Pyromark reagents

Wild-type and heterozygous embryos with expressed polymorphisms at Chromosome 12 were obtained from reciprocal crosses between del^L1rep heterozygous mutants (C57BL/6 background) and Mus musculus castaneus (strain CAST/EiJ, abbreviated Cast) (Jackson laboratories) or Mus musculus molossinus 12 (strain B6.MOLF12A, abbreviated Mol 12) (Supplemental Table S7). The B6.MOLF12A congenic strain carries an 8 cM region of Chromosome 12 from the Mus musculus molossinus strain MOLF/Ei on a C57BL/6 genetic background. Allelic expression of eight transcripts was assessed by PCR amplification of fetal (E16.5) brain, liver, and placenta cDNA with biotinylated primers, followed by pyrosequencing. Primer sequences for PCR amplification of the analyzed polymorphisms and amplification conditions are shown in Supplemental Table S7. Pyrosequencing was carried out with up to 15 µL of PCR product. The biotinylated strand was purified using Strepdavidin Sepharose High Performance Beads (GE Healthcare), washed, denatured, and annealed to the pyrosequencing primer (0.3 µM) using PyroMark reagents (Qiagen), as described (Tost and Gut 2007 (link)). Pyrosequencing was performed on a PyroMark MD pyrosequencer (Biotage) using PyroMark Gold Qp6 SQA reagents (Roche), and the data were analyzed with the PyroMark MD version 1.0 software (Biotage).

Peripheral venous blood samples were collected into EDTA-anticoagulate tubes. Genomic DNA was extracted using a E.Z.N.A.^® SQ Blood DNA Kit II (Omega Bio-Tek, USA) according to the manufacturer’s instructions and stored at − 80 °C until use. Genotyping of UGT1A1 polymorphisms was carried out by polymerase chain reaction (PCR) and pyrosequencing as described previously [32 (link)]. Briefly, The DNA fragments flanking the UGT1A1*28 or *6 polymorphisms were amplified using Mastercycler (Eppendorf, Germany) in a final reaction volume of 50 µl, which contained 2 µl DNA, 5 µl PCR buffer, 1.5 µl dNTP, 0.5 ul DNA polymerase, 0.05 nM of each amplification primer (Additional file 1: Table S1), and 40 μl sterile double-distilled water. The thermal cycling of PCR was used as follows: degeneration at 94 °C for 5 min; 35 cycles of 94 °C for 30 s, 58 °C or 66 °C for 30 s respectively, and 72 °C for 30 s; and a final extension 72 °C for 7 min. The PCR products were verified by agarose electrophoresis, followed by pyrosequencing on the PyroMark Q24 Advanced platform (Qiagen, Germany) using PyroMark Reagents (Qiagen, Germany) with the pyrosequencing primer (Additional file 1: Table S1). Genotyping results of each SNP were verified in 5% samples selected randomly using Sanger sequencing.

DNA was extracted from cells using the Puregene system (Qiagen) or Wizard SV Genomic DNA Purification System (Promega), and the proportion of wild-type mtDNA and m.3243A>G was determined by pyrosequencing, which has been validated for quantification of m.3243A>G heteroplasmy^{34 (link)}. Briefly, a 155 base pair region of human mtDNA encompassing the m.3243A>G site was amplified using the PyroMark PCR kit (Qiagen) (all primers are listed in Table S1). Pyrosequencing reactions were performed using a sequencing primer and PyroMark reagents (Qiagen) on a PSQ 96MA pyrosequencer and analysed with PSQ 96MA 2.1 software. Pyrosequencing exhibited a standard deviation range of 0.06–4.64% change in heteroplasmy across 359 samples measured in triplicate. Last-cycle PCR of sequence spanning 1155–1725 bp of human mtDNA that includes an invariant ApaI site was used as a positive control to confirm complete digestion. Alternatively, heteroplasmy was measured by restriction fragment length polymorphism analysis, using amplified mtDNA spanning 2966–3572 bp; and the mutant load was estimated from the proportion of DNA cleaved by ApaI, after separation of digested PCR product via agarose gel electrophoresis^{12 (link)}.

Sodium bisulphite mutagenesis was carried out on 1 µg gDNA per sample using the two-step conversion protocol of the Imprint DNA Modification Kit (Sigma). Two samples with no template were included to confirm contamination had not occurred during bisulphite treatment. Five microliters of bisulphite-treated sample were PCR amplified with biotinylated primers (Forward 5′-GTGGTTTGTTATGGGTAAGTTT, Reverse 5′-Btn-CCCTTCCCTCACTCCAAAAATTAA, 54°C annealing for 36 cycles) and quality controlled by gel electrophoresis of 5 µL of each PCR reaction. A volume of up to 15 µL PCR product was then used for pyrosequencing, which was carried out as previously described (Tost and Gut 2007 (link)). The biotinylated strand was purified using Strepdavidin Sepharose High Performance Beads (GE Healthcare), washed, denatured, and annealed to the pyrosequencing primer (0.3 µM 5′-TGGTTTATTGTATATAATGT) using PyroMark reagents (Qiagen). Pyrosequencing was performed on a PyroMark MD pyrosequencer (Biotage) using PyroMark Gold Qp6 SQA reagents (Roche), and the data were analyzed with the Pyro Q-CpG 1.0.9 software (Biotage).