title: Finding MAPT Mutations in Frontotemporal Dementia and Other Tauopathies.
abstract: Sanger sequencing is a classic technique in molecular genetics to detect single nucleotide DNA variants in genomic DNA. Here we describe the detection of MAPT mutations by polymerase chain reaction amplification of patient genomic DNA followed by bidirectional Sanger sequencing. Exon trapping is a technique whereby genomic DNA covering the exon of interest and flanking intronic sequence is cloned into the intron of an expression vector and transfected into human cell lines. RNA is extracted and splicing products are examined by reverse-transcriptase PCR and agarose gel electrophoresis. We outline the application of this technique to assess the effect of novel DNA variants on the splicing efficiency of MAPT exon 10, a common mechanism of disease for pathogenic MAPT mutations.
authors.affiliation: Neuroscience Research Australia, Barker St., Randwick, Sydney, NSW, 2031, Australia.|School of Medical Sciences, University of New South Wales, Sydney, NSW, Australia.
authors.forename: Carol
authors.initials: C
authors.lastname: Dobson-Stone
authors.affiliation: Neuroscience Research Australia, Barker St., Randwick, Sydney, NSW, 2031, Australia. j.kwok@neura.edu.au.|School of Medical Sciences, University of New South Wales, Sydney, NSW, Australia. j.kwok@neura.edu.au.
authors.forename: John B J
authors.initials: JB
authors.lastname: Kwok
mesh_terms: D000328:Adult; D004252:DNA Mutational Analysis / Q000379:methods*; D005091:Exons / Q000235:genetics; D005260:Female; D057180:Frontotemporal Dementia / Q000235:genetics*; D006801:Humans; D007438:Introns / Q000235:genetics; D009154:Mutation / Q000235:genetics*; D016133:Polymerase Chain Reaction; D011247:Pregnancy; D012326:RNA Splicing / Q000235:genetics; D024801:Tauopathies / Q000235:genetics*
keywords: Exon trapping; Heterozygote detection; Polymerase chain reaction; Sanger sequencing; pSPL3