Viable (DAPI−) germinal centre B cells (CD19-APC+, PNAhigh-FITC, CD95-PE+) were sorted from Peyer's patches of 8-week old PrimPolΔ/Δ and their wild-type littermates on an Aria® sorter (Becton Dickinson). DNA was extracted using proteinase K treatment and ethanol precipitation. The JH4 intronic sequence of rearranged VHJ558 family members were amplified by PCR using Pfu Ultra® polymerase (Stratagene) (31 (link)). PCR products were purified using the QIAquick® Gel Extraction kit (Qiagen), cloned into the TOPO II blunt vector (Invitrogen Life Technologies). Plasmid DNA was isolated using High Pure Plasmid kit (Roche) and sequenced on a 3730 DNA analyzer (Applied Biosystems). Sequence alignments were performed using Seqman software (DNAStar version 12,2,0).
Calculations exclude non-mutated sequences, insertions and deletions. Clonally related sequences with identical mutations and complementary determining region 3 and duplicated sequences were counted only once. Statistic analysis of the mutation spectra was performed using the χ2-test, with the number of mutations of base x and number sequenced base x. For the corrected percentage, the number of mutations were adjusted such that each base contributes 25% of the sequence. Mutation frequencies are expressed as the percentage of a defined nucleotide substitution at base (X) relative to all sequenced bases (X).
Calculations exclude non-mutated sequences, insertions and deletions. Clonally related sequences with identical mutations and complementary determining region 3 and duplicated sequences were counted only once. Statistic analysis of the mutation spectra was performed using the χ2-test, with the number of mutations of base x and number sequenced base x. For the corrected percentage, the number of mutations were adjusted such that each base contributes 25% of the sequence. Mutation frequencies are expressed as the percentage of a defined nucleotide substitution at base (X) relative to all sequenced bases (X).