain-truncation mutations in the APC gene using DNA or RNA from cancer cell lines as well as DNA of individuals diagnosed with familial adenomatous polyposis (FAP). HTS-PTT can also provide a high-throughput method for noninvasive colorectal cancer screening when used in conjunction with methods of enriching and amplifying low-abundance mutant DNA 4 .