Navigation Links
Protein Truncation Tests Using the DCode System

Helen Chun, Milhan Telatar and Richard A. Gatti Department of Pathology, UCLA School of Medicine, Los Angeles, California


Introduction
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.


Method
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 product which encoded a 45 kD protein. PTT analysis of the 12 samples identified two samples having premature termination mutations. These truncated proteins were detected by having a different migration pattern compared to the full-length protein product when run on an SDS-PAGE using the DCode system (Figure 1).

Lane 2 shows a protein product smaller than the normal protein. The fact that no normal product is observed in this lane usually indicates a homozygous mutation. Sequencing of cDNA indicated a deletion of 174 bp, corresponding to the entire exon 26. Because this cDNA mutation would result in an in-frame deletion, the final protein product would lack only 58 amino acids. Genomic sequencing analysis revealed a homozygous G to A substitution at the last nucleotide of exon 26 (nt 3576). This mutation is referred to as 3576 G > A (c1135del174nt).

Sample 10 showed the presence of two protein products, one of normal length and a second of smaller size, suggesting this patient to be heterozygous for a mutation in this region. Sequence analysis identified a nonsense mutation at 5971 nt substituting a G to T (5971G>T[c1990]).

PTT analysis has proven to be advantageous in initial attempts to screen for mutations in a large gene such as ATM. This method allows for detection of any out-of-frame insertions or deletions resulting in a truncated protein, as well as for substitutions creating a nonsense mutation. PTT will also reveal large in-frame insertions or deletions producing a gross protein product alteration.


Summary
The versatile DCode system can be used for multiple mutation detection techniques, including single-stranded conformational polymorphism ( SSCP), conformation-sensitive gel electrophoresis (CSGE) and denaturing gradient gel electrophoresis (DGGE). Protein truncation test analysis can also be performed on the DCode system, producing results equivalent to other electrophoretic systems.


References
1. Savitsky, K., et al., Science, 268, 1749-1753 (1995).

2. Telatar, M., et al., Am. J. Hum. Genet., 59, 40-44 (1996).

*The Polymerase Chain Reaction (PCR) process is covered by patents owned by
Hoffmann-LaRoche. Use of the PCR process requires a license.


back to top
'"/>

Source:


Page: All 1 2 3

Related biology technology :

1. A New C-Terminal GST Vector for Protein Production in S. pombe
2. New Mammalian Two-Hybrid System Detects Protein-Protein Interactions
3. Antibodies for Studying NMDA Receptor Protein Expression and Synapse-Specific Immunolabeling
4. Simple, Sensitive, and Rapid Detection of FLAG -Tagged Fusion Proteins
5. New Yeast Cloning System for Producing Proteins with Native Amino Acid Sequences
6. Optimized Imaging of Protein Gels Stained with Coomassie Brilliant Blue Dye
7. High-Level Protein Expression, One-Column Purification, and FLAG Epitope Tagging in E. coli
8. Transfection of Green Fluorescent Protein into Human Adrenalcarcinoma Cells
9. Yeast Protein Production System Features High Yields and One-Step Purification
10. Efficient Cleavage of Fusion Proteins to Yield Native Amino Termini
11. Expression and Purification of Recombinant Proteins That Have Native Amino Acid Sequence
Post Your Comments:
*Name:
*Comment:
*Email:


(Date:5/24/2017)... As Ebola resurfaces in the Democratic ... suspected cases now reported, a new analysis of the Ebola ... correlation between the 2014 and 2017 outbreaks of the disease.  ... 2012-13, which preceded the 2014 outbreak. An analysis of the ... counts in 2014-15, which again precedes the current outbreak in ...
(Date:5/23/2017)... ... May 23, 2017 , ... ... month its 20th anniversary, marking the occasion with a strong presence at Bio-IT ... Welcome Reception and further extends an invitation to all attendees to view ...
(Date:5/23/2017)... ... May 23, 2017 , ... ... technology applications, has announced a facility expansion to accommodate its rapid growth. , ... of new workspace and renovation of the existing areas. The expansion includes, a ...
(Date:5/23/2017)... Switzerland (PRWEB) , ... May 23, 2017 , ... ... forces machine manufacturers to re-engineer their control technology again and again. METTLER TOLEDO ... problem for machine manufacturers. The videos illustrate how integration of the ACT350 into ...
Breaking Biology Technology:
(Date:3/24/2017)... Controller General of Immigration from Maldives Mr. Mohamed Anwar and ... international IAIR Award for the most innovative high security ePassport and eGates  ... ... Maldives Immigration Controller General, Mr. Mohamed Anwar ... right) have received the IAIR award for the "Most innovative high security ...
(Date:3/22/2017)... VILNIUS, Lithuania , March 21, 2017 /PRNewswire/ ... identification and object recognition technologies, today announced the ... development kit (SDK), which provides improved facial recognition ... safety cameras on a single computer. The new ... algorithms to improve accuracy, and it utilizes a ...
(Date:3/20/2017)... PMD Healthcare announces the release of its ... System (WMS), a remote, real-time lung health monitoring and ... is a Medical Device, Digital Health, and Chronic Care ... innovative solutions that empower people to improve their healthcare ... developed the first ever personal spirometer, Spiro PD, which ...
Breaking Biology News(10 mins):