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    New England Biolabs xbai site located
    (GAA•TTC) n constructs used to analyze effect of DSB repair on triplet-repeat instability. pUC19 based constructs are shown containing either uninterrupted (GAA•TTC) 79 or (GAA•TTC) n sequences engineered to contain a <t>XbaI</t> recognition sequence at specific locations within the repeat tract (see Materials and Methods section for details). Repeat tracts were cloned in both orientations relative to the origin of replication (arrow indicates direction of replication). Repeat-containing plasmids are depicted either in the <t>‘GAA</t> orientation’ (e.g. GAA-79) or ‘TTC’ orientation (e.g. TTC-79), based on whether (GAA) n or (TTC) n serves as the lagging strand template, respectively. Numbers within the boxes indicate the length of the uninterrupted (GAA•TTC) n sequence.
    Xbai Site Located, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Repair of DNA double-strand breaks within the (GAAoTTC)n sequence results in frequent deletion of the triplet-repeat sequence"

    Article Title: Repair of DNA double-strand breaks within the (GAAoTTC)n sequence results in frequent deletion of the triplet-repeat sequence

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkm1066

    (GAA•TTC) n constructs used to analyze effect of DSB repair on triplet-repeat instability. pUC19 based constructs are shown containing either uninterrupted (GAA•TTC) 79 or (GAA•TTC) n sequences engineered to contain a XbaI recognition sequence at specific locations within the repeat tract (see Materials and Methods section for details). Repeat tracts were cloned in both orientations relative to the origin of replication (arrow indicates direction of replication). Repeat-containing plasmids are depicted either in the ‘GAA orientation’ (e.g. GAA-79) or ‘TTC’ orientation (e.g. TTC-79), based on whether (GAA) n or (TTC) n serves as the lagging strand template, respectively. Numbers within the boxes indicate the length of the uninterrupted (GAA•TTC) n sequence.
    Figure Legend Snippet: (GAA•TTC) n constructs used to analyze effect of DSB repair on triplet-repeat instability. pUC19 based constructs are shown containing either uninterrupted (GAA•TTC) 79 or (GAA•TTC) n sequences engineered to contain a XbaI recognition sequence at specific locations within the repeat tract (see Materials and Methods section for details). Repeat tracts were cloned in both orientations relative to the origin of replication (arrow indicates direction of replication). Repeat-containing plasmids are depicted either in the ‘GAA orientation’ (e.g. GAA-79) or ‘TTC’ orientation (e.g. TTC-79), based on whether (GAA) n or (TTC) n serves as the lagging strand template, respectively. Numbers within the boxes indicate the length of the uninterrupted (GAA•TTC) n sequence.

    Techniques Used: Construct, Sequencing, Clone Assay

    DSB repair mediated instability of the (GAA•TTC) n sequence is independent of RecA. ( A ) Repair of linear templates obtained by XbaI digestion of GAA-70X, TTC-70X, GAA-30X and TTC-30X resulted in similar levels of instability when transformed into E. coli MM28 [wild-type (WT)] and its isogenic recA mutant strain, M152. There was no difference in the level of repeat instability between the RecA-proficient and RecA-deficient strains, regardless of repeat length or the orientation with respect to the origin of replication. ( B ) Instability of GAA-30X constructs either uncut or following repair at the PstI, XbaI or KpnI restriction sites in isogenic E. coli strains AB1157 (RecA-proficient) and JC10287 (RecA-deficient) showing that DSB repair-mediated instability is independent of RecA. Note that there is no difference in the level of instability with any of the four constructs in AB1157 versus JC10287. DSB repair, when specifically within the repeat tract, causes significantly increased instability in both strains. All error bars represent +/−2 SEM derived from triplicate experiments.
    Figure Legend Snippet: DSB repair mediated instability of the (GAA•TTC) n sequence is independent of RecA. ( A ) Repair of linear templates obtained by XbaI digestion of GAA-70X, TTC-70X, GAA-30X and TTC-30X resulted in similar levels of instability when transformed into E. coli MM28 [wild-type (WT)] and its isogenic recA mutant strain, M152. There was no difference in the level of repeat instability between the RecA-proficient and RecA-deficient strains, regardless of repeat length or the orientation with respect to the origin of replication. ( B ) Instability of GAA-30X constructs either uncut or following repair at the PstI, XbaI or KpnI restriction sites in isogenic E. coli strains AB1157 (RecA-proficient) and JC10287 (RecA-deficient) showing that DSB repair-mediated instability is independent of RecA. Note that there is no difference in the level of instability with any of the four constructs in AB1157 versus JC10287. DSB repair, when specifically within the repeat tract, causes significantly increased instability in both strains. All error bars represent +/−2 SEM derived from triplicate experiments.

