Uracil dna glycosylase

Lambda exonuclease, RNase H, RNase HII and uracil-DNA glycosylase were purchased from New England Biolabs and TURBO DNase from Invitrogen and were used at the recommended concentrations and with supplied buffers. RNase A was obtained from Sigma-Aldrich and applied at 100 nM concentration in 0.1 M MES buffer. For each biochip cleavage assay, appropriate nucleic acid substrates were synthesized. In the case of double-stranded substrates, the biochip was synthesized with oligonucleotides capable of self-annealing to form hairpin-loop structures. Cleavage was detected through either the loss of fluorescence of Cy3-terminally labeled sequences, or loss of fluorescence from Cy3-labeled oligonucleotides hybridized to the nuclease substrate. In time course experiments, the enzymatic reaction was stopped by washing the surface followed by drying with argon.

First-strand complementary DNA (cDNA) was synthesized using random hexamer primers and M-MuLV reverse transcriptase (RNase H-) [73 (link)], with rRNA-depleted RNA used as a template. Second-strand cDNA was then synthesized with dNTPs, DNA polymerase I and RNase H. Next, T4 DNA polymerase and Klenow DNA polymerase were used to repair and modify the ends to add an A base and ligate the sequencing adapter. The cDNA products were then purified using AMPure XP beads (Beckman Coulter, Brea, CA, USA). Finally, uracil DNA glycosylase (NEB, Ipswich, MA, USA) was used to degrade the U-containing chain to remove second-strand cDNA. The purified first-strand cDNA was enriched by PCR to obtain a cDNA library. The quality of the libraries was assessed using an Agilent 2100 Bioanalyzer, and sequencing was performed using paired-end sequencing (2*150 bp) with the Illumina HiSeq 4000 platform (LC Sciences, Houston, TX, USA).

To characterize the frequencies of random mutations in RASFC and synovial biopsy samples, we used the mitochondrial Random Mutation Capture assay as described previously73 (link). Mitochondrial DNA was extracted using a previously reported protocol21 (link). Following extraction, 10 μg of mtDNA was digested with 100 units of Taq αI restriction enzyme (New England Biolabs), 1X bovine serum albumin, and a Taq αI–specific digestion buffer (10 mM Tris HCl, 10 mM MgCl_2, 100 mM NaCl [pH 8.4]) for 10 hrs, with 100units of Taq αI added to the reaction mixture every hour. PCR amplification was performed in 25 μl reaction mixtures containing 12.5 μl 2X SYBR Green Brilliant Mastermix (Stratagene), 0.1 μl uracil DNA glycosylase (New England Biolabs), 0.7 μl forward and reverse primers (10pM/μl; IDT), and 6.7 μl H₂O. The samples were amplified using a Roche LightCycler 480, according to the following protocol; 37 °C for 10 mins, 95 °C for 10 mins, followed by 45 cycles of 95 °C for 15secs and 60 °C for 1 min. Samples were kept at 72 °C for 7 mins and following melting-curve analysis, immediately stored at −80 °C. The primer sequences used were as follows: for mtDNA copy number 5′-ACAGTTTATGTAGCTTACCTCC-3′ (forward) and 5′-TTGCTGCGTGCTTGATGCTTGT-3′ (reverse); for random mutations 5′-CCTCAACAGTTAAATCAACAAAACTGC-3′ (forward) and 5′-GCGCTTACTTTGTAGCCTTCA-3′ (reverse).

mtDNA mutation frequency was detected by random mutation capture assay as described in our published article (Wu et al., 2015 (link)). In summary, two primer pairs were designed for PCR reactions. One is flanking the Taq1 sites at mtDNA positions 1427 and 8335, the other primer pair does not flank a TaqI restriction sites as controls. PCR amplification was performed in 25 μL reactions, containing 2 μL of 10 μM forward and reverse primers, 0.2 μL of uracil DNA glycosylase (New England Biolabs, Beverly, MA, USA), 12.5 μL 2× Brilliant SYBR Green I Master Mix (Stratagene, La Jolla, CA, USA) and 3.3 μL H₂O. Then the PCR product was digested with TaqI restriction enzyme and the mtDNA mutation frequency was determined by analysis of the band size of gel electrophoresis. The mutation frequency was showed as the mutation number per million bases.
LX-PCR assay was performed to determine DNA damage using GeneAmp XL PCR kit (Applied Biosystems, CA, USA) as previously described (Wu et al., 2015 (link)). DNA damage was quantified by comparing the ratio between the long and short fragments of PCR amplicons (mtDNA = 210 bp/13.4 kb).