Journal: Nucleic Acids Research

Article Title: Pathways and products of base excision DNA repair in Xenopus laevis eggs: contrast with human cell pathways

doi: 10.1093/nar/gkaf1326

Figure Lengend Snippet: The isotopic labeling plasmid approach to determine BER patch size. ( A ) The target DNA fragment for “oligo swapping” was inserted into the multicloning site of pUC19, and one of the Nt.BspQI sites was deleted. ( B ) The DNA sequence flanking the target site, indicating the cut sites of key restriction enzymes. The incoming biotin-labeled synthetic oligonucleotide is shown above the target sequence; the *G indicates isotopically labeled dGMP residues (fully substituted with 13 C and 15 N). The segment highlighted in green is used for mass spectrometry analysis. ( C ) Overview of the oligo swapping procedure (see the “Materials and methods” section for details). ( D ) Stepwise products from the substrate synthesis procedure in panel (C) were loaded on an ethidium bromide-containing agarose gel (lanes 2–5) and subjected to electrophoresis. A DNA ladder was loaded in lane 1. To determine the oligo swapping efficiency, the plasmid substrate was digested with UDG and NdeI (lane 6): removal of U prevents cleavage by NdeI. ( E ) Mass spectrometry analysis confirming the target fragment released from plasmid substrate by NdeI and HaeII. ( F ) A sample ethidium bromide-containing agarose gel image showing the repair product of plasmid substrate by Xenopus HSS at the indicated time points, with the repaired substrate susceptible to UDG and NdeI cleavage (lanes 6–9). A DNA ladder was loaded in lane 1. Native pUC19 was digested with PstI (lane 2) or NdeI (lane 3) for size markers. Plasmid substrate was digested with PstI (lane 4) or UDG + NdeI (lane 5) to confirm the presence of uracil. P, repaired product; U, unrepaired substrate. ( G ) Repair rates for U:A and rAP:A substrates incubated with Xenopus HSS. Gel images were quantified using ImageJ. * P < .05. n = 3 biological replicates and the data are means ± SD.

Article Snippet: Subsequently, 50 ng of sample DNA and 10 ng of pUC19 control DNA (NEB, N3041S) were separately added to the thawed competent cells, and the tubes were flicked gently.

Techniques: Isotopic Labeling, Plasmid Preparation, Sequencing, Labeling, Mass Spectrometry, Agarose Gel Electrophoresis, Electrophoresis, Incubation

Journal: Nucleic Acids Research

Article Title: Pathways and products of base excision DNA repair in Xenopus laevis eggs: contrast with human cell pathways

doi: 10.1093/nar/gkaf1326

Figure Lengend Snippet: The isotopic labeling plasmid approach to determine BER patch size. ( A ) The target DNA fragment for “oligo swapping” was inserted into the multicloning site of pUC19, and one of the Nt.BspQI sites was deleted. ( B ) The DNA sequence flanking the target site, indicating the cut sites of key restriction enzymes. The incoming biotin-labeled synthetic oligonucleotide is shown above the target sequence; the *G indicates isotopically labeled dGMP residues (fully substituted with 13 C and 15 N). The segment highlighted in green is used for mass spectrometry analysis. ( C ) Overview of the oligo swapping procedure (see the “Materials and methods” section for details). ( D ) Stepwise products from the substrate synthesis procedure in panel (C) were loaded on an ethidium bromide-containing agarose gel (lanes 2–5) and subjected to electrophoresis. A DNA ladder was loaded in lane 1. To determine the oligo swapping efficiency, the plasmid substrate was digested with UDG and NdeI (lane 6): removal of U prevents cleavage by NdeI. ( E ) Mass spectrometry analysis confirming the target fragment released from plasmid substrate by NdeI and HaeII. ( F ) A sample ethidium bromide-containing agarose gel image showing the repair product of plasmid substrate by Xenopus HSS at the indicated time points, with the repaired substrate susceptible to UDG and NdeI cleavage (lanes 6–9). A DNA ladder was loaded in lane 1. Native pUC19 was digested with PstI (lane 2) or NdeI (lane 3) for size markers. Plasmid substrate was digested with PstI (lane 4) or UDG + NdeI (lane 5) to confirm the presence of uracil. P, repaired product; U, unrepaired substrate. ( G ) Repair rates for U:A and rAP:A substrates incubated with Xenopus HSS. Gel images were quantified using ImageJ. * P < .05. n = 3 biological replicates and the data are means ± SD.

Article Snippet: The process commenced with the expansion of pUC19 (NEB, N3041S).

Techniques: Isotopic Labeling, Plasmid Preparation, Sequencing, Labeling, Mass Spectrometry, Agarose Gel Electrophoresis, Electrophoresis, Incubation