    Techniques Used: Sequencing, Transformation Assay, Mutagenesis, Construct, Derivative Assay

    DSB repair at the center of the (GAA•TTC) n sequence results in the preferential deletion of approximately half (or less than half) of the total repeat length. The residual tract lengths of the (GAA•TTC) n sequence are shown (as a percentage of full-length) after transformation in E. coli MM28 of ( A ) EcoRI and HindIII linearized GAA-79 and TTC-79; ( B ) XbaI linearized GAA-70X and TTC-70X; ( C ) XbaI linearized GAA-30X and TTC-30X, ( D ) XbaI linearized (3′ recessed) and after end filling (blunt-ended) GAA-70X, ( E ) XbaI linearized GAA-30X in MM28 (RecA-proficient) and M152 (RecA-deficient) isogenic strains, and ( F ) XbaI linearized GAA-30X in AB1157 (RecA-proficient) and JC10287 (RecA-deficient) isogenic strains. Note that the size distribution of deletion products is random when DSB repair occurs outside the repeat (EcoRI, HindIII); however, approximately half of the repeat tract, or less, is preferentially deleted when DSB repair occurs at the center of the repeat tract. This is irrespective of the initial length of the repeat tract (70 or 30 triplets), the nature of the termini being repaired (staggered or blunt), or the presence/absence of RecA.
    Figure Legend Snippet: DSB repair at the center of the (GAA•TTC) n sequence results in the preferential deletion of approximately half (or less than half) of the total repeat length. The residual tract lengths of the (GAA•TTC) n sequence are shown (as a percentage of full-length) after transformation in E. coli MM28 of ( A ) EcoRI and HindIII linearized GAA-79 and TTC-79; ( B ) XbaI linearized GAA-70X and TTC-70X; ( C ) XbaI linearized GAA-30X and TTC-30X, ( D ) XbaI linearized (3′ recessed) and after end filling (blunt-ended) GAA-70X, ( E ) XbaI linearized GAA-30X in MM28 (RecA-proficient) and M152 (RecA-deficient) isogenic strains, and ( F ) XbaI linearized GAA-30X in AB1157 (RecA-proficient) and JC10287 (RecA-deficient) isogenic strains. Note that the size distribution of deletion products is random when DSB repair occurs outside the repeat (EcoRI, HindIII); however, approximately half of the repeat tract, or less, is preferentially deleted when DSB repair occurs at the center of the repeat tract. This is irrespective of the initial length of the repeat tract (70 or 30 triplets), the nature of the termini being repaired (staggered or blunt), or the presence/absence of RecA.

    Techniques Used: Sequencing, Transformation Assay

    DSB repair results in dramatically increased instability of the (GAA•TTC) n sequence when the break is located within the repeat tract. ( A ) Representative agarose gels with products of colony PCR showing transformants of GAA-79 [circular (uncut), or linearized with HindIII or EcoRI], and GAA-70X [circular (uncut), or linearized with XbaI]. Arrowheads indicate the position of the full-length repeat tract. DSB repair outside the repeat tract showed levels of instability that were similar to the uncut plasmids; however, DSB repair within the repeat tract resulted in a very high frequency of deletions. The first lane in each gel contains the 1 kb Plus ladder (Invitrogen) with bands from the bottom of the gel representing 0.2, 0.3, 0.4, 0.5 and 0.65 kb (note: the full-length products of GAA-79 and GAA-70X are 423 and 312 bp, respectively, due to the presence of some flanking intron 1 sequence from the human FXN gene in the former). ( B ) Representative agarose gels with products of colony PCR showing transformants of GAA-30X [circular (uncut), or linearized with PstI, XbaI or KpnI]. Arrowheads indicate the position of the full-length repeat tract. DSB repair outside the repeat tract showed levels of instability that were similar to the uncut plasmids; however, DSB repair within the repeat tract resulted in a very high frequency of deletions. Note that even the (GAA•TTC) 30 sequence, which was otherwise extremely stable, showed a dramatic rise in the frequency of deletions. The first lane of every gel contains a DNA size marker; the markers used in the gels containing repair products of templates GAA-30X PstI and KpnI are different from all other gels. The first lane in each gel contains either the 1 kb Plus ladder (Invitrogen) (GAA-30X; PstI and KpnI with bands from the bottom of the gel representing 0.1, 0.2 and 0.3 kb, or the 50 bp DNA ladder (Invitrogen) (GAA-30X; uncut and XbaI) with bands from the bottom of the gel representing 0.1, 0.15, 0.2, 0.25, 0.3 and 0.35 kb. ( C ) DSB repair within a slightly unstable (GAA•TTC) 70–79 sequence produced a dramatic rise in instability. Note that the (GAA•TTC) n sequence was equally unstable in the GAA and TTC orientations. ( D ) DSB repair within a highly stable (GAA•TTC) 30 sequence produced a dramatic rise in instability. Note that the (GAA•TTC) n sequence was equally unstable in the GAA and TTC orientations. All error bars represent +/−2 SEM derived from triplicate experiments.
    Figure Legend Snippet: DSB repair results in dramatically increased instability of the (GAA•TTC) n sequence when the break is located within the repeat tract. ( A ) Representative agarose gels with products of colony PCR showing transformants of GAA-79 [circular (uncut), or linearized with HindIII or EcoRI], and GAA-70X [circular (uncut), or linearized with XbaI]. Arrowheads indicate the position of the full-length repeat tract. DSB repair outside the repeat tract showed levels of instability that were similar to the uncut plasmids; however, DSB repair within the repeat tract resulted in a very high frequency of deletions. The first lane in each gel contains the 1 kb Plus ladder (Invitrogen) with bands from the bottom of the gel representing 0.2, 0.3, 0.4, 0.5 and 0.65 kb (note: the full-length products of GAA-79 and GAA-70X are 423 and 312 bp, respectively, due to the presence of some flanking intron 1 sequence from the human FXN gene in the former). ( B ) Representative agarose gels with products of colony PCR showing transformants of GAA-30X [circular (uncut), or linearized with PstI, XbaI or KpnI]. Arrowheads indicate the position of the full-length repeat tract. DSB repair outside the repeat tract showed levels of instability that were similar to the uncut plasmids; however, DSB repair within the repeat tract resulted in a very high frequency of deletions. Note that even the (GAA•TTC) 30 sequence, which was otherwise extremely stable, showed a dramatic rise in the frequency of deletions. The first lane of every gel contains a DNA size marker; the markers used in the gels containing repair products of templates GAA-30X PstI and KpnI are different from all other gels. The first lane in each gel contains either the 1 kb Plus ladder (Invitrogen) (GAA-30X; PstI and KpnI with bands from the bottom of the gel representing 0.1, 0.2 and 0.3 kb, or the 50 bp DNA ladder (Invitrogen) (GAA-30X; uncut and XbaI) with bands from the bottom of the gel representing 0.1, 0.15, 0.2, 0.25, 0.3 and 0.35 kb. ( C ) DSB repair within a slightly unstable (GAA•TTC) 70–79 sequence produced a dramatic rise in instability. Note that the (GAA•TTC) n sequence was equally unstable in the GAA and TTC orientations. ( D ) DSB repair within a highly stable (GAA•TTC) 30 sequence produced a dramatic rise in instability. Note that the (GAA•TTC) n sequence was equally unstable in the GAA and TTC orientations. All error bars represent +/−2 SEM derived from triplicate experiments.

