Journal: Nucleic Acids Research
Article Title: Targeted detection of in vivo endogenous DNA base damage reveals preferential base excision repair in the transcribed strand
Figure Lengend Snippet: Schematic representation of PADDA and identified DNA damage. ( A ) A single non-cycled primer extension performed with a 5′-biotin-tagged primer and Vent exo– DNA polymerase identifies damaged nucleotides (inverted triangles), and generates a pool of highly specific biotin-tagged extended products (EP), each of them derived from a single DNA molecule and strand. Each extended product has a stop (indicated by a red star), which represents replicative arrest by a damaged nucleotide, a nick or random polymerase stalling. Some extended products will contain misincorporations (indicated by a solid red circle) that represent polymerase lesion by-pass with misincorporation. After several purification steps that include the use of biotin-binding, streptavidin-coated, paramagnetic beads, the strand-specific, highly purified, biotin-bound extended products can be used for damage fingerprinting or quantification. ( B ) For strand specific fingerprinting analysis (f-PADDA) of damage and repair at the single-nucleotide level, the purified pool of EPs is ligated to a chemically modified oligonucleotide-adapter using a highly efficient single-strand DNA ligase. The pool of adapter-linked EPs can be diluted to one EP per well and each EP is directly amplified and sequenced, or the EPs can be amplified as a pool, cloned and sequenced. In either case high fidelity amplification is performed using an oligonucleotide complementary to the adapter–primer and a nested gene-specific primer. Data is obtained in
Article Snippet: Vent exo–, a truncated form of a native DNA polymerase (Vent, NEB), was chosen for this assay instead of the currently used Sequenase ( ) or Taq DNA polymerase , because of its reported high sensitivity to detect DNA damage ( ) and primer-extension efficiency ( ).
Techniques: Derivative Assay, Purification, Binding Assay, Modification, Amplification, Clone Assay