Properties and efficient scrap-and-build repairing of mechanically sheared 3’ DNA ends

Abstract

Repairing of DNA termini is a crucial step in a variety of DNA handling techniques. In this study, we investigated mechanically-sheared DNA 3’-ends (MSD3Es) to establish an efficient repair method. As opposed to the canonical view of DNA terminus generated by sonication, we showed that approximately 47% and 20% of MSD3Es carried a phosphate group and a hydroxyl group, respectively. The others had unidentified abnormal terminal structures. Notably, a fraction of the abnormal 3’ termini (about 20% of the total) was not repaired after the removal of 3’ phosphates and T4 DNA polymerase (T4DP) treatment. To overcome this limitation, we devised a reaction, in which the 3’− > 5’ exonuclease activity of exonuclease III (3’− > 5’ exonuclease, insensitive to the 3’ phosphate group) was counterbalanced by the 5’− > 3’ polymerase activity of T4DP. This combined reaction, termed “SB-repairing” (for scrap-and-build repairing), will serve as a useful tool for the efficient repair of MSD3Es.

Local states of chromatin compaction at transcription start sites control transcription levels

“…To examine nucleosome arrays in Fr-5 chromatin particles, DNA fragments in the chromatin were extended using terminal deoxynucleotidyl transferase (TdT), and observed by HS-AFM. Briefly, Fr-5 chromatin was immunoprecipitated as described above, and treated with T4 DNA polymerase (#311–02481, Nippon Gene) and exonuclease III (#2170A, Takara Bio) to repair DNA termini as described previously ( 41 ). After the chromatin was collected by re-immunoprecipitation, biotin-16-dUTP (#11093070910, Roche) was added to the DNA termini using TdT (#M828A, Promega).…”

RNA Viral Metagenome Analysis of Subnanogram dsRNA Using Fragmented and Primer Ligated dsRNA Sequencing (FLDS)

“…Therefore, we‍ ‍compared five types of oligonucleotide adaptors (U2-DNA-5P3P, U2-DNA-5P3H, U2-DNA-5P3am, U2-DNA-5OH3H, and U2-RNA-5P3H) for cDNA synthesis in the FLDS library construction. Based on the chemical structures of the 3′ end of mechanically-sheared dsDNA ( Ohtsubo et al , 2019 ), oligonucleotides with phosphorylated or hydroxylated 5′-ends were prepared. To prevent ligation among the oligonucleotide adaptors, appropriate chemical modifications were prepared for the 3′-ends.…”

“…In the initial test, 1‍ ‍ng of mechanically-sheared dsRNA was used as a template, and 25 and 35 PCR cycles were used for cDNA amplification. Unexpectedly, insufficient cDNA amplification was obtained using U2-DNA-5OH3H despite the expected dominance of a phosphate group in the 3′ end of mechanically-sheared dsRNA, as in the case of dsDNA ( Ohtsubo et al , 2019 ). In the sequencing libraries constructed using other U2 adaptors, the abundance of high-quality reads (depicted as Clean in Fig.…”

“…2 ( Urayama et al , 2018 ). A recent study reported that the 3′-end of mechanically-sheared dsDNA carries a phosphate group, hydroxyl group, and unidentified structures ( Ohtsubo et al , 2019 ). This finding suggests that a reassessment of the terminal residue modifications in oligonucleotide adaptors will increase the efficiency of adaptor ligation and subsequent cDNA synthesis in FLDS if similar structures occur in mechanically-sheared dsRNA.…”

The integrity and assay performance of tissue mitochondrial DNA is considerably affected by choice of isolation method

“…Single-strand DNA breaks can be inflicted by numerous factors, of which DNA shearing, oxidative damage and various endonucleases appear most relevant to our isolation procedure. Shearing and oxidative damage to DNA often generate ‘dirty’ DNA ends that require further processing before ligation can occur (Bertoncini and Meneghini, 1995; Caldecott, 2008; Ohtsubo et al , 2019), whereas nucleases can leave ‘clean’ DNA ends that are directly ligatable. To determine whether the ssDNA breaks observed in our samples contained ligatable ends, we treated the DNA preparations from adult skeletal muscle with T4 DNA ligase and separated ligase-treated and untreated samples on a denaturing gel (Fig.…”