Cell-free cloning of highly expanded CTG repeats by amplification of dimerized expanded repeats.
We describe conditions for producing uninterrupted expanded CTG repeats consisting of up to 2000 repeats using 29 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 29 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. 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.
Osborne
Robert J
RJ
Department of Neurology, University of Rochester School of Medicine and Dentistry, 601 Elmwood Avenue, Rochester, NY 14642, USA. robert.osborne@dpag.ox.ac.uk
Thornton
Charles A
CA
D017103:Bacillus Phages / Q000201:enzymology; D002460:Cell Line; D002474:Cell-Free System; D003001:Cloning, Molecular / Q000379:methods*; D004259:DNA-Directed DNA Polymerase; D019281:Dimerization; D006801:Humans; D065636:Myotonin-Protein Kinase; D021141:Nucleic Acid Amplification Techniques; D010957:Plasmids / Q000235:genetics; D017346:Protein Serine-Threonine Kinases / Q000235:genetics; D019680:Trinucleotide Repeat Expansion
C585517:DMPK protein, human; D065636:Myotonin-Protein Kinase; D017346:Protein Serine-Threonine Kinases; D004259:DNA-Directed DNA Polymerase