Markerless modification of trinucleotide repeat loci in BACs

Research output: Chapter in Book/Report/Conference proceedingChapter

Abstract

Transcription and splicing of human genes are regulated by nucleotide sequences encoded across large segments of our genome, and trinucleotide repeat expansion mutations can have both profound and subtle effects on these processes. In the course of our work to understand the impact of the Spinocerebellar Ataxia type 8 (SCA8) CTG repeat expansion on the transcription and splicing of the RNAs encoded near the SCA8 locus, we have developed a set of reagents and protocols for modifying large genomic BAC clones of this region. We describe the two-step procedure that allows us to precisely replace unexpanded trinucleotide repeats with expanded variants of these repeat sequences without leaving any exogenous sequences in the final constructs, and we discuss how this approach can be adapted to make other desired sequence changes to these genomic clones.

Original languageEnglish (US)
Title of host publicationTrinucleotide Repeat Protocols
PublisherHumana Press Inc.
Pages265-276
Number of pages12
ISBN (Print)9781627034104
DOIs
StatePublished - 2013

Publication series

NameMethods in Molecular Biology
Volume1010
ISSN (Print)1064-3745

Keywords

  • Bacterial Artificial Chromosomes (BACs)
  • Electroporation
  • Homologous recombination
  • Pulsed field gel electrophoresis (PFGE)
  • Trinucleotide Repeat Expansion

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