    Techniques Used: Sequencing, Polymerase Chain Reaction, Marker, Produced, Derivative Assay

    DSB repair-mediated instability of the (GAA•TTC) n sequence is independent of the length of the intervening sequence at the center of the repeat tract. ( A ) GAA-70-spacer construct containing a 28 bp spacer in the XbaI site of GAA-70X, such that BamHI would cut in the center of the spacer (indicated by the black box). ( B ) DSB repair at the BamHI site in GAA-70-spacer construct produced a dramatic rise in instability. Note that the (GAA•TTC) n sequence was equally unstable when transformed into E. coli MM28 [wild-type (WT)] and its isogenic recA mutant strain, M152. All error bars represent +/−2 SEM derived from triplicate experiments. ( C ) DSB repair at the center of the (GAA•TTC) n sequence results in the preferential deletion of approximately half of the total repeat length. The residual tract lengths of the (GAA•TTC) n sequence are shown (as a percentage of full-length) after transformation of BamHI-linearized GAA-70-spacer vector in E. coli MM28 (WT) and M152 ( recA ).
    Figure Legend Snippet: DSB repair-mediated instability of the (GAA•TTC) n sequence is independent of the length of the intervening sequence at the center of the repeat tract. ( A ) GAA-70-spacer construct containing a 28 bp spacer in the XbaI site of GAA-70X, such that BamHI would cut in the center of the spacer (indicated by the black box). ( B ) DSB repair at the BamHI site in GAA-70-spacer construct produced a dramatic rise in instability. Note that the (GAA•TTC) n sequence was equally unstable when transformed into E. coli MM28 [wild-type (WT)] and its isogenic recA mutant strain, M152. All error bars represent +/−2 SEM derived from triplicate experiments. ( C ) DSB repair at the center of the (GAA•TTC) n sequence results in the preferential deletion of approximately half of the total repeat length. The residual tract lengths of the (GAA•TTC) n sequence are shown (as a percentage of full-length) after transformation of BamHI-linearized GAA-70-spacer vector in E. coli MM28 (WT) and M152 ( recA ).

    Techniques Used: Sequencing, Construct, Produced, Transformation Assay, Mutagenesis, Derivative Assay, Plasmid Preparation

    Related Articles

    Clone Assay:

    Article Title: Repair of DNA double-strand breaks within the (GAAoTTC)n sequence results in frequent deletion of the triplet-repeat sequence
    Article Snippet: Plasmid construction A (GAA•TTC)79 repeat sequence, isolated by PCR from the FXN locus of a human subject, was cloned into pUC19 as previously described ( ). .. The GAA-70X construct, which contains the (GAA•TTC)70 sequence with an XbaI site located exactly at the center of the repeat tract , was created using four synthetic oligonucleotides, as follows: Oligo #1: 5′-GGCGCTCCGCTGCAGCC(GAA)35 TCTAGACGCATCGCC-3′ and Oligo #2: 5′GGCGATGCGTCTAGA(TTC)35 GGCTGCAGCGGAGCGCC-3′ were annealed together in 10 mM Tris, pH 8.0, and digestion buffer 3 (100 mM NaCl, 10 mM MgCl2 ) (New England Biolabs), followed by incubating with PstI and XbaI restriction enzymes to digest both ends of the annealed oligos.

    Isolation:

    Article Title: Repair of DNA double-strand breaks within the (GAAoTTC)n sequence results in frequent deletion of the triplet-repeat sequence
    Article Snippet: Plasmid construction A (GAA•TTC)79 repeat sequence, isolated by PCR from the FXN locus of a human subject, was cloned into pUC19 as previously described ( ). .. The GAA-70X construct, which contains the (GAA•TTC)70 sequence with an XbaI site located exactly at the center of the repeat tract , was created using four synthetic oligonucleotides, as follows: Oligo #1: 5′-GGCGCTCCGCTGCAGCC(GAA)35 TCTAGACGCATCGCC-3′ and Oligo #2: 5′GGCGATGCGTCTAGA(TTC)35 GGCTGCAGCGGAGCGCC-3′ were annealed together in 10 mM Tris, pH 8.0, and digestion buffer 3 (100 mM NaCl, 10 mM MgCl2 ) (New England Biolabs), followed by incubating with PstI and XbaI restriction enzymes to digest both ends of the annealed oligos.

