DNA substrates and hairpin repair assays. (A) DNA hairpin substrates. Circular DNA hairpin heteroduplexes were constructed using the M13mp18 bacterial phage series as described  (also see Methods for details). The blue and red types/lines represent CAG and CTG repeats, respectively, which are located between HindIII and EcoRI restriction enzyme sites of the plasmid. Each substrate contains a (CAG)25 or (CTG)25 hairpin either in the complementary (C) or viral (V) strand and a strand break (at the BglI recognition position) 5' to the hairpin. Substrates with a (CAG)25 or (CTG)25 hairpin in the V strand were named V-(CAG)25 or V-(CTG)25, respectively, while substrates with a (CAG)25 or (CTG)25 hairpin in the C strand were referred to as C-(CAG)25 or C-(CTG)25, respectively. Blue and red bars represent oligonucleotide probes that anneal to the nicked strand near the BglI and BsmBI sites, respectively. (B and C) Schematic diagrams of hairpin repair (HPR) assays. Given that CAG/CTG HPR only occurs in the nicked strand via incisions, followed by DNA resynthesis using the continuous strand as a template [12, 13], the repair would result in either the hairpin sequence removal (B) or addition (C) depending on if the hairpin is located in C or V strand, respectively. The change in DNA length can be detected by Southern hybridization using an oligonucleotide probe (i.e., the blue or red bar) specifically annealing to the nicked strand.