A high-throughput nonisotopic protein truncation test

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.

Transforming single DNA molecules into fluorescent magnetic particles for detection and enumeration of genetic variations

“…Beads generated from random fragments of whole genomes (26) rather than from individual genes as described above could be used to identify gene segments that bind to specific DNA-binding proteins (27). If the beads made by BEAMing were used in compartmentalized in vitro transcription-translation reactions, variant proteins would be bound to beads containing the corresponding variant DNA sequences (23), which could allow facile flow-cytometric evaluation of rare mutations by using antibodies that distinguish between wild-type and mutant gene products (28).…”

ACMG technical standards and guidelines for genetic testing for inherited colorectal cancer (Lynch syndrome, familial adenomatous polyposis, and MYH-associated polyposis)

“…More recently, a DNA-based protein truncation test has been published, and some laboratories continue to use it. 91,92 Other, less popular methods include scanning methods, followed by limited sequencing of aberrant fragments, as described in section 2.13.8. However, none of these methods has detection sensitivity as high as that of direct sequencing, which is a standard method in most clinical laboratories.…”

Cell-Free Protein Synthesis Systems: Biotechnological Applications

“…Although non-isotopic methods have clear advantages, they still suffer from the intrinsic throughput problems associated with electrophoresis . In order to overCOlne these liltlitations, ELISA-PlT has been developed, which provides higher through.. put and lower cost because it circunlvents electrophoresi s (Gite et al, 2003). The basic ELISA-PTT approach is illustrated in Figure 8.2.…”

“…Although this process can be multiplexed and is amenable to automation, the small test sequence size reduces its effective throughput compared to an electrophoresi s-based PTT or ELISA-PIT. (Gite et al, 2003).…”