High-throughput in vitro translation.
Extract: Cell-free systems for the expression of proteins are widely used in basic and clinical research. In molecular diagnostics, the method is used for the detection of mutations in a variety of disease-related genes that lead to premature translation termination caused by nonsense substitutions or frameshift mutations. The use of in vitro transcription and translation (IVTT) assays for population-wide disease screening demands the development of nonisotopic, automatable, and sensitive assay systems. Several recent papers have begun to address these issues. All approaches are based on the protein truncation test, also referred to as in vitro synthesized protein (IVSP) assay, which was first reported by Powell et al. (1993) and Roest et al. (1993). The IVTT assay begins with the purification of genomic RNA or DNA from clinical samples, followed by either RT-PCR or PCR. The forward primer includes all the cis-elements for transcription and translation, i.e., the RNA polymerase binding site (T7 promoter), ribosome binding site (kozak) and a start codon. The amplified DNA is then directly added to the IVTT reaction.
Diehl
Frank
F
Sidney Kimmel Comprehensive Cancer Center, The Johns Hopkins Medical Institutions, 1650 Orleans Street, Baltimore, MD 21231, USA and Trinity College, Cambridge CB2 1TQ, U.K.
Traverso
Giovanni
G