    Low Copy Number:

    Article Title: Repair of DNA double-strand breaks within the (GAAoTTC)n sequence results in frequent deletion of the triplet-repeat sequence
    Article Snippet: The pUC19 vector contains a portion of the ROP gene, making it a low copy number plasmid. .. The GAA-70X construct, which contains the (GAA•TTC)70 sequence with an XbaI site located exactly at the center of the repeat tract , was created using four synthetic oligonucleotides, as follows: Oligo #1: 5′-GGCGCTCCGCTGCAGCC(GAA)35 TCTAGACGCATCGCC-3′ and Oligo #2: 5′GGCGATGCGTCTAGA(TTC)35 GGCTGCAGCGGAGCGCC-3′ were annealed together in 10 mM Tris, pH 8.0, and digestion buffer 3 (100 mM NaCl, 10 mM MgCl2 ) (New England Biolabs), followed by incubating with PstI and XbaI restriction enzymes to digest both ends of the annealed oligos.

    Construct:

    Article Title: Repair of DNA double-strand breaks within the (GAAoTTC)n sequence results in frequent deletion of the triplet-repeat sequence
    Article Snippet: .. The GAA-70X construct, which contains the (GAA•TTC)70 sequence with an XbaI site located exactly at the center of the repeat tract , was created using four synthetic oligonucleotides, as follows: Oligo #1: 5′-GGCGCTCCGCTGCAGCC(GAA)35 TCTAGACGCATCGCC-3′ and Oligo #2: 5′GGCGATGCGTCTAGA(TTC)35 GGCTGCAGCGGAGCGCC-3′ were annealed together in 10 mM Tris, pH 8.0, and digestion buffer 3 (100 mM NaCl, 10 mM MgCl2 ) (New England Biolabs), followed by incubating with PstI and XbaI restriction enzymes to digest both ends of the annealed oligos. .. The purified insert was subcloned into the PstI and XbaI sites of pUC19, and confirmed by sequencing.

    Purification:

    Article Title: Repair of DNA double-strand breaks within the (GAAoTTC)n sequence results in frequent deletion of the triplet-repeat sequence
    Article Snippet: The GAA-70X construct, which contains the (GAA•TTC)70 sequence with an XbaI site located exactly at the center of the repeat tract , was created using four synthetic oligonucleotides, as follows: Oligo #1: 5′-GGCGCTCCGCTGCAGCC(GAA)35 TCTAGACGCATCGCC-3′ and Oligo #2: 5′GGCGATGCGTCTAGA(TTC)35 GGCTGCAGCGGAGCGCC-3′ were annealed together in 10 mM Tris, pH 8.0, and digestion buffer 3 (100 mM NaCl, 10 mM MgCl2 ) (New England Biolabs), followed by incubating with PstI and XbaI restriction enzymes to digest both ends of the annealed oligos. .. The purified insert was subcloned into the PstI and XbaI sites of pUC19, and confirmed by sequencing.

    Sequencing:

    Article Title: Repair of DNA double-strand breaks within the (GAAoTTC)n sequence results in frequent deletion of the triplet-repeat sequence
    Article Snippet: .. The GAA-70X construct, which contains the (GAA•TTC)70 sequence with an XbaI site located exactly at the center of the repeat tract , was created using four synthetic oligonucleotides, as follows: Oligo #1: 5′-GGCGCTCCGCTGCAGCC(GAA)35 TCTAGACGCATCGCC-3′ and Oligo #2: 5′GGCGATGCGTCTAGA(TTC)35 GGCTGCAGCGGAGCGCC-3′ were annealed together in 10 mM Tris, pH 8.0, and digestion buffer 3 (100 mM NaCl, 10 mM MgCl2 ) (New England Biolabs), followed by incubating with PstI and XbaI restriction enzymes to digest both ends of the annealed oligos. .. The purified insert was subcloned into the PstI and XbaI sites of pUC19, and confirmed by sequencing.

    Polymerase Chain Reaction:

    Article Title: Repair of DNA double-strand breaks within the (GAAoTTC)n sequence results in frequent deletion of the triplet-repeat sequence
    Article Snippet: Plasmid construction A (GAA•TTC)79 repeat sequence, isolated by PCR from the FXN locus of a human subject, was cloned into pUC19 as previously described ( ). .. The GAA-70X construct, which contains the (GAA•TTC)70 sequence with an XbaI site located exactly at the center of the repeat tract , was created using four synthetic oligonucleotides, as follows: Oligo #1: 5′-GGCGCTCCGCTGCAGCC(GAA)35 TCTAGACGCATCGCC-3′ and Oligo #2: 5′GGCGATGCGTCTAGA(TTC)35 GGCTGCAGCGGAGCGCC-3′ were annealed together in 10 mM Tris, pH 8.0, and digestion buffer 3 (100 mM NaCl, 10 mM MgCl2 ) (New England Biolabs), followed by incubating with PstI and XbaI restriction enzymes to digest both ends of the annealed oligos.

