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Title: Mutational analysis of NF2 by in vitro expression assay

Abstract

Neurofibromatosis 2 (NF2) is an autosomal dominant disorder characterized by the development of multiple nervous system tumors. The recently cloned NF2 tumor suppressor gene encodes a novel member of a family of cytoskeleton associated proteins. Because the majority of germline mutational events of the NF2 gene cause gross truncation of the protein product, we investigated the feasibility of a single step protein-based screen for mutation. Total cellular RNA extracted from blood or cell lines was used to synthesize cDNA from mRNA using reverse transcriptase. Two rounds of PCR amplification were carried out. The 5{prime} primer contained an in-frame T7 promoter followed by an initiation methionine within a Kozak consensus sequence. The antisense 3{prime} primer contained the native stop codon followed by a poly (A) tail. The resulting product was used in a cell-free coupled transcription/translation reaction which was visualized on a standard protein separating gel. We were able to amplify 95% of the coding sequence of the NF2 gene with a single set of primers which produced a 1724 basepair product. Normal transcripts produced an approximately 66 KDa protein product while transcripts which contained known nonsense or splice site mutations produced truncated protein products in addition to the normal sizedmore » product. Estimation of the location of the mutation could be determined by the extent of the protein shift. This system may improve both efficiency and sensitivity of mutational analysis of the NF2 gene.« less

Authors:
; ; ;
Publication Date:
OSTI Identifier:
134294
Report Number(s):
CONF-941009-
Journal ID: AJHGAG; ISSN 0002-9297; TRN: 95:005313-1027
Resource Type:
Journal Article
Resource Relation:
Journal Name: American Journal of Human Genetics; Journal Volume: 55; Journal Issue: Suppl.3; Conference: 44. annual meeting of the American Society of Human Genetics, Montreal (Canada), 18-22 Oct 1994; Other Information: PBD: Sep 1994
Country of Publication:
United States
Language:
English
Subject:
55 BIOLOGY AND MEDICINE, BASIC STUDIES; BIOASSAY; EFFICIENCY; SENSITIVITY; GENES; GENE MUTATIONS; DNA-CLONING; TRANSCRIPTION; SPLICING; GENETIC MAPPING; SCREENING; PATIENTS; HEREDITARY DISEASES; NERVOUS SYSTEM DISEASES; CENTRAL NERVOUS SYSTEM; NEOPLASMS; PROTEINS; DOMINANT MUTATIONS; POLYMERASE CHAIN REACTION; MESSENGER-RNA; GENE REPRESSORS; CODONS

Citation Formats

Pulaski, K., Pettingell, W., MacCollin, M., and Gusella, J.F. Mutational analysis of NF2 by in vitro expression assay. United States: N. p., 1994. Web.
Pulaski, K., Pettingell, W., MacCollin, M., & Gusella, J.F. Mutational analysis of NF2 by in vitro expression assay. United States.
Pulaski, K., Pettingell, W., MacCollin, M., and Gusella, J.F. 1994. "Mutational analysis of NF2 by in vitro expression assay". United States. doi:.
@article{osti_134294,
title = {Mutational analysis of NF2 by in vitro expression assay},
author = {Pulaski, K. and Pettingell, W. and MacCollin, M. and Gusella, J.F.},
abstractNote = {Neurofibromatosis 2 (NF2) is an autosomal dominant disorder characterized by the development of multiple nervous system tumors. The recently cloned NF2 tumor suppressor gene encodes a novel member of a family of cytoskeleton associated proteins. Because the majority of germline mutational events of the NF2 gene cause gross truncation of the protein product, we investigated the feasibility of a single step protein-based screen for mutation. Total cellular RNA extracted from blood or cell lines was used to synthesize cDNA from mRNA using reverse transcriptase. Two rounds of PCR amplification were carried out. The 5{prime} primer contained an in-frame T7 promoter followed by an initiation methionine within a Kozak consensus sequence. The antisense 3{prime} primer contained the native stop codon followed by a poly (A) tail. The resulting product was used in a cell-free coupled transcription/translation reaction which was visualized on a standard protein separating gel. We were able to amplify 95% of the coding sequence of the NF2 gene with a single set of primers which produced a 1724 basepair product. Normal transcripts produced an approximately 66 KDa protein product while transcripts which contained known nonsense or splice site mutations produced truncated protein products in addition to the normal sized product. Estimation of the location of the mutation could be determined by the extent of the protein shift. This system may improve both efficiency and sensitivity of mutational analysis of the NF2 gene.},
doi = {},
journal = {American Journal of Human Genetics},
number = Suppl.3,
volume = 55,
place = {United States},
year = 1994,
month = 9
}
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