The library of 240×CAG repeat-containing plasmids with variable flanking sequences was generated as follows. Double-stranded DNA oligonucleotides (~300 bases) were purchased from Quintara Biosciences. These DNA fragments were inserted in plasmids with 47× or 240×CAG repeats between EcoRI and MluI sites using standard restriction digestion and ligation procedures. To construct variants of CAGRAN with 5× or 22× CAG repeats, we purchased CAG repeat-containing single-stranded DNA (Integrated DNA Technologies, IDT), annealed and incorporated them between EcoRI and SgrDI sites downstream of the flanking sequence in CAGRAN. To construct CAGRANBFP, EBFP2 was obtained as a double-stranded DNA fragment (IDT) and was cloned between BamHI and NotI sites in CAGRAN. All cloning and plasmid preparations were performed in Stbl3 Escherichia coli cells (Invitrogen, C7373-03) grown at 30 °C. Since repeat number can spontaneously change during the cloning process, for each construct we verified the repeat tract in two ways. One, we optimized a Sanger sequencing protocol (in collaboration with Quintara Biosciences), which used betaine and 7-deaza-dGTP. This optimized sequencing protocol provided ~800 base long reads from each end. In constructs with 240×CAG repeats, Sanger sequencing did not provide sufficient read length to unambiguously determine the number of repeats, so the repeat number was verified by examining the size of the insert after restriction digestion and gel electrophoresis. Sanger sequencing revealed eight unintended interruptions in the CAG repeat track in our constructs with 240×CAG repeats (sequences in
Plasmid Construction for Trinucleotide Repeat Studies
The library of 240×CAG repeat-containing plasmids with variable flanking sequences was generated as follows. Double-stranded DNA oligonucleotides (~300 bases) were purchased from Quintara Biosciences. These DNA fragments were inserted in plasmids with 47× or 240×CAG repeats between EcoRI and MluI sites using standard restriction digestion and ligation procedures. To construct variants of CAGRAN with 5× or 22× CAG repeats, we purchased CAG repeat-containing single-stranded DNA (Integrated DNA Technologies, IDT), annealed and incorporated them between EcoRI and SgrDI sites downstream of the flanking sequence in CAGRAN. To construct CAGRANBFP, EBFP2 was obtained as a double-stranded DNA fragment (IDT) and was cloned between BamHI and NotI sites in CAGRAN. All cloning and plasmid preparations were performed in Stbl3 Escherichia coli cells (Invitrogen, C7373-03) grown at 30 °C. Since repeat number can spontaneously change during the cloning process, for each construct we verified the repeat tract in two ways. One, we optimized a Sanger sequencing protocol (in collaboration with Quintara Biosciences), which used betaine and 7-deaza-dGTP. This optimized sequencing protocol provided ~800 base long reads from each end. In constructs with 240×CAG repeats, Sanger sequencing did not provide sufficient read length to unambiguously determine the number of repeats, so the repeat number was verified by examining the size of the insert after restriction digestion and gel electrophoresis. Sanger sequencing revealed eight unintended interruptions in the CAG repeat track in our constructs with 240×CAG repeats (sequences in
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Corresponding Organization :
Other organizations : Whitehead Institute for Biomedical Research, Massachusetts Institute of Technology, University of California, San Francisco
Variable analysis
- Plasmid sequences with different CAG repeat lengths (47x, 240x, 5x, 22x)
- Plasmids with different flanking sequences from ATXN3, ATXN8, and HTT genes
- Plasmids with or without EBFP2 reporter
- Toxicity of the constructs
- Cytoplasmic RNA aggregation
- RAN translation
- Bacterial strain (Stbl3 Escherichia coli) used for cloning and plasmid preparation
- Temperature (30°C) used for bacterial growth
- Plasmid constructs containing 47x CAG repeats were used as a control for comparison with 240x CAG repeat constructs
- Plasmid constructs without unintended interruptions in the CAG repeat tract were used as a control for comparison with constructs containing interruptions
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