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

Osborne RJ, Thornton CA - Nucleic Acids Res. (2008)

Bottom Line: Correctly ligated products generating a dimerized repeat tract formed substrates for rolling circle amplification (RCA).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.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurology, University of Rochester School of Medicine and Dentistry, 601 Elmwood Avenue, Rochester, NY 14642, USA. robert.osborne@dpag.ox.ac.uk

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.

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RCA of pHC or pHC120–130 plasmid DNA. (A) Restriction endonuclease digest of plasmid pHC generated a linear 1796 bp fragment with XhoI (lane 2), or 771 bp and 1025 bp fragments with HindIII (lane 3). RCA of plasmid pHC is shown in lanes 4–11. No RCA product was observed in the absence of template (lane 4) or ϕ29 DNA polymerase (lane 5). RCA products from duplicate reactions containing template and ϕ29 DNA polymerase migrated as high molecular weight DNA (lanes 6 and 7), slower than the largest marker DNA (10 kb). Restriction endonuclease digest of duplicate amplification products using XhoI (lanes 8 and 10) or HindIII (lanes 9 and 11) generated the same pattern as plasmid pHC template. Lanes 1 and 12 contain 1 kb DNA ladder (NEB). (B) Restriction endonuclease digest of plasmid pHC120–130 generated a linear 2194–2224 bp fragment with XhoI (lane 2), 1169–1199 bp and 1025 bp fragments with HindIII (lane 3) and 379–409 bp and 1815 bp with Acc65I and BsrGI (lane 4). Acc65I and BsrGI cleave on either side of the repeat tract. RCA products were observed from duplicate reactions containing both template and ϕ29 polymerase (lanes 7 and 8) but not in the absence of template (lane 5) or ϕ29 polymerase (lane 6). Restriction endonuclease digest of duplicate RCA products using XhoI (lanes 9 and 12), HindIII (lanes 10 and 13) or Acc65I-BsrGI (lanes 11 and 14) generated the same pattern as plasmid pHC120–130. Fragments containing repeats are marked with arrows. Lanes 1 and 15 contain 1 kb DNA ladder (NEB).
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Figure 2: RCA of pHC or pHC120–130 plasmid DNA. (A) Restriction endonuclease digest of plasmid pHC generated a linear 1796 bp fragment with XhoI (lane 2), or 771 bp and 1025 bp fragments with HindIII (lane 3). RCA of plasmid pHC is shown in lanes 4–11. No RCA product was observed in the absence of template (lane 4) or ϕ29 DNA polymerase (lane 5). RCA products from duplicate reactions containing template and ϕ29 DNA polymerase migrated as high molecular weight DNA (lanes 6 and 7), slower than the largest marker DNA (10 kb). Restriction endonuclease digest of duplicate amplification products using XhoI (lanes 8 and 10) or HindIII (lanes 9 and 11) generated the same pattern as plasmid pHC template. Lanes 1 and 12 contain 1 kb DNA ladder (NEB). (B) Restriction endonuclease digest of plasmid pHC120–130 generated a linear 2194–2224 bp fragment with XhoI (lane 2), 1169–1199 bp and 1025 bp fragments with HindIII (lane 3) and 379–409 bp and 1815 bp with Acc65I and BsrGI (lane 4). Acc65I and BsrGI cleave on either side of the repeat tract. RCA products were observed from duplicate reactions containing both template and ϕ29 polymerase (lanes 7 and 8) but not in the absence of template (lane 5) or ϕ29 polymerase (lane 6). Restriction endonuclease digest of duplicate RCA products using XhoI (lanes 9 and 12), HindIII (lanes 10 and 13) or Acc65I-BsrGI (lanes 11 and 14) generated the same pattern as plasmid pHC120–130. Fragments containing repeats are marked with arrows. Lanes 1 and 15 contain 1 kb DNA ladder (NEB).

Mentions: Initial experiments using the pHC vector without CTG repeats were used to establish conditions for the RCA. Using 100 ng of plasmid DNA as template we were able to detect reaction product in an RCA reaction at 37°C but not at 30°C (data not shown). Restriction endonuclease digestions of amplification products with XhoI or HindIII yielded fragments at the expected size, but no products were produced when template or ϕ29 DNA polymerase were omitted from the reaction (Figure 2A). We then used RCA to amplify 100 ng of plasmid DNA prepared from a vector containing CTG repeats, pHC120–130. Digestion of amplification products with XhoI, HindIII or Acc65I-BsrGI verified that the repeats were not deleted during the RCA reaction (Figure 2B). Digestions also yielded some minor bands which correspond in size to digested amplification products that terminate at one of the primers used for RCA. We confirmed that the (CTG)120–130 repeats were faithfully copied by sequencing plasmid DNA and amplification products. These sequencing reads included the entire CTG repeat tract, confirming that the plasmid DNA template and duplicate RCA products contained the same number (n = 124) of uninterrupted repeats.Figure 2.


