Mutational analysis of NF2 by in vitro expression assay

Journal Article · Thu Sep 01 04:00:00 EDT 1994 · American Journal of Human Genetics

OSTI ID:134294

Pulaski, K; Pettingell, W; MacCollin, M; Gusella, J F

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.

• OSTI ID: 134294
• Report Number(s): CONF-941009--
• Journal Information: American Journal of Human Genetics, Journal Name: American Journal of Human Genetics Journal Issue: Suppl.3 Vol. 55; ISSN AJHGAG; ISSN 0002-9297
• Country of Publication: United States
• Language: English

Related Subjects

55 BIOLOGY AND MEDICINE
BASIC STUDIES
BIOASSAY
CENTRAL NERVOUS SYSTEM
CODONS
DNA-CLONING
DOMINANT MUTATIONS
EFFICIENCY
GENE MUTATIONS
GENE REPRESSORS
GENES
GENETIC MAPPING
HEREDITARY DISEASES
MESSENGER-RNA
NEOPLASMS
NERVOUS SYSTEM DISEASES
PATIENTS
POLYMERASE CHAIN REACTION
PROTEINS
SCREENING
SENSITIVITY
SPLICING
TRANSCRIPTION