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A high-throughput nonisotopic protein truncation test
Sadanand Gite1, Mark Lim, Rick Carlson
1AmberGen, Inc., 1106 Commonwealth Avenue, Boston, MA 02215, USA. sadanand@ambergen.com
Abstract:
Nonsense or frameshift mutations, which result in a truncated gene product, are prevalent in a variety of disease-related genes, including APC (implicated in colorectal cancer), BRCA1 and BRCA2 (breast and ovarian cancer), PKD1 (polycystic kidney disease), NF1 and NF2 (neurofibromatosis), and DMD (Duchenne muscular dystrophy). 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, 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.
Insights
A new high-throughput protein truncation test (HTS-PTT) simplifies detecting chain-truncating mutations in disease genes like APC. This method enables faster, more accurate genetic analysis for cancer screening and diagnosis.
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Background:
- Chain-truncating mutations (nonsense or frameshift) are common in disease-related genes such as APC, BRCA1/2, PKD1, NF1/2, and DMD.
- The conventional protein truncation test (PTT) detects these mutations but is difficult to scale for high-throughput applications due to reliance on SDS-PAGE and manual analysis.
- Limitations of conventional PTT include susceptibility to human error and challenges in adapting to automated, large-scale screening.
Purpose of the Study:
- To develop a high-throughput, automated method for detecting chain-truncating mutations.
- To overcome the limitations of conventional PTT, including its low throughput and susceptibility to human error.
- To enable efficient genetic screening for diseases like colorectal cancer and familial adenomatous polyposis.
Main Methods:
- Developed a high-throughput solid-phase protein truncation test (HTS-PTT) utilizing misaminoacylated tRNAs with affinity tags for protein capture.
- Incorporated N- and C-terminal markers via specialized PCR primers to quantify shortened polypeptide fragments.
- Employed a 96-well microtiter plate ELISA format with chemiluminescence detection for single-well capture and analysis of cell-free expressed protein fragments.
Main Results:
- Successfully demonstrated HTS-PTT for detecting chain-truncation mutations in the APC gene.
- Validated the technique using DNA and RNA from cancer cell lines and DNA from individuals with familial adenomatous polyposis.
- Showcased the potential of HTS-PTT for high-throughput, noninvasive colorectal cancer screening when combined with DNA enrichment and amplification methods.
Conclusions:
- HTS-PTT offers a robust, high-throughput alternative to conventional PTT for detecting chain-truncating mutations.
- The developed method streamlines genetic analysis, reducing human error and enabling large-scale screening.
- HTS-PTT holds significant promise for applications in disease diagnosis, genetic screening, and cancer research.
