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Tau Protein pp 307-324 | Cite as

Finding MAPT Mutations in Frontotemporal Dementia and Other Tauopathies

  • Carol Dobson-Stone
  • John B. J. KwokEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1523)

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.

Key words

Polymerase chain reaction Sanger sequencing Heterozygote detection Exon trapping pSPL3 

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Copyright information

© Springer Science+Business Media New York 2017

Authors and Affiliations

  1. 1.Neuroscience Research AustraliaSydneyAustralia
  2. 2.School of Medical SciencesUniversity of New South WalesSydneyAustralia

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