    Recombinant:

    Article Title: Repair of DNA double-strand breaks within the (GAAoTTC)n sequence results in frequent deletion of the triplet-repeat sequence
    Article Snippet: The GAA-70X construct, which contains the (GAA•TTC)70 sequence with an XbaI site located exactly at the center of the repeat tract , was created using four synthetic oligonucleotides, as follows: Oligo #1: 5′-GGCGCTCCGCTGCAGCC(GAA)35 TCTAGACGCATCGCC-3′ and Oligo #2: 5′GGCGATGCGTCTAGA(TTC)35 GGCTGCAGCGGAGCGCC-3′ were annealed together in 10 mM Tris, pH 8.0, and digestion buffer 3 (100 mM NaCl, 10 mM MgCl2 ) (New England Biolabs), followed by incubating with PstI and XbaI restriction enzymes to digest both ends of the annealed oligos. .. Oligo #3: 5′-GGCGCTCCGTCTAGA(GAA)35 CCGGTACCCGCATCGCC-3′ and Oligo #4: 5′-GGCGATGCGGGTACCGG(TTC)35 TCTAGACGCATCGCC-3′were annealed in 10 mM Tris, pH 8.0 and digestion buffer 2 (50 mM NaCl, 10 mM MgCl2 ) (New England Biolabs), digested with XbaI and KpnI, and ligated into the similarly digested recombinant pUC19 vector containing the first (GAA•TTC)35 insert in the PstI and XbaI restriction sites (described above).

    Plasmid Preparation:

    Article Title: Repair of DNA double-strand breaks within the (GAAoTTC)n sequence results in frequent deletion of the triplet-repeat sequence
    Article Snippet: Paragraph title: Plasmid construction ... The GAA-70X construct, which contains the (GAA•TTC)70 sequence with an XbaI site located exactly at the center of the repeat tract , was created using four synthetic oligonucleotides, as follows: Oligo #1: 5′-GGCGCTCCGCTGCAGCC(GAA)35 TCTAGACGCATCGCC-3′ and Oligo #2: 5′GGCGATGCGTCTAGA(TTC)35 GGCTGCAGCGGAGCGCC-3′ were annealed together in 10 mM Tris, pH 8.0, and digestion buffer 3 (100 mM NaCl, 10 mM MgCl2 ) (New England Biolabs), followed by incubating with PstI and XbaI restriction enzymes to digest both ends of the annealed oligos.

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    New England Biolabs xbai site located
    (GAA•TTC) n constructs used to analyze effect of DSB repair on triplet-repeat instability. pUC19 based constructs are shown containing either uninterrupted (GAA•TTC) 79 or (GAA•TTC) n sequences engineered to contain a <t>XbaI</t> recognition sequence at specific locations within the repeat tract (see Materials and Methods section for details). Repeat tracts were cloned in both orientations relative to the origin of replication (arrow indicates direction of replication). Repeat-containing plasmids are depicted either in the <t>‘GAA</t> orientation’ (e.g. GAA-79) or ‘TTC’ orientation (e.g. TTC-79), based on whether (GAA) n or (TTC) n serves as the lagging strand template, respectively. Numbers within the boxes indicate the length of the uninterrupted (GAA•TTC) n sequence.
    Xbai Site Located, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/xbai site located/product/New England Biolabs
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    xbai site located - by Bioz Stars, 2020-04
    86/100 stars
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    (GAA•TTC) n constructs used to analyze effect of DSB repair on triplet-repeat instability. pUC19 based constructs are shown containing either uninterrupted (GAA•TTC) 79 or (GAA•TTC) n sequences engineered to contain a XbaI recognition sequence at specific locations within the repeat tract (see Materials and Methods section for details). Repeat tracts were cloned in both orientations relative to the origin of replication (arrow indicates direction of replication). Repeat-containing plasmids are depicted either in the ‘GAA orientation’ (e.g. GAA-79) or ‘TTC’ orientation (e.g. TTC-79), based on whether (GAA) n or (TTC) n serves as the lagging strand template, respectively. Numbers within the boxes indicate the length of the uninterrupted (GAA•TTC) n sequence.

    Journal: Nucleic Acids Research

    Article Title: Repair of DNA double-strand breaks within the (GAAoTTC)n sequence results in frequent deletion of the triplet-repeat sequence

    doi: 10.1093/nar/gkm1066

    Figure Lengend Snippet: (GAA•TTC) n constructs used to analyze effect of DSB repair on triplet-repeat instability. pUC19 based constructs are shown containing either uninterrupted (GAA•TTC) 79 or (GAA•TTC) n sequences engineered to contain a XbaI recognition sequence at specific locations within the repeat tract (see Materials and Methods section for details). Repeat tracts were cloned in both orientations relative to the origin of replication (arrow indicates direction of replication). Repeat-containing plasmids are depicted either in the ‘GAA orientation’ (e.g. GAA-79) or ‘TTC’ orientation (e.g. TTC-79), based on whether (GAA) n or (TTC) n serves as the lagging strand template, respectively. Numbers within the boxes indicate the length of the uninterrupted (GAA•TTC) n sequence.

