Cell-free cloning of highly expanded CTG repeats by amplification of dimerized expanded repeats
Open Access
- 20 December 2007
- journal article
- research article
- Published by Oxford University Press (OUP) in Nucleic Acids Research
- Vol. 36 (4) , e24
- https://doi.org/10.1093/nar/gkn025
Abstract
We describe conditions for producing uninterrupted expanded CTG repeats consisting of up to 2000 repeats using ϕ29 DNA polymerase. Previously, generation of such repeats was hindered by CTG repeat instability in plasmid vectors maintained in Escherichia coli and poor in vitro ligation of CTG repeat concatemers due to strand slippage. Instead, we used a combination of in vitro ligation and ϕ29 DNA polymerase to amplify DNA. Correctly ligated products generating a dimerized repeat tract formed substrates for rolling circle amplification (RCA). In the presence of two non-complementary primers, hybridizing to either strand of DNA, ligations can be amplified to generate microgram quantities of repeat containing DNA. Additionally, expanded repeats generated by rolling circle amplification can be produced in vectors for expression of expanded CUG (CUG exp ) RNA capable of sequestering MBNL1 protein in cell culture. A mplification of d imerized e xpanded r epeats (ADER) opens new possibilities for studies of repeat instability and pathogenesis in myotonic dystrophy, a neurological disorder caused by an expanded CTG repeat.Keywords
This publication has 27 references indexed in Scilit:
- Reversible model of RNA toxicity and cardiac conduction defects in myotonic dystrophyNature Genetics, 2006
- MBNL1 and CUGBP1 modify expanded CUG-induced toxicity in a Drosophila model of myotonic dystrophy type 1Human Molecular Genetics, 2006
- Failure of MBNL1-dependent post-natal splicing transitions in myotonic dystrophyHuman Molecular Genetics, 2006
- Insights into Strand Displacement and Processivity from the Crystal Structure of the Protein-Primed DNA Polymerase of Bacteriophage φ29Molecular Cell, 2004
- Myotonic Dystrophy in Transgenic Mice Expressing an Expanded CUG RepeatScience, 2000
- Recruitment of human muscleblind proteins to (CUG)n expansions associated with myotonic dystrophyThe EMBO Journal, 2000
- Disruption of Splicing Regulated by a CUG-Binding Protein in Myotonic DystrophyScience, 1998
- Expansion and deletion of CTG repeats from human disease genes are determined by the direction of replication in E. coliNature Genetics, 1995
- Correlation between CTG trinucleotide repeat length and frequency of severe congenital myotonic dystrophyNature Genetics, 1992
- Expression of transfected DNA depends on DNA topologyCell, 1986