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A high-throughput nonisotopic protein truncation test

Nature Biotechnology volume 21, pages 194–197 (2003)Cite this article

A Corrigendum to this article was published on 01 September 2003

Abstract

Nonsense or frameshift mutations, which result in a truncated gene product, are prevalent in a variety of disease-related genes1, 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 tRNAs14,15, 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 DNA4.

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Figure 1
Figure 2: HTS-PTT for APC.
Figure 3: Detection by HTS-PTT of truncation mutation in APC gene at various dilutions.

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Acknowledgements

We wish to thank S. Mamaev, U. RajBhandary, and J. Hecht for helpful discussions. This work was funded by a Phase II SBIR grant (CA83396) from the National Cancer Institute to AmberGen, Inc.

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Authors and Affiliations

  1. AmberGen, Inc., 1106 Commonwealth Avenue, Boston, 02215, MA

    Sadanand Gite, Mark Lim, Rick Carlson, Jerzy Olejnik & Kenneth Rothschild

  2. Departments of Pathology and Immunology and Pediatrics, Molecular Diagnostic Laboratory, Washington University School of Medicine, St. Louis, 63110, MO

    Barbara Zehnbauer

  3. Department of Physics and Photonics Center, Molecular Biophysics Laboratory, Boston University, Boston, 02215, MA

    Kenneth Rothschild

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  1. Sadanand Gite
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Correspondence to Sadanand Gite.

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S.G., M.L., R.C., J.O., and K.J.R. are employees of AmberGen, Inc.

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Gite, S., Lim, M., Carlson, R. et al. A high-throughput nonisotopic protein truncation test. Nat Biotechnol 21, 194–197 (2003). https://doi.org/10.1038/nbt779

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