    Article Snippet: The GAA-70X construct, which contains the (GAA•TTC)70 sequence with an XbaI site located exactly at the center of the repeat tract , was created using four synthetic oligonucleotides, as follows: Oligo #1: 5′-GGCGCTCCGCTGCAGCC(GAA)35 TCTAGACGCATCGCC-3′ and Oligo #2: 5′GGCGATGCGTCTAGA(TTC)35 GGCTGCAGCGGAGCGCC-3′ were annealed together in 10 mM Tris, pH 8.0, and digestion buffer 3 (100 mM NaCl, 10 mM MgCl2 ) (New England Biolabs), followed by incubating with PstI and XbaI restriction enzymes to digest both ends of the annealed oligos.

    Techniques: Construct, Sequencing, Clone Assay

    DSB repair mediated instability of the (GAA•TTC) n sequence is independent of RecA. ( A ) Repair of linear templates obtained by XbaI digestion of GAA-70X, TTC-70X, GAA-30X and TTC-30X resulted in similar levels of instability when transformed into E. coli MM28 [wild-type (WT)] and its isogenic recA mutant strain, M152. There was no difference in the level of repeat instability between the RecA-proficient and RecA-deficient strains, regardless of repeat length or the orientation with respect to the origin of replication. ( B ) Instability of GAA-30X constructs either uncut or following repair at the PstI, XbaI or KpnI restriction sites in isogenic E. coli strains AB1157 (RecA-proficient) and JC10287 (RecA-deficient) showing that DSB repair-mediated instability is independent of RecA. Note that there is no difference in the level of instability with any of the four constructs in AB1157 versus JC10287. DSB repair, when specifically within the repeat tract, causes significantly increased instability in both strains. All error bars represent +/−2 SEM derived from triplicate experiments.

    Journal: Nucleic Acids Research

    Article Title: Repair of DNA double-strand breaks within the (GAAoTTC)n sequence results in frequent deletion of the triplet-repeat sequence

    doi: 10.1093/nar/gkm1066

    Figure Lengend Snippet: DSB repair mediated instability of the (GAA•TTC) n sequence is independent of RecA. ( A ) Repair of linear templates obtained by XbaI digestion of GAA-70X, TTC-70X, GAA-30X and TTC-30X resulted in similar levels of instability when transformed into E. coli MM28 [wild-type (WT)] and its isogenic recA mutant strain, M152. There was no difference in the level of repeat instability between the RecA-proficient and RecA-deficient strains, regardless of repeat length or the orientation with respect to the origin of replication. ( B ) Instability of GAA-30X constructs either uncut or following repair at the PstI, XbaI or KpnI restriction sites in isogenic E. coli strains AB1157 (RecA-proficient) and JC10287 (RecA-deficient) showing that DSB repair-mediated instability is independent of RecA. Note that there is no difference in the level of instability with any of the four constructs in AB1157 versus JC10287. DSB repair, when specifically within the repeat tract, causes significantly increased instability in both strains. All error bars represent +/−2 SEM derived from triplicate experiments.

    Article Snippet: The GAA-70X construct, which contains the (GAA•TTC)70 sequence with an XbaI site located exactly at the center of the repeat tract , was created using four synthetic oligonucleotides, as follows: Oligo #1: 5′-GGCGCTCCGCTGCAGCC(GAA)35 TCTAGACGCATCGCC-3′ and Oligo #2: 5′GGCGATGCGTCTAGA(TTC)35 GGCTGCAGCGGAGCGCC-3′ were annealed together in 10 mM Tris, pH 8.0, and digestion buffer 3 (100 mM NaCl, 10 mM MgCl2 ) (New England Biolabs), followed by incubating with PstI and XbaI restriction enzymes to digest both ends of the annealed oligos.

    Techniques: Sequencing, Transformation Assay, Mutagenesis, Construct, Derivative Assay

    DSB repair at the center of the (GAA•TTC) n sequence results in the preferential deletion of approximately half (or less than half) of the total repeat length. The residual tract lengths of the (GAA•TTC) n sequence are shown (as a percentage of full-length) after transformation in E. coli MM28 of ( A ) EcoRI and HindIII linearized GAA-79 and TTC-79; ( B ) XbaI linearized GAA-70X and TTC-70X; ( C ) XbaI linearized GAA-30X and TTC-30X, ( D ) XbaI linearized (3′ recessed) and after end filling (blunt-ended) GAA-70X, ( E ) XbaI linearized GAA-30X in MM28 (RecA-proficient) and M152 (RecA-deficient) isogenic strains, and ( F ) XbaI linearized GAA-30X in AB1157 (RecA-proficient) and JC10287 (RecA-deficient) isogenic strains. Note that the size distribution of deletion products is random when DSB repair occurs outside the repeat (EcoRI, HindIII); however, approximately half of the repeat tract, or less, is preferentially deleted when DSB repair occurs at the center of the repeat tract. This is irrespective of the initial length of the repeat tract (70 or 30 triplets), the nature of the termini being repaired (staggered or blunt), or the presence/absence of RecA.

