Helen Chun, Milhan Telatar and Richard A. Gatti Department of Pathology, UCLA School of Medicine, Los Angeles, California
Protein Truncation Testing (PTT) is a method used to screen large coding regions of DNA to detect translation termination mutations. The gene responsible for the autosomal recessive disease ataxia-telangiectasia, ATM (ataxia-telangiectasia, mutated),1 is a large transcript, approximately 10 kb, which lends itself well to PTT by analysis of seven overlapping regions (ag) for truncated in vitro translation products.2 Here, we report our PTT analysis of ATM mutations. Approximately 80% result in truncation.
Twelve samples were RT-PCR* amplified for region b of ATM using specialized PTT modified primers containing a T7 promoter and a eukaryotic translation initiation sequence. One hundred nanograms of amplified product was used to produce protein in the coupled transcriptiontranslation reaction of the TNT Coupled Reticulocyte Lysate System (Promega). Reactions were performed in 12.5 ml with 6 Ci of 35S-methionine. Protein products were run on a 16 x 20 cm, 14% SDS-PAGE denaturing polyacrylamide gel using the DCode universal mutation detection system. The samples were run at room temperature in 1x Tris/Glycine/SDS buffer (25 mM Tris, 192 mM glycine, 0.1% SDS) at 200 V for 3 hours. The gel was fixed for 15 minutes, washed in Amplify (Amersham Life Science) for an additional 15 minutes, dried and exposed to film (Kodak XOMAT- AR) overnight.
Results and Discussion
Region b amplification of 12 different AT patient samples resulted in a 1,300 bp