Abstract:
: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.
journal_name
Nucleic Acids Resjournal_title
Nucleic acids researchauthors
Osborne RJ,Thornton CAdoi
10.1093/nar/gkn025subject
Has Abstractpub_date
2008-03-01 00:00:00pages
e24issue
4eissn
0305-1048issn
1362-4962pii
gkn025journal_volume
36pub_type
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