    Journal: Nucleic Acids Research

    Article Title: Repair of DNA double-strand breaks within the (GAAoTTC)n sequence results in frequent deletion of the triplet-repeat sequence

    doi: 10.1093/nar/gkm1066

    Figure Lengend Snippet: DSB repair at the center of the (GAA•TTC) n sequence results in the preferential deletion of approximately half (or less than half) of the total repeat length. The residual tract lengths of the (GAA•TTC) n sequence are shown (as a percentage of full-length) after transformation in E. coli MM28 of ( A ) EcoRI and HindIII linearized GAA-79 and TTC-79; ( B ) XbaI linearized GAA-70X and TTC-70X; ( C ) XbaI linearized GAA-30X and TTC-30X, ( D ) XbaI linearized (3′ recessed) and after end filling (blunt-ended) GAA-70X, ( E ) XbaI linearized GAA-30X in MM28 (RecA-proficient) and M152 (RecA-deficient) isogenic strains, and ( F ) XbaI linearized GAA-30X in AB1157 (RecA-proficient) and JC10287 (RecA-deficient) isogenic strains. Note that the size distribution of deletion products is random when DSB repair occurs outside the repeat (EcoRI, HindIII); however, approximately half of the repeat tract, or less, is preferentially deleted when DSB repair occurs at the center of the repeat tract. This is irrespective of the initial length of the repeat tract (70 or 30 triplets), the nature of the termini being repaired (staggered or blunt), or the presence/absence of RecA.

    Article Snippet: The GAA-70X construct, which contains the (GAA•TTC)70 sequence with an XbaI site located exactly at the center of the repeat tract , was created using four synthetic oligonucleotides, as follows: Oligo #1: 5′-GGCGCTCCGCTGCAGCC(GAA)35 TCTAGACGCATCGCC-3′ and Oligo #2: 5′GGCGATGCGTCTAGA(TTC)35 GGCTGCAGCGGAGCGCC-3′ were annealed together in 10 mM Tris, pH 8.0, and digestion buffer 3 (100 mM NaCl, 10 mM MgCl2 ) (New England Biolabs), followed by incubating with PstI and XbaI restriction enzymes to digest both ends of the annealed oligos.

    Techniques: Sequencing, Transformation Assay

    DSB repair results in dramatically increased instability of the (GAA•TTC) n sequence when the break is located within the repeat tract. ( A ) Representative agarose gels with products of colony PCR showing transformants of GAA-79 [circular (uncut), or linearized with HindIII or EcoRI], and GAA-70X [circular (uncut), or linearized with XbaI]. Arrowheads indicate the position of the full-length repeat tract. DSB repair outside the repeat tract showed levels of instability that were similar to the uncut plasmids; however, DSB repair within the repeat tract resulted in a very high frequency of deletions. The first lane in each gel contains the 1 kb Plus ladder (Invitrogen) with bands from the bottom of the gel representing 0.2, 0.3, 0.4, 0.5 and 0.65 kb (note: the full-length products of GAA-79 and GAA-70X are 423 and 312 bp, respectively, due to the presence of some flanking intron 1 sequence from the human FXN gene in the former). ( B ) Representative agarose gels with products of colony PCR showing transformants of GAA-30X [circular (uncut), or linearized with PstI, XbaI or KpnI]. Arrowheads indicate the position of the full-length repeat tract. DSB repair outside the repeat tract showed levels of instability that were similar to the uncut plasmids; however, DSB repair within the repeat tract resulted in a very high frequency of deletions. Note that even the (GAA•TTC) 30 sequence, which was otherwise extremely stable, showed a dramatic rise in the frequency of deletions. The first lane of every gel contains a DNA size marker; the markers used in the gels containing repair products of templates GAA-30X PstI and KpnI are different from all other gels. The first lane in each gel contains either the 1 kb Plus ladder (Invitrogen) (GAA-30X; PstI and KpnI with bands from the bottom of the gel representing 0.1, 0.2 and 0.3 kb, or the 50 bp DNA ladder (Invitrogen) (GAA-30X; uncut and XbaI) with bands from the bottom of the gel representing 0.1, 0.15, 0.2, 0.25, 0.3 and 0.35 kb. ( C ) DSB repair within a slightly unstable (GAA•TTC) 70–79 sequence produced a dramatic rise in instability. Note that the (GAA•TTC) n sequence was equally unstable in the GAA and TTC orientations. ( D ) DSB repair within a highly stable (GAA•TTC) 30 sequence produced a dramatic rise in instability. Note that the (GAA•TTC) n sequence was equally unstable in the GAA and TTC orientations. All error bars represent +/−2 SEM derived from triplicate experiments.

    Journal: Nucleic Acids Research

    Article Title: Repair of DNA double-strand breaks within the (GAAoTTC)n sequence results in frequent deletion of the triplet-repeat sequence

