Sanger dna sequencing

PCR amplification of CAG repeats located in exon 1 of HTT in HD cell lines used forward (5′-ATGAAGGCCTTCGAGTCCCTCAAGT CCTTC-3′) and reverse (5′-CTGAGGCAGCAGCGG CTGTGCCTGCG-3′) primers. PCRs were performed in a volume of 25 μl containing 100 ng genomic DNA, 1.6 mM of each dNTP, 4 pmol of each primer, and 0.5 U Q5 DNA polymerase (New England Biolabs). After an initial denaturation of 4 min at 98 °C, 40 cycles of 45 s at 98 °C, 1 min at 68 °C, and 3 min at 72 °C were carried out, followed by a final extension of 10 min. PCR products were resolved in a 7.5% (wt/vol) denaturing polyacrylamide gel, followed by Southern blot analysis using a ³²P-labeled (CTG)₅ oligonucleotide probe. PCR products were then visualized by an Amersham Typhoon phosphor imager as described (49 (link)).
To analyze the expanded CTG repeats derived from Polθ-catalyzed primer extension, DNA bands (³²P-labeled) were excised and eluted from gels, then PCR-reamplified for 35 cycles by REDTaq DNA polymerase (Sigma-Aldrich) using primers 5′-ACGTTGTAAAACGACGGCCA-3′ (forward) and 5′-CATGATTACGAATTC-3′ (reverse), essentially as described above. PCR products were cloned into a pGEM-T vector (Promega) and transfected into E. coli DH5-Alpha (Thermo Fisher). Plasmid DNAs were isolated and subjected to Sanger DNA sequencing (Source BioScience).

Recombinant plasmids were transformed into BL1 (DE3) cells for protein expression. They were then verified via Sanger DNA sequencing (Source Bioscience Ltd). The bacterial cells were grown as previously reported [28 (link)]. The GST-tagged recombinant protein was purified by affinity chromatography (columns) and quantified by the Bradford assay. Purified mutant proteins were analyzed on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS–PAGE) gels to verify protein production.

MRE11 sequences were inserted into the vector pLenti-puro-CMV (P100022; Vigene, Rockville, MD). A QuickChange II XL Site-Directed Mutagenesis Kit (#200521; Agilent, Santa Clara, CA) and XL10-Gold ultracompetent cells were used for all site-directed mutagenesis according to the manufacturers’ instructions. Sanger DNA sequencing (Source Bioscience, Nottingham, UK) was used to confirm the mutated nucleotides and the deleted sequences.