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

Osborne RJ, Thornton CA - Nucleic Acids Res. (2008)

RCA of pHC or pHC120–130 plasmid DNA. (A) Restriction endonuclease digest of plasmid pHC generated a linear 1796 bp fragment with XhoI (lane 2), or 771 bp and 1025 bp fragments with HindIII (lane 3). RCA of plasmid pHC is shown in lanes 4–11. No RCA product was observed in the absence of template (lane 4) or ϕ29 DNA polymerase (lane 5). RCA products from duplicate reactions containing template and ϕ29 DNA polymerase migrated as high molecular weight DNA (lanes 6 and 7), slower than the largest marker DNA (10 kb). Restriction endonuclease digest of duplicate amplification products using XhoI (lanes 8 and 10) or HindIII (lanes 9 and 11) generated the same pattern as plasmid pHC template. Lanes 1 and 12 contain 1 kb DNA ladder (NEB). (B) Restriction endonuclease digest of plasmid pHC120–130 generated a linear 2194–2224 bp fragment with XhoI (lane 2), 1169–1199 bp and 1025 bp fragments with HindIII (lane 3) and 379–409 bp and 1815 bp with Acc65I and BsrGI (lane 4). Acc65I and BsrGI cleave on either side of the repeat tract. RCA products were observed from duplicate reactions containing both template and ϕ29 polymerase (lanes 7 and 8) but not in the absence of template (lane 5) or ϕ29 polymerase (lane 6). Restriction endonuclease digest of duplicate RCA products using XhoI (lanes 9 and 12), HindIII (lanes 10 and 13) or Acc65I-BsrGI (lanes 11 and 14) generated the same pattern as plasmid pHC120–130. Fragments containing repeats are marked with arrows. Lanes 1 and 15 contain 1 kb DNA ladder (NEB).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Figure 2: RCA of pHC or pHC120–130 plasmid DNA. (A) Restriction endonuclease digest of plasmid pHC generated a linear 1796 bp fragment with XhoI (lane 2), or 771 bp and 1025 bp fragments with HindIII (lane 3). RCA of plasmid pHC is shown in lanes 4–11. No RCA product was observed in the absence of template (lane 4) or ϕ29 DNA polymerase (lane 5). RCA products from duplicate reactions containing template and ϕ29 DNA polymerase migrated as high molecular weight DNA (lanes 6 and 7), slower than the largest marker DNA (10 kb). Restriction endonuclease digest of duplicate amplification products using XhoI (lanes 8 and 10) or HindIII (lanes 9 and 11) generated the same pattern as plasmid pHC template. Lanes 1 and 12 contain 1 kb DNA ladder (NEB). (B) Restriction endonuclease digest of plasmid pHC120–130 generated a linear 2194–2224 bp fragment with XhoI (lane 2), 1169–1199 bp and 1025 bp fragments with HindIII (lane 3) and 379–409 bp and 1815 bp with Acc65I and BsrGI (lane 4). Acc65I and BsrGI cleave on either side of the repeat tract. RCA products were observed from duplicate reactions containing both template and ϕ29 polymerase (lanes 7 and 8) but not in the absence of template (lane 5) or ϕ29 polymerase (lane 6). Restriction endonuclease digest of duplicate RCA products using XhoI (lanes 9 and 12), HindIII (lanes 10 and 13) or Acc65I-BsrGI (lanes 11 and 14) generated the same pattern as plasmid pHC120–130. Fragments containing repeats are marked with arrows. Lanes 1 and 15 contain 1 kb DNA ladder (NEB).
Mentions: Initial experiments using the pHC vector without CTG repeats were used to establish conditions for the RCA. Using 100 ng of plasmid DNA as template we were able to detect reaction product in an RCA reaction at 37°C but not at 30°C (data not shown). Restriction endonuclease digestions of amplification products with XhoI or HindIII yielded fragments at the expected size, but no products were produced when template or ϕ29 DNA polymerase were omitted from the reaction (Figure 2A). We then used RCA to amplify 100 ng of plasmid DNA prepared from a vector containing CTG repeats, pHC120–130. Digestion of amplification products with XhoI, HindIII or Acc65I-BsrGI verified that the repeats were not deleted during the RCA reaction (Figure 2B). Digestions also yielded some minor bands which correspond in size to digested amplification products that terminate at one of the primers used for RCA. We confirmed that the (CTG)120–130 repeats were faithfully copied by sequencing plasmid DNA and amplification products. These sequencing reads included the entire CTG repeat tract, confirming that the plasmid DNA template and duplicate RCA products contained the same number (n = 124) of uninterrupted repeats.Figure 2.

Bottom Line: Correctly ligated products generating a dimerized repeat tract formed substrates for rolling circle amplification (RCA).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.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurology, University of Rochester School of Medicine and Dentistry, 601 Elmwood Avenue, Rochester, NY 14642, USA. robert.osborne@dpag.ox.ac.uk

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.

Show MeSH
Related in: MedlinePlus