    doi: 10.1093/nar/gkm1066

    Figure Lengend Snippet: DSB repair results in dramatically increased instability of the (GAA•TTC) n sequence when the break is located within the repeat tract. ( A ) Representative agarose gels with products of colony PCR showing transformants of GAA-79 [circular (uncut), or linearized with HindIII or EcoRI], and GAA-70X [circular (uncut), or linearized with XbaI]. Arrowheads indicate the position of the full-length repeat tract. DSB repair outside the repeat tract showed levels of instability that were similar to the uncut plasmids; however, DSB repair within the repeat tract resulted in a very high frequency of deletions. The first lane in each gel contains the 1 kb Plus ladder (Invitrogen) with bands from the bottom of the gel representing 0.2, 0.3, 0.4, 0.5 and 0.65 kb (note: the full-length products of GAA-79 and GAA-70X are 423 and 312 bp, respectively, due to the presence of some flanking intron 1 sequence from the human FXN gene in the former). ( B ) Representative agarose gels with products of colony PCR showing transformants of GAA-30X [circular (uncut), or linearized with PstI, XbaI or KpnI]. Arrowheads indicate the position of the full-length repeat tract. DSB repair outside the repeat tract showed levels of instability that were similar to the uncut plasmids; however, DSB repair within the repeat tract resulted in a very high frequency of deletions. Note that even the (GAA•TTC) 30 sequence, which was otherwise extremely stable, showed a dramatic rise in the frequency of deletions. The first lane of every gel contains a DNA size marker; the markers used in the gels containing repair products of templates GAA-30X PstI and KpnI are different from all other gels. The first lane in each gel contains either the 1 kb Plus ladder (Invitrogen) (GAA-30X; PstI and KpnI with bands from the bottom of the gel representing 0.1, 0.2 and 0.3 kb, or the 50 bp DNA ladder (Invitrogen) (GAA-30X; uncut and XbaI) with bands from the bottom of the gel representing 0.1, 0.15, 0.2, 0.25, 0.3 and 0.35 kb. ( C ) DSB repair within a slightly unstable (GAA•TTC) 70–79 sequence produced a dramatic rise in instability. Note that the (GAA•TTC) n sequence was equally unstable in the GAA and TTC orientations. ( D ) DSB repair within a highly stable (GAA•TTC) 30 sequence produced a dramatic rise in instability. Note that the (GAA•TTC) n sequence was equally unstable in the GAA and TTC orientations. All error bars represent +/−2 SEM derived from triplicate experiments.

    Article Snippet: The GAA-70X construct, which contains the (GAA•TTC)70 sequence with an XbaI site located exactly at the center of the repeat tract , was created using four synthetic oligonucleotides, as follows: Oligo #1: 5′-GGCGCTCCGCTGCAGCC(GAA)35 TCTAGACGCATCGCC-3′ and Oligo #2: 5′GGCGATGCGTCTAGA(TTC)35 GGCTGCAGCGGAGCGCC-3′ were annealed together in 10 mM Tris, pH 8.0, and digestion buffer 3 (100 mM NaCl, 10 mM MgCl2 ) (New England Biolabs), followed by incubating with PstI and XbaI restriction enzymes to digest both ends of the annealed oligos.

    Techniques: Sequencing, Polymerase Chain Reaction, Marker, Produced, Derivative Assay

    DSB repair-mediated instability of the (GAA•TTC) n sequence is independent of the length of the intervening sequence at the center of the repeat tract. ( A ) GAA-70-spacer construct containing a 28 bp spacer in the XbaI site of GAA-70X, such that BamHI would cut in the center of the spacer (indicated by the black box). ( B ) DSB repair at the BamHI site in GAA-70-spacer construct produced a dramatic rise in instability. Note that the (GAA•TTC) n sequence was equally unstable when transformed into E. coli MM28 [wild-type (WT)] and its isogenic recA mutant strain, M152. All error bars represent +/−2 SEM derived from triplicate experiments. ( C ) DSB repair at the center of the (GAA•TTC) n sequence results in the preferential deletion of approximately half of the total repeat length. The residual tract lengths of the (GAA•TTC) n sequence are shown (as a percentage of full-length) after transformation of BamHI-linearized GAA-70-spacer vector in E. coli MM28 (WT) and M152 ( recA ).

    Journal: Nucleic Acids Research

    Article Title: Repair of DNA double-strand breaks within the (GAAoTTC)n sequence results in frequent deletion of the triplet-repeat sequence

    doi: 10.1093/nar/gkm1066

    Figure Lengend Snippet: DSB repair-mediated instability of the (GAA•TTC) n sequence is independent of the length of the intervening sequence at the center of the repeat tract. ( A ) GAA-70-spacer construct containing a 28 bp spacer in the XbaI site of GAA-70X, such that BamHI would cut in the center of the spacer (indicated by the black box). ( B ) DSB repair at the BamHI site in GAA-70-spacer construct produced a dramatic rise in instability. Note that the (GAA•TTC) n sequence was equally unstable when transformed into E. coli MM28 [wild-type (WT)] and its isogenic recA mutant strain, M152. All error bars represent +/−2 SEM derived from triplicate experiments. ( C ) DSB repair at the center of the (GAA•TTC) n sequence results in the preferential deletion of approximately half of the total repeat length. The residual tract lengths of the (GAA•TTC) n sequence are shown (as a percentage of full-length) after transformation of BamHI-linearized GAA-70-spacer vector in E. coli MM28 (WT) and M152 ( recA ).

    Article Snippet: The GAA-70X construct, which contains the (GAA•TTC)70 sequence with an XbaI site located exactly at the center of the repeat tract , was created using four synthetic oligonucleotides, as follows: Oligo #1: 5′-GGCGCTCCGCTGCAGCC(GAA)35 TCTAGACGCATCGCC-3′ and Oligo #2: 5′GGCGATGCGTCTAGA(TTC)35 GGCTGCAGCGGAGCGCC-3′ were annealed together in 10 mM Tris, pH 8.0, and digestion buffer 3 (100 mM NaCl, 10 mM MgCl2 ) (New England Biolabs), followed by incubating with PstI and XbaI restriction enzymes to digest both ends of the annealed oligos.

    Techniques: Sequencing, Construct, Produced, Transformation Assay, Mutagenesis, Derivative Assay, Plasmid Preparation