Cell-free cloning of highly expanded CTG repeats by amplification of dimerized expanded repeats

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 (CUGexp) RNA capable of sequestering MBNL1 protein in cell culture. Amplification of dimerized expanded repeats (ADER) opens new possibilities for studies of repeat instability and pathogenesis in myotonic dystrophy, a neurological disorder caused by an expanded CTG repeat. © 2008 The Author(s).