Qiasymphony dna midi kit

DNA samples were extracted from buffy coats using the QIAsymphony DNA Midi Kit (Qiagen, Crawley, UK). Bisulfite treatment of 2 mg of each sample was performed using the EZ DNA Methylation Kit (Zymo Research, Orange, CA, USA). The converted DNA was eluted in 50 ml of 0.1X TE buffer and pyrosequenced as previously described [17 (link)].

Genomic DNA was extracted from all blood samples using a Qiagen kit (QIAsymphony DNA midi Kit, Cat # 931255) as per protocols provided (Qiagen, Germantown, MD, USA). To identify the causal gene variant in both families, targeted next generation sequencing (NGS) was performed on two affected and two unaffected family members in Family 1 (Fig. 2A, IV.1, IV.3, V.1 and V.2) and two affected and two unaffected members of Family 2 (Fig. 2B II.1, II.2, II.6 and III.1). Libraries were prepared with the extracted DNA using a Kapa Biosystems kit as per manufacturers instruction (Massachusetts, USA), and hybridized on a custom designed 20MB panel (details of this panel are provided in the Suppl. Table 8) following the manufacturer’s protocol. Libraries were then subjected to paired-end sequencing on an Illumina HiSeq. 2500, leading to the identification of a germline mutation in the MLH1 gene (see above). All family members from both families were then screened for confirmation for the presence of the identified mutation in the MLH1 gene by Sanger sequencing using standard protocols on an ABI 3730xl instrument using the following oligomers purchased from PxLence (Ghent, Belgium) (Cat #: PXL A0182967; context sequence: 3:36996535–36996928).

Blood DNA was extracted using either the QIASymphony robot (Qiagen) together with the QIASymphony DNA Midi kit (Qiagen) or manually by NucleoSpin Blood, Mini DNA extraction kit (Macherey–Nagel). Tissue DNA was extracted using the AllPrep DNA/RNA/miRNA extraction kit (Qiagen). Tissue pieces stored in RNAlater were homogenized in RLT buffer using the BeadBeater (Biospec) with zirconia beads and after cell lysis the RNA and DNA containing fractions were separated. Concentration and purity were measured using Qubit Fluorometric Quantification (ThermoFisher).

DNA was extracted from EDTA blood using either manual salt extraction or the QIASymphony DNA Midi kit (Qiagen, Venlo, Netherlands) on the QIASymphony robot (Qiagen). DNA was extracted from muscle tissue using the DNeasy blood and tissue kit (Qiagen). Buccal swab samples were stored on FTA cards from which DNA subsequently was eluted using a boiling method as follows: first, 2–3 punches of each FTA card were placed in a tube and washed three times in 180 μl ddH₂O; second, elution was done by adding 25 μl ddH₂O to each sample and boiling this for 30 min at 95 °C.

Genomic DNA was extracted from FFPE tissue sections using the QIAamp FFPE Tissue Kit (Qiagen, Manchester, UK) based upon the manufacturer’s protocols for both tumour and non-malignant content. The extraction of genomic DNA from blood samples was completed using the QIAamp Blood mini kit (manual) or QIAsymphony DNA Midi Kit (automated) (Qiagen) using the manufacturer’s protocols. The quality of the extracted DNA was analysed using the Agilent 2200 Tapestation (Agilent, Stockport, UK) and the Qubit Fluorometer (Fisher Scientific, Loughborough, UK). Both DNA extraction and next generation sequencing (NGS) were completed at Good Clinical Laboratory Practice (GCLP)-accredited laboratories.

For skin tissue, 5 cm of a distal tail portion were cut with scissors. Skin was separated and hair removed using scalpel. Tissue was placed in a tube and stored as described for other tissues.
DNA was extracted from blood on an automated nucleic acid extraction platform called QiaSymphony (Qiagen) with a magnetic bead-based extraction kit, QiaSymphony DNA Midi Kit (Qiagen). DNA was extracted from tissue on an automated nucleic acid extraction platform called Anaprep (Biochain) with a magnetic bead-based extraction kit, Tissue DNA Extraction Kit (Biochain). DNA from brain regions was extracted using an automated nucleic acid extraction platform called QIAcube HT (Qiagen) with a column-based extraction kit, QIAamp 96 DNA QIAcube HT Kit (Qiagen).